Patent application title: Method of Reducing The Effects of Chemotherapy Using Flagellin Related Polypeptides
Inventors:
Andrei V. Gudkov (East Aurora, NY, US)
Andrei V. Gudkov (East Aurora, NY, US)
Vadim Krivokrysenko (Orchard Park, NY, US)
Lyudmila Burdelya (Lancaster, NY, US)
IPC8 Class: AA61K3816FI
USPC Class:
514 49
Class name: Peptide (e.g., protein, etc.) containing doai weight regulation affecting appetite or satiation affecting
Publication date: 2013-12-05
Patent application number: 20130324462
Abstract:
The use of flagellin and flagellin related polypeptides for reducing
cancer treatment side effects in mammals is described.Claims:
1.-7. (canceled)
8. A method of reducing a side effect of cancer treatment in a mammal in need thereof, comprising administering a therapeutically effective amount of a composition comprising flagellin, wherein the cancer therapy comprises radiation therapy and cisplatinum.
9. The method of claim 8, wherein the mammal is a human.
10. The method of claim 8, wherein the cisplatinum is administered at 50-270 mg/m2/day.
11. The method of claim 8, wherein the side effect is one or more of myelosuppression, renal toxicity, weight loss pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, numbness, changes in tastes, loss of appetite, thinned or brittle hair, mouth sores, memory loss, hemorrhage, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment.
12. The method of claim 11, wherein the behavioral change is limited mobility.
13. The method of claim 11, wherein the side effect is selected from the group consisting of: myelosuppression, renal toxicity, and weight loss.
14. The method claim 11, wherein the mammal is a human.
15. The method of claim 11, wherein the cisplatinum is administered at 50-270 mg/m2/day.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent application Ser. No. 12/369,704, filed Feb. 11, 2009, which claims the benefit of U.S. Patent Application Ser. No. 61/027,729, filed Feb. 11, 2008, and which also is a Continuation-in-Part of U.S. patent application Ser. No. 11/421,918, filed Jun. 2, 2006, now U.S. Pat. No. 7,638,485. U.S. patent application Ser. No. 11/421,918, filed Jun. 2, 2006, is a Continuation of PCT/US04/40753, filed Dec. 2, 2004, which claims the benefit of U.S. Patent Application Ser. No. 60/526,460, filed Dec. 2, 2003, U.S. Patent Application Ser. No. 60/526,461, filed Dec. 2, 2003, U.S. Patent Application Ser. No. 60/526,666, filed Dec. 2, 2003, and U.S. Patent Application Ser. No. 60/526,496, filed Dec. 2, 2003, the contents of all of which are hereby incorporated by reference in their entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: CLEV--002--09US_SeqList_ST25.txt, date recorded: May 16, 2013, file size 155 kilobytes).
FIELD OF THE INVENTION
[0003] This invention relates to the use of flagellin related polypeptides to reduce the side effects of cancer treatments, such as chemotherapy, in mammals.
BACKGROUND OF THE INVENTION
[0004] The progression from normal cells to tumor cells involves a loss of negative mechanisms of growth regulation, including resistance to growth inhibitory stimuli and a lack of dependence on growth factors and hormones. Traditional cancer treatments that are based on radiation or cytotoxic drugs rely on the differences in growth control of normal and malignant cells. Traditional cancer treatments subject cells to severe genotoxic stress. Under these conditions, the majority of normal cells become arrested and therefore saved, while tumor cells continue to divide and die.
[0005] However, the nature of conventional cancer treatment strategy is such that normal rapidly dividing or apoptosis-prone tissues are at risk. Damage to these normal rapidly dividing cells causes the well-known side effects of cancer treatment (sensitive tissues: hematopoiesis, small intestine, hair follicles). The natural sensitivity of such tissues is complicated by the fact that cancer cells frequently acquire defects in suicidal (apoptotic) machinery and those therapeutic procedures that cause death in normal sensitive tissues may not be that damaging to cancer cells. Conventional attempts to minimize the side effects of cancer therapies are based on (a) making tumor cells more susceptible to treatment, (b) making cancer therapies more specific for tumor cells, or (c) promoting regeneration of normal tissue after treatment (e.g., erythropoietin, GM-CSF, and KGF).
[0006] There continues to be a need for therapeutic agents to mitigate the side effects associated with chemotherapy and radiation therapy in the treatment of cancer. This invention fulfills these needs and provides other related advantages.
SUMMARY OF THE INVENTION
[0007] Provided herein is a method of reducing a side effect of cancer treatment in a mammal, which may comprise administering to a mammal in need thereof a composition comprising flagellin. The mammal may be a human.
[0008] The effect may be selected from the group consisting of: myelosuppression, renal toxicity, weight loss, behavioral changes, pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, mouth sores, memory loss, hemorrhage, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment. The behavioral change may be limited mobility. The composition may be administered prior to, concomitantly with, or after the cancer treatment.
[0009] The cancer treatment may comprise chemotherapy and may also comprise radiation therapy. The chemotherapy may be selected from the group consisting of: cisplatinum, cyclophosphamide, doxorubicin, 5-fluorouracil, camptothecin, methotrexate, melphalan, taxanes, isosfamide, melphalan, hexamethyl-melamine, thiotepa, dacarbazine, cytarabine, 2-fluorodeoxycytidine, idatrexate, trimetrexate, vinblastine, vincristine, navelbine, estramustine, taxoids, etoposide, teniposide, daunorubicine, bleomycin, mitomycin, L-asparaginase, topotecan, procarbazine, mitoxantrone, carboplatinum, interferon, and interleukin.
[0010] Also provided herein is a method of reducing cisplatin-associated toxicity in a mammal, which may comprise administering to a mammal in need thereof a composition comprising flagellin. The cisplatin-associated toxicity may be selected from the group consisting of: myelosuppression, renal toxicity, weight loss, and behavior changes. The mammal may be a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 demonstrates that p53 deficiency accelerated development of GI syndrome in mice. Panel A: I.P. injection of PFTa (10 mg/kg) protects C57B1/6J mice (if not indicated otherwise, here and below 6-8 weeks old males were used) from a single 9 Gy dose of gamma radiation and a fractioned cumulative radiation dose 12.5 Gy (5×2.5 Gy). PFTα has no effect on survival of mice treated with single 12.5 and 25 Gy doses of IR: (results of representative experiments are shown; Shepherd 4000 Ci Cesium 137 source at a dose rate of 4 Gy per minute was used). Panel B: Wild-type and p53-null C57B1/6J mice differ in their relative sensitivity to low (10 Gy) and high (15 Gy) doses of gamma radiation: wild-type mice were more sensitive to 10 Gy but more resistant to 15 Gy as compared to p53-null mice. Panel C: Mice treated with 11 Gy of total body gamma irradiation were injected 12 h later with 1.5×107 bone marrow cells from wild type or p53-null syngeneic C57B1/6J mice. (This dose causes 100% lethality in nonreconstituted controls group of mice). Two months later, after complete recovery of hematopoiesis, animals were treated with 15 Gy of total body gamma radiation and showed no difference in death rates between the two groups differing in the p53 status of their bone marrow. Panel D: Comparison of dynamics of injury to small intestines of wild-type and p53-null mice at the indicated time points after 15 Gy of gamma radiation indicates accelerated damage in p53-null mice (haematoxylin-eosin stained paraffin sections; magnification ×125). 24 h panels include images of TUNEL staining if sections of crypts: massive apoptosis is evident in wild type but not in p53-deficient epithelium.
[0012] FIG. 2 demonstrates the dynamics of cell proliferation and survival in small intestine of wild type and p53-null mice. Panel A: Comparison of proliferation rates in intestines of wild-type and p53 null mice after treatment with IR. (Left) Autoradiographs of whole-body sections (1.7× magnification) of 4-week-old wild-type and p53 null mice injected intraperitoneally with 14C-thymidine (10 μCi per animal) treated or untreated with 15 Gy of gamma radiation. Arrows point at intestines. (Right) Comparison of BrdU incorporation in small intestine of wild-type and p53-null mice at different time points after 15 Gy of gamma radiation. BrdU (50 mg/kg) was injected 2 h before sacrificing mice followed by immunostaining Fragments of 96 h panels are shown at higher magnification (×400). Panel B: Comparison of the number of BrdU positive cells/crypt in small intestine of wild-type and p53-null mice at different time points after 15 Gy of gamma radiation. Three animals were analyzed for each time point, five ileum cross sections were prepared from each animal and analyzed microscopically to estimate the number of crypts and villi. Numbers of BrdU-positive cells in the crypts were counted in 5 random fields under 200× magnification (100-30 crypts) and the average number of BrdU-positive cells was plotted. Panel C: Tracing the number and position of BrdU-labeled cells in small intestine of wild type and p53-null mice during different time points after 15 Gy of gamma radiation. BrdU was injected 30 min. before irradiation and mice were sacrificed at the indicated time points. Accelerated migration from crypts to villi followed by rapid elimination of labeled cells was observed in p53-null mice.
[0013] FIG. 3 demonstrates that recombinant flagellin is capable of NF-κB activation.
[0014] FIG. 4 shows a representative experiment testing the ability of flagellin to protect mice from radiation. C56BL6 mice (6 week old males, 10 animals per group) were injected i.v. with 2.0 μg (0.1 mg/kg) or 5 μg (0.25 mg/kg) of flagellin in PBS. Four hours later, mice were irradiated with 15 Gy and mouse survival was monitored daily.
[0015] FIG. 5 shows histological sections (HE stained) of small intestinal epithelium of mice that were treated with 15 Gy of gamma radiation with or without i.v. injection of 0.25 mg/kg of flagellin. Complete destruction of crypts and villi in control mouse contrasts with close to normal morphology of tissue from flagellin-treated animal.
[0016] FIG. 6 shows the effect of flagellin on mouse sensitivity to 10 Gy of total body gamma radiation.
[0017] FIG. 7 shows the effect of flagellin injected i.v. at indicated times before irradiation on mouse sensitivity to 13 Gy (left) and 10 Gy (right) of total body gamma radiation.
[0018] FIG. 8 shows the effect of flagellin on mouse sensitivity to 10, 13 and 15 Gy of total body gamma radiation.
[0019] FIG. 9 shows the domain structure of bacterial flagellin. The Ca backbone trace, hydrophobic core distribution and structural information of F41. Four distinct hydrophobic cores that define domains D1, D2a, D2b and D3. All the hydrophobic side-chain atoms are displayed with the Ca backbone. Side-chain atoms are color coded: Ala, yellow; Leu, Ile or Val, orange; Phe and Tyr, purple (carbon atoms) and red (oxygen atoms). c, Position and region of various structural features in the amino-acid sequence of flagellin. Shown are, from top to bottom: the F41 fragment in blue; three b-folium folds in brown; the secondary structure distribution with a--helix in yellow, b--structure in green, and b--turn in purple; tic mark at every 50th residue in blue; domains D0, D1, D2 and D3; the axial subunit contact region within the proto-element in cyan; the well-conserved amino-acid sequence in red and variable region in violet; point mutations in F41 that produce the elements of different supercoils. Letters at the bottom indicate the morphology of mutant elements: L (D107E, R124A, R124S, G426A), L-type straight; R (A449V), R-type straight; C (D313Y, A414V, A427V, N433D), curly33.
[0020] FIG. 10 shows a schematic of Salmonella flagellin domains, its fragments, and its interaction with TLR5. Dark bars denote regions of the flagellin gene used to construct fragments comprising A, B, C, A' and B'.
[0021] FIG. 11 shows soluble flagellin (FliC), and two fragments (AA' and BB') after fractionation by SDS-PAGE, with molecular weight markers listed to the left.
[0022] FIG. 12 shows induction of NF-κB nuclear translocation by Salmonella flagellin (FliC) and flagellin fragments.
[0023] FIG. 13 shows activation of NF-κB-regulated luciferase reporter construct by flagellin and flagellin fragments in H116 cells. Concentrations of proteins are given in μg/ml.
[0024] FIG. 14 shows NF-κB DNA binding in HT29 human colon cancer cells induced by flagellin and flagellin fragments.
[0025] FIG. 15 shows the activation of a NF-κB reporter in HCT116 reporter cells by full-length flagellin and flagellin fragments.
[0026] FIG. 16 shows a comparison of the radioprotective properties of flagellin (FliC) and fragments AA' and BB'.
[0027] FIG. 17 shows that the AA' fragment protects intestinal epithelium from degeneration caused by radiation. A: Histological sections (hematoxylin and eosin-stained) of small intestinal epithelium of mice 5 days after 14 Gy irradiation are shown. B: Treatment with the AA' fragment prevents apoptosis ongoing 5 hours after irradiation in endothelial cells of villi (detected by immunostaining for endothelial marker CD31 and marked by arrows), as determined by TUNEL assay. C: Histological sections of skin of mice 5 days after 14 Gy of gamma irradiation demonstrate the protective effect of the AA' fragment for sebaceous glands (red arrows).
[0028] FIG. 18 shows that the AA' fragment provides partial protection and delays death of mice after supralethal irradiation with 17 and 20 Gy total-body gamma radiation.
[0029] FIG. 19 shows anti-flagellin antibody titers induced in mice after 21 and 28 days by flagellin and AA'. For individual mice, the averages of two measurements are shown. Mice were injected with: Fl: flagellin; or AA'. 21d and 28ds--mice injected with first dose 21 and 28 days before second, respectively. PBS: saline buffer (no serum) control; blank: empty well reading control.
[0030] FIG. 20 shows anti-flagellin antibody titers induced in mice after 21 and 28 days by flagellin and AA'. For individual mice, the averages of two measurements are shown. Mice were injected with: Fl: flagellin; or AA'. 21d and 28ds--mice injected with first dose 21 and 28 days before second, respectively. PBS: saline buffer (no serum) control; blank: empty well reading control.
[0031] FIG. 21 shows that flagellin fragment AA' protects mice from multiple successive doses of gamma-irradiation. Arrows denote radiation treatments (days 1-4).
[0032] FIG. 22 shows the effect of AA' on tumor sensitivity to radiation treatment. Left Panel: NIH3T3-derived sarcoma cells were injected s.c. in NIH-Swiss mice. When tumors reached 7-10 mm in diameter, mice received three 4.3 Gy doses of total body irradiation, with or without pretreatment with AA'. The dynamics of tumor growth after radiation treatment is displayed. U/t: untreated; AA': AA' with no irradiation; 3×4 Gy: irradiation only; 3×4 Gy+AA':AA' and irradiation. (The shape of curves reflects slow growth of tumors that is a characteristic of this model). Results are displayed as relative tumor volumes normalized to tumor volume measured at day 7 after last irradiation. Right Panel: The experiment was done in the same way with another syngeneic mouse tumor model: B16 melanoma (C57BL6 background). Treatment was applied when tumors reached 4-5 mm in diameter and involved three subsequent 4 Gy doses of total body gamma radiation applied with or without pretreatment with AA' (30 min. before irradiation, 5 μg/mouse).
[0033] FIG. 23 shows the influence of NS398 on the radioprotection of LPS and AA' in mice after 13 Gy of total-body gamma irradiation.
[0034] FIG. 24 shows a comparison of amino acid sequences of the conserved amino (FIG. 24A, SEQ ID NOS.:1 and 57-76) and carboxy (FIG. 24B, select amino acid residues of SEQ ID NOS.:1 and 57-76) terminus from 21 species of bacteria. The 13 conserved amino acids important for TLR5 activity are shown with shading. The amino acid sequences are identified by their accession numbers from TrEMBL (first letter=Q) or Swiss-Prot (first letter=P).
[0035] FIGS. 25A-D show results of a BLAST search using SEQ ID NO: 1 as the query sequence. The parameters used in all searches was as follows: expected value cutoff=10, matrix=BLOSUM62, gap penalties of existence=11 and extension=1, filtering=none. FIG. 25A: NR_Bacteria (Protein-Protein); FIG. 25B: NR_Bacteria (Protein-DNA); FIG. 25C: Bacterial Genomes (Protein-Protein); FIG. 25D: Bacterial Genomes (Protein-DNA).
[0036] FIG. 26 shows the percentage identities of the amino- and carboxy-terminus of the homologs shown in FIG. 24 compared to SEQ ID NO: 1, as shown in BLAST results from http://www.expasy.org/ using the same search parameters as listed for FIGS. 25A-D.
[0037] FIG. 27 demonstrates that AA' mediates rescue of multiple mouse strains after 10 Gy total-body γ-IR. Cone heights represent fractions of survivors.
[0038] FIG. 28 demonstrates the pharmacokinetics of AA' after intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.) or intramuscular (i.m.) injection.
[0039] FIG. 29 demonstrates the extended pharmacokinetics of AA' after intramuscular (i.m.) injection.
[0040] FIG. 30 demonstrates the influence of AA' on gamma-irradiation induced cell death and growth inhibition in A549 cells.
[0041] FIG. 31 demonstrates the influence of AA' on gamma-irradiation induced cell death and growth inhibition in multiple cell lines.
[0042] FIG. 32 demonstrates the influence of irradiation and AA' on BrdU incorporation in small intestinal crypts of NIH-Swiss mice. A comparison of BrdU incorporation in small intestine of control and AA' treated NIH-Swiss mice, with and without 15 Gy of gamma radiation is shown. BrdU (50 mg/kg) was injected 1.5 h before sacrificing mice and immunostaining was done as previously described (Watson A J & Pritchard D M., Am JPhysiol Gastrointest Liver Physiol. 2000 January; 278(1):G1-5). Red channel of the image is shown (positive signal is bright white on the dark background).
[0043] FIG. 33 demonstrates the duration of AA'-mediated growth arrest and reduced BrdU incorporation in small intestine of mice. BrdU (50 mg/kg) was injected in Balb/c mice i.p., 1 or 4 hrs after CBLB502 (AA') injection. Samples of small intestine were obtained 1.5 hrs after BrdU injection. Immunostaining was done as previously described (Watson A J & Pritchard D M., Am J Physiol Gastrointest Liver Physiol. 2000 January; 278(1):G1-5). Inverted image is shown (positive signal is dark on the light background).
[0044] FIG. 34 demonstrates the influence of AA' on BrdU incorporation in colonic crypts of NIH-Swiss mice. BrdU (50 mg/kg) was injected in NIH-Swiss mice i.p., 1 hr after CBLB502 (AA') injection, Samples of colon were obtained 1.5 hrs after BrdU injection. Immunostaining was done as previously described (Watson A J & Pritchard D M., Am J Physiol Gastrointest Liver Physiol. 2000 January; 278(1):G1-5). Inverted image is shown (positive signal is dark on the light background). Bottom panel shows smaller magnification/larger area of the sample.
[0045] FIG. 35 demonstrates the morphology of small intestine in TLR5 deficient MOLF/Ei and TLR5 wt NIH-Swiss mice after treatment with AA'.
[0046] FIG. 36 depicts flagellin derivatives. The domain structure and approximate boundaries (amino acid coordinates) of selected flagellin derivatives (listed on the right). FliC flagellin of Salmonella dublin is encoded within 505 amino acids (aa).
[0047] FIG. 37 shows the testing of additional flagellin derivatives tested for NF-κB stimulating activity.
[0048] FIG. 38 shows the nucleotide and amino acid sequence for the following flagellin variants: AA' (SEQ ID NO: 7-8), AB' (SEQ ID NO: 9-10), BA' (SEQ ID NO: 11-12), BB' (SEQ ID NO: 13-14), CA' (SEQ ID NO: 15-16), CB' (SEQ ID NO: 17-18), A (SEQ ID NO: 19-20), B (SEQ ID NO: 21-22), C (SEQ ID NO: 23-24), GST-A' (SEQ ID NO: 25-26), GST-B' (SEQ ID NO: 27-28), AA'n1-170 (SEQ ID NO: 29-30), AA'n1-163 (SEQ ID NO: 33-34), AA'n54-170 (SEQ ID NO: 31-32), AA'n54-163 (SEQ ID NO: 335-36), AB'n1-170 (SEQ ID NO: 37-38), AB'n1-163 (SEQ ID NO: 39-40), AA'n1-129 (SEQ ID NO: 41-42), AA'n54-129 (SEQ ID NO: 43-44), AB'n1-129 (SEQ ID NO: 45-46), AB'n54-129 (SEQ ID NO: 47-48), AA'n1-100 (SEQ ID NO: 49-50), AB'n1-100 (SEQ ID NO: 51-52), AA'n1-70 (SEQ ID NO: 53-54) and AB'n1-70 (SEQ ID NO: 55-56). The pRSETb leader sequence is shown in Italic (leader includes Met, which is also amino acid 1 of FliC). The N terminal constant domain is underlined. The amino acid linker sequence is in Bold. The C terminal constant domain is underlined. GST, if present, is highlighted.
[0049] FIG. 39 shows the effects of CLBL502 on toxicity of cisplatinum in mice. Panels A and C show the weights of mice that received 2× maximum tolerable dose (MTD) cisplatinum at either 20 mg/kg (A) or 10 mg/kg (B) and were pretreated with either CBLB502 or PBS. Panel C shows the percentage of animals that died after receiving 20 mg/kg of cisplatinum, with or without CBLB502. Panel D depicts behavioral changes in mice that received cisplatinum with PBS compared to animals that received a similar dose of cisplatinum preceded with a single injection of CBLBL502.
[0050] FIG. 40 shows the weight loss effects in NIH Swiss mice of cisplatinum injected alone at a dose of 10 mg/kg compared with the same dose of cisplatinum when 0.04 mg/kg of CBLB502 is injected 30 min prior to the cisplatinum. Asterisks indicate that the difference between groups at a particular time point is statistically significant at P<0.05.
[0051] FIG. 41 shows the dynamic of survival of NIH-Swiss mice treated with cisplatin alone (10 mg/kg, i.p.) and with the same dose of cisplatin combined with a single s.c. injection of CBLB502 (0.04 mg/kg) done 30 min prior to cisplatin. The gray arrow marks time interval when the difference between control and experimental groups was statistically significant (P<0.05).
[0052] FIG. 42 shows the dynamic of weight loss of FVB mice treated with cisplatin alone (10 mg/kg, i.p.) compared with mice treated with the same dose of cisplatin combined with a single s.c. injection of CBLB502 (0.04 mg/kg) done 30 min prior to cisplatin.
[0053] FIG. 43 shows the results of detection of urea in blood sera of mice treated with cisplatin (i.p., 20 mg/kg) alone compared with mice treated with CBLB502 (s.c., 0.04 mg/kg) injected 30 min prior to cisplatinum.
DETAILED DESCRIPTION
[0054] This invention is related to protecting normal cells and tissues from apoptosis caused by stresses including, but not limited to, chemotherapy, radiation therapy and radiation. There are two major mechanisms controlling apoptosis in the cell: the p53 pathway (pro-apoptotic) and the NF-κB pathway (anti-apoptotic). Both pathways are frequently deregulated in tumors: p53 is usually lost, while NF-κB becomes constitutively active. Hence, inhibition of p53 and activation of NF-κB in normal cells may protect them from death caused by stresses, such as cancer treatment, but would not make tumor cells more resistant to treatment because they have these control mechanisms deregulated. This contradicts the conventional view on p53 and NF-κB, which are considered as targets for activation and repression, respectively.
[0055] This invention relates to inducing NF-κB activity to protect normal cells from apoptosis by using flagellin. Flagellin may protect normal cells in a mammal from apoptosis attributable to conditions such as, but not limited to (1) cellular stress, which results from cancer treatments and hyperthermia; (2) exposure to harmful doses of radiation, for example, workers in nuclear power plants, the defense industry radiopharmaceutical production, or the military; and (3) cell aging. Since NF-κB is constitutively active in many tumor cells, flagellin may also protect normal cells from apoptosis without providing a beneficial effect to tumor cells. Once the normal cells are repaired, NF-κB activity may be restored to normal levels. Flagellin may be used to protect such radiation- and chemotherapy-sensitive tissues as the hematopoietic system (including immune system), the epithelium of the gut, and hair follicles.
[0056] Flagellin may also be used for several other applications. Pathological consequences and death caused by exposure of mammals to a variety of severe conditions including, but not limited to, radiation, wounding, poisoning, infection, aging, and temperature shock, and may result from the activity of normal physiological mechanisms of stress response, such as induction of programmed cell death (apoptosis) or release of bioactive proteins, cytokines
[0057] Apoptosis normally functions to "clean" tissues from wounded and genetically damaged cells, while cytokines serve to mobilize the defense system of the organism against the pathogen. However, under conditions of severe injury, both stress response mechanisms can by themselves act as causes of death. For example, lethality from radiation may result from massive p53-mediated apoptosis occurring in hematopoietic, immune and digestive systems. Rational pharmacological regulation of NF-κB may increase survival under conditions of severe stress. Control over these factors may allow control of both inflammatory response and the life-death decision of cells from the injured organs. Tissues that may be protected from apoptosis by inducing flagellin include the GI tract, lungs, kidneys, liver, cardiovascular system, blood vessel endothelium, central and peripheral neural system, hematopoietic (HP) progenitor cells, immune system, and hair follicles.
[0058] The protective role of flagellin is mediated by transcriptional activation of multiple genes coding for: a) anti-apoptotic proteins that block both major apoptotic pathways, b) cytokines and growth factors that induce proliferation and survival of HP and other stem cells, and c) potent ROS-scavenging antioxidant proteins, such as MnSOD (SOD-2). Thus, by temporal activation of NF-κB for radioprotection, it may be possible to achieve not only suppression of apoptosis in cancer patients, but also reduction of the rate of secondary cancer incidence because of simultaneous immunostimulatory effect. These results may be achieved if NF-κB is activated by Toll-like receptors.
[0059] Another attractive property of the NF-κB pathway as a target is its activation by numerous natural factors that can be considered as candidate radioprotectants. Among these, are multiple pathogen-associated molecular patterns (PAMPs). PAMPs are molecules that are not found in the host organism, are characteristic for large groups of pathogens, and cannot be easily mutated. They are recognized by Toll-like receptors (TLRs), the key sensor elements of innate immunity. TLRs act as a first warning mechanism of immune system by inducing migration and activation of immune cells directly or through cytokine release. TLRs are type I membrane proteins known to work as homo- and heterodimers. Upon ligand binding, TLRs recruit MyD88 protein, an indispensable signaling adaptor for most TLRs. The signaling cascade that follows leads to effects including (i) activation of NF-κB pathway, and (ii) activation of MAPKs, including Jun N-terminal kinease (JNK). The activation of the NF-κB pathway by Toll-like receptor ligands makes the ligands attractive as potential radioprotectors. Unlike cytokines, many PAMPs have little effect besides activating TLRs and thus are unlikely to produce side effects. Moreover, many PAMPs are present in humans.
[0060] Consistently with their function of immunocyte activation, all TLRs are expressed in spleen and peripheral blood leukocytes, with more TLR-specific patterns of expression in other lymphoid organs and subsets of leukocytes. However, TLRs are also expressed in other tissues and organs of the body, e.g., TLR1 is expressed ubiquitously, TLR5 is also found in GI epithelium and endothelium, and TLRs 2, 6, 7 and 8 are known to be expressed in lung.
1. DEFINITIONS
[0061] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.
[0062] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.
[0063] As used herein, the terms "administer" when used to describe the dosage of an agent that induces NF-κB activity, means a single dose or multiple doses of the agent.
[0064] As used herein, the term "analog" when used in the context of a peptide or polypeptide, means a peptide or polypeptide comprising one or more non-standard amino acids or other structural variations from the conventional set of amino acids.
[0065] As used herein, the term "antibody" means an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom. The antibody may also be a chimeric antibody. The antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art. The antibody may be conjugated with a chemical moiety.
[0066] As used herein, "apoptosis" refers to a form of cell death that includes progressive contraction of cell volume with the preservation of the integrity of cytoplasmic organelles; condensation of chromatin (i.e., nuclear condensation), as viewed by light or electron microscopy; and/or DNA cleavage into nucleosome-sized fragments, as determined by centrifuged sedimentation assays. Cell death occurs when the membrane integrity of the cell is lost (e.g., membrane blebbing) with engulfment of intact cell fragments ("apoptotic bodies") by phagocytic cells.
[0067] As used herein, the term "cancer" means any malignant growth or tumor caused by abnormal and uncontrolled cell division; it may spread to other parts of the body through the lymphatic system or the blood stream.
[0068] As used herein, the term "cancer treatment" means any treatment for cancer known in the art including, but not limited to, chemotherapy and radiation therapy.
[0069] As used herein, the term "combination with" when used to describe administration of an agent that induces NF-κB activity and an additional treatment means that the agent may be administered prior to, together with, after, or metronomically with the additional treatment. The term "together with," "simultaneous" or "simultaneously" as used herein, means that the additional treatment and the agent of this invention are administered within 48 hours, preferably 24 hours, more preferably 12 hours, yet more preferably 6 hours, and most preferably 3 hours or less, of each other. The term "metronomically" as used herein means the administration of the agent at times different from the additional treatment and at certain frequency relative to repeat administration and/or the additional treatment.
[0070] The agent may be administered at any point prior to the additional treatment including, but not limited to, about 1-48 hr prior to the additional treatment. The agent may be administered at any point after the additional treatment including, but not limited to, about 1-48 hr after exposure.
[0071] As used herein, the term "derivative", when used in the context of a peptide or polypeptide, means a peptide or polypeptide different other than in primary structure (amino acids and amino acid analogs). By way of illustration, derivatives may differ by being glycosylated, one form of post-translational modification. For example, peptides or polypeptides may exhibit glycosylation patterns due to expression in heterologous systems. If at least one biological activity is retained, then these peptides or polypeptides are derivatives according to the invention. Other derivatives include, but are not limited to, fusion peptides or fusion polypeptides having a covalently modified N- or C-terminus, PEGylated peptides or polypeptides, peptides or polypeptides associated with lipid moieties, alkylated peptides or polypeptides, peptides or polypeptides linked via an amino acid side-chain functional group to other peptides, polypeptides or chemicals, and additional modifications as would be understood in the art.
[0072] As used herein, the term "fragment" when used in the context of a peptide or polypeptide, means a portion of a reference peptide or polypeptide.
[0073] As used herein, the term "homolog" when used in the context of a peptide or polypeptide, means a peptide or polypeptide sharing a common evolutionary ancestor.
[0074] As used herein, the term "treat" or "treating" when referring to protection of a mammal from a condition, means preventing, suppressing, repressing, or eliminating the condition. Preventing the condition involves administering a composition of this invention to a mammal prior to onset of the condition. Suppressing the condition involves administering a composition of this invention to a mammal after induction of the condition but before its clinical appearance. Repressing the condition involves administering a composition of this invention to a mammal after clinical appearance of the condition such that the condition is reduced or maintained. Elimination the condition involves administering a composition of this invention to a mammal after clinical appearance of the condition such that the mammal no longer suffers the condition.
[0075] As used herein, the term "tumor cell" means any cell associated with a cancer.
[0076] As used herein, the term "variant" when used in the context of a peptide or polypeptide, means a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" include, but are not limited to, the ability to bind to TLR5 and to be bound by a specific antibody. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. This is disclosed in U.S. Pat. No. 4,554,101, the contents of which are incorporated herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. In one aspect, substitutions are performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. A variant may also be a polypeptide fragment or a polypeptide derivative.
2. REDUCING A SIDE EFFECT OF CANCER TREATMENT
[0077] This invention relates to a method of reducing a side effect of cancer treatment in a mammal comprising administering to a mammal in need thereof a composition comprising flagellin, which may induce NF-kB. While not being bound by theory, the side effect may be an effect of damage to normal tissue attributable to treatment of a constitutively active NF-kB cancer.
[0078] a. Cancer Treatment
[0079] The cancer treatment may comprise administration of a chemotherapy, which may be a cytotoxic agent or cytostatic agent, or combination thereof. Cytotoxic agents prevent cancer cells from multiplying by: (1) interfering with the cell's ability to replicate DNA and (2) inducing cell death and/or apoptosis in the cancer cells. Cytostatic agents act via modulating, interfering or inhibiting the processes of cellular signal transduction which regulate cell proliferation and sometimes at low continuous levels.
[0080] Classes of compounds that may be used as cytotoxic agents include, but are not limited to, the following: alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard, chlormethine, cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide; antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine, and gemcitabine; natural products and their derivatives (for example, vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins): vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c, paclitaxel (paclitaxel is commercially available as Taxol®), mithramycin, deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons (preferably IFN-α), etoposide, and teniposide. Other proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
[0081] Microtubule affecting agents interfere with cellular mitosis and are well known in the art for their cytotoxic activity. Microtubule affecting agents useful in the invention include, but are not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives (e.g., NSC 608832), thiocolchicine (NSC 361792), trityl cysteine (NSC 83265), vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574), natural and synthetic epothilones including but not limited to epothilone A, epothilone B, and discodermolide (see Service, (1996) Science, 274:2009) estramustine, nocodazole, MAP4, and the like. Examples of such agents are also described in Bulinski (1997) J. Cell Sci. 110:3055 3064; Panda (1997) Proc. Natl. Acad. Sci. USA 94:10560-10564; Muhlradt (1997) Cancer Res. 57:3344-3346; Nicolaou (1997) Nature 387:268-272; Vasquez (1997) Mol. Biol. Cell. 8:973-985; and Panda (1996) J. Biol. Chem 271:29807-29812.
[0082] Also suitable are cytotoxic agents such as epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin (cisplatinum) and carboplatin (carboplatinum); biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; and haematopoietic growth factors.
[0083] Cytostatic agents that may be used include, but are not limited to, hormones and steroids (including synthetic analogs): 17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, hlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesteroneacetate, leuprolide, flutamide, toremifene, zoladex.
[0084] Other cytostatic agents are antiangiogenics such as matrix metalloproteinase inhibitors, and other VEGF inhibitors, such as anti-VEGF antibodies and small molecules such as ZD6474 and SU6668 are also included. Anti-Her2 antibodies from Genetech may also be utilized. A suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Also included are Imclone antibody C225 immunospecific for the EGFR, and src inhibitors.
[0085] Also suitable for use as an cytostatic agent is Casodex® (bicalutamide, Astra Zeneca) which renders androgen-dependent carcinomas non-proliferative. Yet another example of a cytostatic agent is the antiestrogen Tamoxifen® which inhibits the proliferation or growth of estrogen dependent breast cancer. Inhibitors of the transduction of cellular proliferative signals are cytostatic agents. Representative examples include epidermal growth factor inhibitors, Her-2 inhibitors, MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Src kinase inhibitors, and PDGF inhibitors.
[0086] A variety of cancers may be treated according to this invention including, but not limited to, the following: carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), head and neck cancer, oral cancer, islet cell carcinoma, adrenocortical carcinoma, astrocytoma, urethral cancer, uterine cancer, vaginal cancer, basal cell carcinoma, bile duct cancer, bone cancer, brain tumor (including glioma, astrocytoma, ependymoma and others), osteosarcoma, malignant fibrous histiocytoma, burkitt lymphoma, cervical cancers, childhood cancers, eye cancer, mouth cancer, mesothelioma, nasal cavity and paranasal sinus cancer, kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemias, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; solid tumor cancers; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyoscarcoma, and osteosarcoma; and other tumors including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, teratocarcinoma, and cancers of the gastrointestinal tract or the abdominopelvic cavity. The cancer may also be bladder cancer, ovarian cancer, head and neck cancer including squamous cell cariconoma, non-small lung cancer, oral cancer, islet cell carcinoma, adrenocortical carcinoma, astrocytoma, urethral cancer, uterine cancer, vaginal cancer, testicular cancer, basal cell carcinoma, bile duct cancer, bone cancer, brain tumor (including glioma, astrocytoma, ependymoma and others), osteosarcoma, malignant fibrous histiocytoma, burkitt lymphoma, Hodgkin's and non-Hodgkin's lymphoma, neuroblastoma, sarcomas, myeloma, melanoma, osteosarcoma, cervical cancers, childhood cancers, eye cancer, mouth cancer, mesothelioma, nasal cavity and paranasal sinus cancer.
[0087] (1) Cisplatinum Treatments
[0088] As described above, the cancer treatment may be cisplatinum, which is a platinum-containing compound that slows or stops the growth of cancer cells. Cisplatinum inhibits DNA synthesis by the formation of DNA cross-links, which denatures the double helix. Cisplatinum also binds to DNA bases such as two adjacent guanines and disrupts DNA function because cisplatinum damaged DNA is not easily recognized by cell enzymes. Cisplatinum may also bind proteins.
[0089] The length of treatment of cisplatinum may depend upon the type of drugs a subject is taking, how well the body responds to them, and the type of cancer. Cisplatinum may be rapidly distributed into the tissue and may be found in high concentrations in the kidneys, liver, ovaries, uterus, and lungs. The half-life of cisplatinum initially is 20-30 minutes. A beta deterioration may be 60 minutes and terminal greater than 24 hours. Cisplatinum may have a secondary half-life of 44-73 hours. Cisplatinum may be administered in daily dosages of 50-270 mg/m2/day. Cisplatinum may also be administered a daily dosage schedule for 15-20 mg/m2/day, 60-100 mg/m2/day, 50-70 mg/m2/day, 75-100 mg/m2/day or 100-120 mg/m2/day for 3-4 weeks.
[0090] Cisplatinum may be combined in regimens with other cancer related therapies and drugs including, but not limited to, CAP, CISCA, cisplatin-docetaxel, CMV, gemcitabine, M-VAC, CDDP/VP-16, COPE, docetaxel-trastuzumab, fluorouracil, vinorelbine, BIP, PFL, topotecan, AP, irinotecan, TCF, TIP, EAP, ECF, EFP, FAP, FUP, EP/EMA, CABO, cetuximab, CF, interferon (IFN), IPA, GC, EP, bevacizumab, gemcitabine, DHAP, ESHAP, pemetrexed, CCDT, dacarbazine-carmustine, deacarbazine-interferon-α2-aldesleukin, and IL-2-IFN. After treatment with cisplatinum, the following parameters may be monitored such as renal function, electrolytes, audiography, neurological exam, liver function tests, CBC with differential and platelet count, urine output, and urinalysis.
[0091] (2) Cancer Treatment Side Effect
[0092] The cancer treatment may cause or be associated with a side effect selected from the group consisting of: myelosuppression, renal toxicity, pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, mouth sores, memory loss, hemorrhage, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment. The cancer treatment may also cause a side effect such as weight loss or behavioral changes.
[0093] Side effects from cisplatinum may include those described above as well as thinned or brittle hair, loss of appetite or weight, diarrhea, nausea and vomiting, change in taste, numbness or tingling in the body including the fingertips and toes, fatigue, unusual bruising or bleeding, black tarry stools, fever, chills, dizziness or feeling of faintness, fever, chills, renal toxicity, mild alopecia, myelosuppression, hearing impairment, pain in one's side or back, shortness of breath or wheezing, swelling of feet or ankles, seizures and rash.
[0094] (3) Radiation
[0095] The chemotherapy may also be used to treat cancer in conjunction with radiation therapy. Injury and death of normal cells from ionizing radiation is a combination of direct radiation-induced damage to the exposed cells and an active genetically programmed cell reaction to radiation-induced stress resulting in suicidal death or apoptosis. Apoptosis plays a key role in massive cell loss occurring in several radiosensitive organs (i.e., hematopoietic and immune systems, epithelium of digestive tract, etc.), the failure of which determines general radiosensitivity of the organism.
[0096] Exposure to ionizing radiation (IR) may be short- or long-term, it may be applied as a single or multiple doses, to the whole body or locally. Thus, nuclear accidents or military attacks may involve exposure to a single high dose of whole body irradiation (sometimes followed by a long-term poisoning with radioactive isotopes). The same is true (with strict control of the applied dose) for pretreatment of patients for bone marrow transplantation when it is necessary to prepare hematopoietic organs for donor's bone marrow by "cleaning" them from the host blood precursors. Cancer treatment may involve multiple doses of local irradiation that greatly exceeds lethal dose if it were applied as a total body irradiation. Poisoning or treatment with radioactive isotopes results in a long-term local exposure to radiation of targeted organs (e.g., thyroid gland in the case of inhalation of 125I). Finally, there are many physical forms of ionizing radiation differing significantly in the severity of biological effects.
[0097] At the molecular and cellular level, radiation particles are able to produce breakage and cross-linking in the DNA, proteins, cell membranes and other macromolecular structures. Ionizing radiation also induces secondary damage to the cellular components by giving rise to the free radicals and reactive oxygen species (ROS). Multiple repair systems counteract this damage, such as several DNA repair pathways that restore the integrity and fidelity of the DNA, and antioxidant chemicals and enzymes that scavenge the free radicals and ROS and reduce the oxidized proteins and lipids. Cellular checkpoint systems detect the DNA defects and delay cell cycle progression until damage is repaired or decision to commit cell to growth arrest or programmed cell death (apoptosis) is reached
[0098] Radiation can cause damage to mammalian organism ranging from mild mutagenic and carcinogenic effects of low doses to almost instant killing by high doses. Overall radiosensitivity of the organism is determined by pathological alterations developed in several sensitive tissues that include hematopoietic system, reproductive system and different epithelia with high rate of cell turnover.
[0099] The acute pathological outcome of gamma irradiation leading to death is different for different doses and is determined by the failure of certain organs that define the threshold of the organism's sensitivity to each particular dose. Thus, lethality at lower doses occurs from bone marrow aplasia, while moderate doses kill faster by inducing gastrointestinal (GI) syndrome. Very high doses of radiation can cause almost instant death eliciting neuronal degeneration.
[0100] Organisms that survive a period of acute toxicity of radiation can suffer from long-term remote consequences that include radiation-induced carcinogenesis and fibrosis developing in exposed organs (e.g., kidney, liver or lungs) months and years after irradiation.
[0101] Cellular DNA is the major target of IR causing a variety of types of DNA damage (genotoxic stress) by direct and indirect (free radical-based) mechanisms. All organisms maintain DNA repair system capable of effective recovery of radiation-damaged DNA; however, errors in the DNA repair process may lead to mutations.
[0102] Tumors are generally more sensitive to gamma radiation and can be treated with multiple local doses that cause relatively low damage to normal tissue. Nevertheless, in some instances, damage of normal tissues is a limiting factor in application of gamma radiation for cancer treatment. The use of gamma-irradiation during cancer therapy by conventional, three-dimensional conformal or even more focused BeamCath delivery has also dose-limiting toxicities caused by cumulative effect of irradiation and inducing the damage of the stem cells of rapidly renewing normal tissues, such as bone marrow and gastrointestinal (GI) tract.
[0103] At high doses, radiation-induced lethality is associated with so-called hematopoietic and gastrointestinal radiation syndromes. Hematopoietic syndrome is characterized by loss of hematopoietic cells and their progenitors making it impossible to regenerate blood and lymphoid system. Death usually occurs as a consequence of infection (result of immunosuppression), hemorrhage and/or anemia. GI syndrome is caused by massive cell death in the intestinal epithelium, predominantly in the small intestine, followed by disintegration of intestinal wall and death from bacteriemia and sepsis. Hematopoietic syndrome usually prevails at the lower doses of radiation and leads to a more delayed death than GI syndrome.
[0104] In the past, radioprotectants were typically antioxidants--both synthetic and natural. More recently, cytokines and growth factors have been added to the list of radioprotectants. The mechanism of their radioprotection is considered to be a result of a facilitating effect on regeneration of sensitive tissues. There is no clear functional distinction between both groups of radioprotectants, however, since some cytokines induce the expression of cellular antioxidant proteins, such as manganese superoxide dismutase (MnSOD) and metallothionein.
[0105] The measure of protection for a particular agent is expressed by dose modification factor (DMF or DRF). DMF is determined by irradiating the radioprotector treated subject and untreated control subjects with a range of radiation doses and then comparing the survival or some other endpoints. DMF is commonly calculated for 30-day survival (LD50/30 drug-treated divided by LD50/30 vehicle-treated) and quantifies the protection of the hematopoietic system. In order to estimate gastrointestinal system protection, LD50 and DMF are calculated for 6- or 7-day survival. DMF values provided herein are 30-day unless indicated otherwise.
[0106] As shown below, flagellin possesses strong pro-survival activity at the cellular level and on the organism as a whole. In response to super-lethal doses of radiation, flagellin inhibits both gastrointestinal and hematopoietic syndromes, which are the major causes of death from acute radiation exposure. As a result of these properties, flagellin may be used to treat the effects of natural radiation events and nuclear accidents. Moreover, since flagellin acts through mechanisms different from all presently known radioprotectants, flagellin can be used in combination with other radioprotectants, thereby, dramatically increasing the scale of protection from ionizing radiation.
[0107] As opposed to conventional radioprotective agents (e.g., scavengers of free radicals), flagellin activity may not reduce primary radiation-mediated damage but may act against secondary events involving active cell reaction to primary damage, therefore complementing the existing lines of defense. Pifithrin-alpha, a pharmacological inhibitor of p53 (a key mediator of radiation response in mammalian cells), is an example of this new class of radioprotectants. However, the activity of p53 inhibitors is limited to protection of the hematopoietic system and has no protective effect in digestive tract (gastrointestinal syndrome), therefore, reducing therapeutic value of these compounds. Anti-apoptotic pharmaceuticals with broader range of activity are desperately needed.
[0108] Flagellin may be used as a radioprotective agent to extend the range of tolerable radiation doses by increasing radioresistance beyond the levels achievable by currently available measures (shielding and application of existing bioprotective agents) and drastically increase the chances of survival, for example, in case of onboard nuclear accidents or large-scale solar particle events. With an approximate DMF (30-day survival) greater than 1.5, the NF-κB inducer flagellin is more effective than any currently reported natural compound.
[0109] Flagellin may be also useful for treating irreplaceable cell loss caused by low-dose irradiation, for example, in the central nervous system and reproductive organs. Flagellin may also be used during cancer chemotherapy to treat the side effects associated with chemotherapy, including alopecia.
[0110] In one embodiment, a mammal is treated for exposure to radiation, comprising administering to the mammal a composition comprising a therapeutically effective amount of a composition comprising flagellin. The composition comprising flagellin may be administered in combination with one or more radioprotectants. The one or more radioprotectants may be any agent that treats the effects of radiation exposure including, but not limited to, antioxidants, free radical scavengers and cytokines
[0111] Flagellin may inhibit radiation-induced programmed cell death in response to damage in DNA and other cellular structures; however, flagellin may not deal with damage at the cellular level and may not prevent mutations. Free radicals and reactive oxygen species (ROS) are the major cause of mutations and other intracellular damage. Antioxidants and free radical scavengers are effective at preventing damage by free radicals. The combination of flagellin and an antioxidant or free radical scavenger may result in less extensive injury, higher survival, and improved health for exposure. Antioxidants and free radical scavengers that may be used in the practice of the invention include, but are not limited to, thiols, such as cysteine, cysteamine, glutathione and bilirubin; amifostine (WR-2721); vitamin A; vitamin C; vitamin E; and flavonoids such as orientin and vicenin derived from Indian holy basil (Ocimum sanctum).
[0112] Flagellin may also be administered in combination with a number of cytokines and growth factors that confer radioprotection by replenishing and/or protecting the radiosensitive stem cell populations. Radioprotection with minimal side effects may be achieved by the use of stem cell factor (SCF, c-kit ligand), Flt-3 ligand, and interleukin-1 fragment IL-1b-rd. Protection may be achieved through induction of proliferation of stem cells (all mentioned cytokines), and prevention of their apoptosis (SCF). The treatment allows accumulation of leukocytes and their precursors prior to irradiation thus enabling quicker reconstitution of the immune system after irradiation. SCF efficiently rescues lethally irradiated mice with DMF in the range of 1.3-1.35 and is also effective against gastrointestinal syndrome. Flt-3 ligand also provides strong protection in mice (70-80% 30-day survival at LD100/30, equivalent to DMF>1.2) and rabbits.
[0113] In addition, flagellin may be used with a combination of cytokines which may provide enhanced radioprotection. The cytokine combination may be TPO combined with interleukin 4 (IL-4) and/or interleukin 11 (IL-11); GM-CSF combined with IL-3; G-CSF combined with Flt-3 ligand; 4F combination: SCF, Flt-3 ligand, TPO and IL-3; or 5F combination: 4F with addition of SDF-1.
[0114] In addition, gastrointestinal radioprotectors may be used in combination with flagellin, including transforming growth factor beta3 (TGFb3), interleukin 11 (IL-11), and mentioned keratinocyte growth factor (KGF). While these radioprotectors also protect the intestine, they are likely to synergize with flagellin or flagellin related polypeptides since the results below show that flagellin and flagellin related polypeptides protect endothelium, while these gastrointestinal radioprotectors protect epithelium of GI tract.
[0115] Several factors, while not cytokines by nature, stimulate the proliferation of immunocytes and may be used in combination with flagellin. For example, 5-AED (5-androstenediol) is a steroid that stimulates the expression of cytokines and increases resistance to bacterial and viral infections. A subcutaneous injection of 5-AED in mice 24 h before irradiation improved survival with DMF=1.26. Synthetic compounds, such as ammonium tri-chloro(dioxoethylene-O,O'--) tellurate (AS-101), may also be used to induce secretion of numerous cytokines and for combination with flagellin. Additional radioprotectors include, growth hormone (GH), thrombopoietin (TPO), interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and stromal derived factor-1 (SDF-1).
[0116] Growth factors and cytokines may also be used in combination with flagellin to provide protection against gastrointestinal syndrome. Keratinocyte growth factor (KGF) promotes proliferation and differentiation in the intestinal mucosa, and increases the post-irradiation cell survival in the intestinal crypts. Hematopoietic cytokine and radioprotectant SCF may also increase intestinal stem cell survival and associated short-term organism survival.
[0117] Flagellin may offer protection against both gastrointestinal (GI) and hematopoietic syndromes. Since mice exposed to 15 Gy of whole-body lethal irradiation die mostly from GI syndrome, a composition comprising flagellin and one or more inhibitors of GI syndrome may be more effective Inhibitors of GI syndrome that may be used in the practice of the invention include, but are not limited to, cytokines such as SCF and KGF.
[0118] The composition comprising flagellin may be administered at any point prior to exposure to radiation including, but not limited to, about 1-48 hr prior to exposure. The composition comprising flagellin may be administered at any point after exposure to radiation including, but not limited to, about 1-48 hr after exposure to radiation.
3. AGENT
[0119] a. Flagellin
[0120] This invention also relates to an agent, which may be flagellin. Flagellin may possess strong pro-survival activity at the cellular level and for the organism as a whole. Flagellin may also stimulate natural killer (NK) cells and T-lymphocytes, which are the major components of anti-tumor immunity (Tsujimoto H, et. al., J Leukoc Biol. 2005 October; 78(4):888-97; Caron G., et. al., J Immunol. 2005 Aug. 1; 175(3):1551-7; Honko A N & Mizel S B, Immunol Res. 2005; 33(1):83-101). As a result, flagellin may be used to reduce the side effects of cancer treatments.
[0121] The flagellin may be a flagellin-related polypeptide. The flagellin may be a flagellin or flagellin-related polypeptide from any source, including a variety of Gram-positive and Gram-negative bacterial species. Flagellin may have the amino acid sequence of one of 23 flagellins from bacterial species that are depicted in FIG. 7 of U.S. Patent Publication No. 2003/0044429, the contents of which are incorporated herein by reference. The nucleotide sequences encoding the flagellin polypeptides listed in FIG. 7 of U.S. 2003/0044429 are publicly available at sources including the NCBI Genbank database.
[0122] Flagellin may be the major component of bacterial flagellum. Flagellin may be composed of three domains (FIG. 9). Domain 1 (D1) and domain 2 (D2) may be discontinuous and may be formed when residues in the amino terminus and carboxy terminus are juxtaposed by the formation of a hairpin structure. The amino and carboxy terminus comprising the D1 and D2 domains may be most conserved, whereas the middle hypervariable domain (D3) may be highly variable. Studies with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicate that D1 and D2 may be bioactive when coupled to an ECH element. This chimera, but not the hinge alone, may include I.sub.κB.sub.α degradation, NF-κB activation, and NO and IL-8 production in two intestinal epithelial cell lines. The non-conserved D3 domain may be on the surface of the flagellar filament and may contain the major antigenic epitopes. The potent proinflammatory activity of flagellin may reside in the highly conserved N and C D1 and D2 regions.
[0123] Flagellin may include NF-κB activity by binding to Toll-like receptor 5 (TLR5). The TLR family may be composed of at least 10 members and is essential in innate immune defense against pathogens. The innate immune system may recognize pathogen-associated molecular patterns (PAMPs) that are conserved on microbial pathogens. TLR may recognize a conserved structure that is particular to bacterial flagellin. The conserved structure may be composed of a large group of residues that are somewhat permissive to variation in amino acid content. Smith et al., Nat Immunol. 4:1247-53 (2003) have identified 13 conserved amino acids in flagellin that are part of the conserved structure recognized by TLR5. The 13 conserved amino acids of flagellin that may be important for TLR5 activity are shown in FIG. 24.
[0124] The flagellin may be from a species of Salmonella, a representative example of which is S. dublin (encoded by GenBank Accession Number M84972) (SEQ ID NO: 1). The flagellin related-polypeptide may be a fragment, variant, analog, homolog, or derivative of SEQ ID NO: 1, or combination thereof, that binds to TLR5 and induces TLR5-mediated activity, such as activation of NF-κB activity. A fragment, variant, analog, homolog, or derivative of flagellin may be obtained by rational-based design based on the domain structure of Flagellin and the conserved structure recognized by TLR5.
[0125] The flagellin may comprise at least 10, 11, 12, or 13 of the 13 conserved amino acids shown in FIG. 24 (positions 89, 90, 91, 95, 98, 101, 115, 422, 423, 426, 431, 436 and 452). The flagellin may be at least 30-99% identical to amino acids 1-174 and 418-505 of SEQ ID NO: 1. FIG. 26 lists the percentage identity of the amino- and carboxy-terminus of flagellin with known TLR-5 stimulating activity, as compared to SEQ ID NO: 1.
[0126] The flagellin may be a flagellin polypeptide from any Gram-positive or Gram-negative bacterial species including, but not limited to, the flagellin polypeptides disclosed in U.S. Pat. Pub. 2003/000044429, the contents of which are incorporated herein, and the flagellin peptides corresponding to the Accession numbers listed in the BLAST results shown in FIGS. 25A-D, or variants thereof.
[0127] The flagellin may stimulate TLR5 activity. Numerous deletional mutants of flagellin have been made that retain at least some TLR5 stimulating activity. The flagellin may be a deletional mutant disclosed in the Examples herein, and may comprise a sequence translated from GenBank Accession number D13689 missing amino acids 185-306 or 444-492, or from GenBank Accession number M84973 missing amino acids 179-415, or a variant thereof.
[0128] The flagellin may comprise transposon insertions and changes to the variable D3 domain. The D3 domain may be substituted in part, or in whole, with a hinge or linker polypeptide that allows the D1 and D2 domains to properly fold such that the variant stimulates TLR5 activity. The variant hinge elements may be found in the E. coli MukB protein and may have a sequence as set forth in SEQ ID NOS: 3 and 4, or a variant thereof
4. COMPOSITION
[0129] This invention also relates to a composition comprising a therapeutically effective amount of the agent. The composition may be a pharmaceutical composition, which may be produced using methods well known in the art. As described above, the composition comprising the agent may be administered to a mammal for the treatment of conditions associated with apoptosis including, but not limited to, exposure to radiation, side effect from cancer treatments, stress and cell aging. The composition may also comprise additional agents including, but not limited to, a radioprotectant or a chemotherapeutic drug.
[0130] a. Administration
[0131] Compositions of this invention may be administered in any manner including, but not limited to, orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, or combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, and intraarticular. Transmucosally administration includes, but is not limited to intranasal. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the dosing regimen and route of administration that is most appropriate for a particular animal.
[0132] The composition may be administered prior to, after or simultaneously with a stress that triggers apoptosis, or a combination thereof. The composition may be administered from about 1 hour to about 48 hours prior to or after exposure to a stress that triggers apoptosis.
[0133] b. Formulation
[0134] Compositions of this invention may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate). Tablets may be coated according to methods well known in the art.
[0135] Compositions of this invention may also be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, nonaqueous vehicles and preservatives. Suspending agent include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid.
[0136] Compositions of this invention may also be formulated as suppositories, which may contain suppository bases including, but not limited to, cocoa butter or glycerides. Compositions of this invention may also be formulated for inhalation, which may be in a form including, but not limited to, a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane. Compositions of this invention may also be formulated transdermal formulations comprising aqueous or nonaqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
[0137] Compositions of this invention may also be formulated for parenteral administration including, but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents. The composition may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.
[0138] Compositions of this invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
[0139] c. Dosage
[0140] A therapeutically effective amount of the agent required for use in therapy varies with the nature of the condition being treated, the length of time that induction of NF-κB activity is desired, and the age and the condition of the patient, and is ultimately determined by the attendant physician. In general, however, doses employed for adult human treatment typically are in the range of 0.001 mg/kg to about 200 mg/kg per day. The dose may be about 1 μg/kg to about 100 μg/kg per day. The desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more subdoses per day. Multiple doses often are desired, or required, because NF-κB activity in normal cells may be decreased once the agent is no longer administered.
[0141] The dosage of flagellin may be at any dosage including, but not limited to, about 1 μg/kg-1 mg/kg.
[0142] This invention has multiple aspects, illustrated by the following non-limiting examples.
Example 1
P53 Deficiency Accelerated Development of GI Syndrome in Mice
[0143] The primary cause of death from ionizing radiation (IR) of mammals depends on the radiation dose. At doses of up to 9-10 Gy, mice die 12-20 days later, primarily from lethal bone marrow depletion-hematopoietic (HP) syndrome. At this dose, irradiated mice can be rescued from lethality by bone marrow transplantation. Animals that receive >15 Gy die between 7-12 days after treatment (before hematopoietic syndrome can kill them) from complications of damage to the small intestine-gastrointestinal (GI) syndrome. In both cases of HP and GI syndromes, lethal damage of tissues starts from massive p53-dependent apoptosis. This observation allowed us earlier to suggest that p53 could be a determinant of radiation-induced death. Consistently, p53-deficient mice were resistant to doses of radiation that kill through HP syndrome, and lethality of wild type animals receiving 6-11 Gy of gamma radiation could be reduced by temporary pharmacological inhibition of p53 by the small molecule p53 inhibitor pifithrin-alpha (PFT) (Komarov et al 1999). Identification of p53 as a factor sensitizing tissues to genotoxic stress was further strengthened by demonstrating the p53 dependence of hair loss (alopecia) occurring as a result of experimental chemotherapy or radiation. Hence, based on previous observations, one would expect that p53 continues to play an important role in development of lethal GI syndrome after higher doses of IR. Surprisingly, p53-deficiency sensitizes mice to higher doses of IR causing lethal gastro-intestinal syndrome (FIG. 1). Continuous cell proliferation in the crypts of p53-deficient epithelium after IR correlates with accelerated death of damaged cells of crypt and rapid destruction of villi. p53 prolongs survival by inducing growth arrest in the crypts of small intestine thereby preserving integrity of the guts (FIG. 2). Thus, proapoptotic function of p53 promotes hematopoietic syndrome while its growth arrest function delays development of gastro-intestinal syndrome.
[0144] The dynamics of cell population in the small intestine have been analyzed in great detail. Cell proliferation in the epithelia of the gut is limited to the crypts where stem cells and early proliferating progenitors are located. After a couple of cell divisions, already differentiated descendants of crypt stem cells move up the villi to be shed at the villar tip. In the small intestine of the mouse, the entire "trip" of the cell (the proliferative compartment to the tip of the villus) normally takes between 3 and 5 days. Although reaction of the small intestine to gamma radiation has been well examined at a pathomorphological level, it still remains unclear what is the exact cause of GI lethality, including the primary event. Death may occur as a direct consequence of the damage of epithelial crypt cells and followed denudation of villi leading to fluid and electrolyte imbalance, bacteremia and endotoxemia. Besides inflammation and stromal responses, endothelial dysfunctions seem to be the important factors contributing to lethality. In summary, pharmacological suppression of p53 that was shown to be so effective as a method of protection from IR-induced HP syndrome, is useless (if not detrimental) against GI syndrome. Therefore, it is necessary to develop alternative approaches to radioprotection of epithelium of small intestine that will rely on another mechanism, such as, for example, activation of NF-κB and subsequent inhibition of cell death.
Example 2
Flagellin Delays Mouse Death Caused by IR-Induced GI Syndrome
[0145] Whole body irradiation of mice with 15 Gy gamma radiation caused death within 8 days from GI syndrome providing a conventional model of radiation induced damage of GI tract. To test whether flagellin was capable of protecting GI epithelium from IR, we tested the effect of i.v.-injected flagellin on the dynamics of mouse lethality after 15 Gy of radiation. We used a range of flagellin doses, all of which were significantly lower than the highest tolerable dose known from literature (300 μg/mouse). Irradiation was done 4 hours post treatment. The results of a representative experiment are shown in FIG. 4. As expected, control irradiated mice (that received PBS i.v.) died between 5 and 8 days post-treatment. Animals that received flagellin lived significantly longer; the extension of animal survival correlated with the dose of flagellin. Pathomorphological analysis of the small intestine on day 7 after irradiation revealed dramatic differences between flagellin-treated and control groups (FIG. 5). Intravenous, intraperitoneal and subcutaneous delivery of 0.2 mg/kg of flagellin followed by 13 Gy irradiation afforded similar degree of protection, leading to 85-90% 30-day survival of mice (data not shown). While not being bound by theory, flagellin may be a radioprotectant due to its activation of NF-κB, which presumably acts as an inhibitor of apoptotic death.
Example 3
Flagellin Rescues Mice from Lethal IR-Induced Hematopoietic Syndrome
[0146] We next tested whether flagellin had an effect on mouse IR-induced death from HP syndrome that was experimentally induced by lower radiation doses (usually up to 11 Gy) that are incapable of causing lethal GI toxicity. The experiments were done similarly to the above-described ones (FIGS. 14 and 15), however, instead of 15 Gy, mice received 10 Gy, the dose that caused 100% killing in control group by day 13 (FIG. 6). Flagellin-treated group (5 μg/mouse) showed complete protection from this dose of IR surprisingly indicating that flagellin-mediated radioprotection acts not only against GI but also against HP IR-induced syndromes.
Example 4
Time Dependence on the Protective Effect of Flagellin
[0147] Mice were next administered flagellin at different times prior to 13 Gy of gamma irradiation. The results of one of such experiments is shown in FIG. 7. The obtained results show that flagellin is effective as a radioprotectant from 13 Gy if injected 1-4 h before treatment.
[0148] In order to further estimate the dependence of radioprotective activity of flagellin on the time of treatment, mice were injected at several time points relative to the moment of gamma-irradiation. Experiments were performed essentially as explained above, using intraperitoneal injection of 5 μg/mouse (0.2 mg/kg) of full-length flagellin or, for control mice, 5 μg/mouse (0.2 mg/kg) of bacterial RNA polymerase. The experiments were performed using the NIH-Swiss mouse strain. The results show that flagellin provides ˜90% survival after 13 Gy irradiation if injected at 1 or 2 hours before treatment (FIG. 7). Only -1 h graph is shown for clarity, however, both timepoints (-1 and -2 h) provide similar degree and dynamics of survival. The 4 h timepoint shows somewhat lower protection. Flagellin injected 24 hours before irradiation had no protective effect against 13 Gy induced death.
[0149] Interestingly, administration of flagellin 24 hours before 10 Gy gamma-irradiation provided 100% protection. While 13 Gy irradiation in mice primarily induces death from GI syndrome, 10 Gy-induced death is mostly mediated by hematopoietic syndrome. Accordingly, such long-term protection from 10 Gy irradiation may be mediated by enhanced proliferation or survival of hematopoietic stem cell induced by flagellin and/or long-living secondary cytokines
Example 5
Determination of LD50/30, LD50/7 and DMF for Flagellin
[0150] We next obtained an estimate of radiation dose-dependent protection for flagellin. As shown above (FIG. 7), treatment with flagellin was sufficient for 100% protection against 10 Gy gamma-irradiation (this dose causes death from hematopoietic syndrome) and 90% 30-day survival at 13 Gy (both hematopoietic and GI syndromes). Experiments were performed as described above, using flagellin 5 μg/mouse (0.2 mg/kg), intraperitoneally injected 1 h before irradiation.
[0151] At 15 Gy, however, 100% 7-day survival was followed by delayed death after 13 days (0% 30-day survival), while control group had fully succumbed to GI syndrome by day 7 (FIG. 8). The kinetics of the flagellin-treated group mortality after 15 Gy irradiation is reminiscent of such of control group at 10 Gy, hinting at death caused by hematopoietic syndrome. The results provide an estimate of flagellin LD50/30 around 13.5-14 Gy and DMF30 of about 1.75-1.8. This degree of radioprotection is significantly higher than any reported for a natural compound.
Example 6
Rational Design and Cloning of Flagellin Fragments
[0152] Salmonella flagellin, encoded by the FliC gene (SEQ ID NO: 2), is a strong activator of pro-survival NF-κB pathway. This is the most likely mechanism of its radioprotective action. Previous studies have shown that binding of flagellin to Toll-like receptor 5 (TLR5) on the cell surface is a necessary step that triggers activation of NF-κB. The domain structure of Salmonella flagellin is described in sufficient detail in the literature (FIG. 9). Moreover, previous structural studies of flagellin-TLR5 complex (FIG. 10) provide the ability to distinguish between domains that are essential or dispensable for binding and thus NF-κB activation. Protein minimization may provide reduced immune response after repeated administration of flagellin-related polypeptides. This may be achieved, in part, due to lower immunogenicity of low molecular weight proteins and smaller number of immunogenic epitopes available.
[0153] The domains needed for TLR5 binding may be located exclusively in the evolutionary conserved N- and C-terminal domains of bacterial flagellins. The hypervariable domain (amino acids 178-402) does not come into close contact with TLR5. As was demonstrated previously, replacement of this domain with a flexible linker peptide did not disrupt binding to TLR5. In addition, N-terminal and C-terminal coiled-coil polymerization domains (amino acids 1-55, 456-505) do not bind to TLR5 and likely are dispensable (see modified N and C termini B and B', respectively, as shown herein). Also, another fragment N-terminus lacking all domains but major N-terminal α-helix that actually binds TLR5 (amino acids 56-100) may be sufficient for binding.
[0154] Accordingly, three types of N-termini (A, B, C) and two types of C-termini (A',B'), connected with a flexible linker (SEQ ID NOS: 3 and 4) taken from pGEX-KG cloning vector (SEQ ID NOS: 5 and 6) were combined into expression constructs to produce several possible flagellin fragments (Table 1). In addition, constructs representing separate N-termini (A, B, C) and glutathione-S-transferase (GST)-fusions of C-termini (GST-A', GST-B') were prepared. All constructs were cloned in the pRSETb bacterial expression vector and 6×His-tagged proteins were produced and purified for further experiments (FIG. 11).
TABLE-US-00001 TABLE 1 Name Structure DNA Protein AA' (1-177)-Linker-(402-505) SEQ ID NO: 7 SEQ ID NO: 8 AB' (1-177)-Linker-(402-450) SEQ ID NO: 9 SEQ ID NO: 10 BA' (56-177)-Linker-(402-505) SEQ ID NO: 11 SEQ ID NO: 12 BB' (56-177)-Linker-(402-450) SEQ ID NO: 13 SEQ ID NO: 14 CA' (56-100)-Linker-(402-505) SEQ ID NO: 15 SEQ ID NO: 16 CB' (56-100)-Linker-(402-450) SEQ ID NO: 17 SEQ ID NO: 18 A (1-177) SEQ ID NO: 19 SEQ ID NO: 20 B (56-177) SEQ ID NO: 21 SEQ ID NO: 22 C (56-100) SEQ ID NO: 23 SEQ ID NO: 24 GST- GST-Linker-(402-505) SEQ ID NO: 25 SEQ ID NO: 26 A' GST- GST-Linker-(402-450) SEQ ID NO: 27 SEQ ID NO: 28 B'
Example 7
Selection of Biologically Active Flagellin Fragments
[0155] Since the radioprotective activities of flagellin appear to be NF-κB dependent, we tested the ability of the flagellin fragments to induce NF-κB translocation to the nucleus and binding to its target sites in DNA. This was tested by electrophoretic mobility shift assay (EMSA) using nuclear extracts from flagellin- and fragment-treated A549 lung cancer cells and labeled synthetic NF-κB binding kB oligonucleotide.
[0156] Only flagellin itself and fragments AA', AB', and BA' were capable of inducing NF-κB translocation (FIG. 12). The level of translocation is comparable for flagellin and fragments AA', AB', and BA'. The hypervariable domain does not appear to be necessary for NF-κB translocation, while the presence of at least one polymerization domain, N- or C-terminal, is required. Mixtures of the N- and C-terminal fragments (A+A', A+B') were inactive.
[0157] While translocation of NF-κB to the nucleus is a crucial step in induction of NF-κB-regulated inhibitors of apoptosis, it is not sufficient in itself. To directly test the ability of selected fragments to induce expression of NF-κB-regulated genes, we performed reporter assay experiments. Flagellin and the AA', BB', A' and B' fragments were used for treatment of H116 human colon cancer cells carrying luciferase gene under a NF-κB-responsive promoter. The reporter construct contained three NF-κB-binding sites from the E-selectin promoter combined with a Hsp70 minimal promoter that is routinely used for the detection of NF-κB status of cells. Luciferase activity was measured in cell lysates six hours after addition of flagellin or its truncated fragments into the medium. TNF was used as positive control. The results of a representative experiment are shown in FIG. 13 and indicates that flagellin and fragment AA' are capable of NF-κB activation, whereas fragments BB', GST-A' and GST-B' are not.
Example 8
Further Optimization of Flagellin Fragments
[0158] We further minimized the AA' flagellin fragment by producing additional fragments through stepwise removal of peptide fragments from its N-terminal half (Table 2). Electrophoretic mobility shift assays were performed as described above using nuclear extracts from flagellin- and fragment-treated HT29 human colon cancer cells and labeled synthetic NF-κB binding κB oligonucleotide. NF-κB binding activity in HT29 cells was stimulated with TNFα (10 ng/ml), or flagellin fragments (1 mg/ml) for 15 min. As shown in FIG. 14, fragments AA'n1-170, AA'n54-170, AA'n1-163 and AA'n54-163 each induce NF-κB translocation, with levels comparable to that of flagellin for AA'n1-170, AA'n54-170 and AA'n1-163.
TABLE-US-00002 TABLE 2 Name Structure DNA Protein AA'n54-177 (54-177)-Linker- SEQ ID NO: 11 SEQ ID NO: 12 (402-505) AA'n1-170 (1-170)-Linker- SEQ ID NO: 29 SEQ ID NO: 30 (402-505) AA'n54-170 (54-170)-Linker- SEQ ID NO: 31 SEQ ID NO: 32 (402-505) AA'n1-163 (1-163)-Linker- SEQ ID NO: 33 SEQ ID NO: 34 (402-505) AA'n54-163 (54-163)-Linker- SEQ ID NO: 35 SEQ ID NO: 36 (402-505)
[0159] In order to study the ability of the AA' fragments to directly activate NF-κB-regulated transcription, we performed reporter assay experiments as described above for a wide range of concentrations of flagellin, original AA' and AA'-derived fragments. As shown above, AA' and AA'n1-170 induce NF-κB-regulated transcription at the level comparable to such of flagellin over the studied range of concentrations (FIG. 15, left). AA' and AA'n1-170 are more active than flagellin in the very low concentration range (FIG. 15, right), possibly due to their reduced molecular weight. The results with fragment AA'n1-170 show that AA'-derived flagellin fragments may be made with a portion of the N-terminal domain removed without significant loss of activity and may be used as effective radioprotectors.
[0160] The above experiments (EMSA and reporter activation assay) were repeated with flagellin and AA' fragments subjected to 30 minutes boiling and renaturation before being applied to cells. The results were comparable to those obtained without boiling (data not shown). This shows that the observed differences in flagellin fragment activity may not be caused by changes in protein stability.
Example 9
In Vivo Comparison of Radioprotective Properties of Flagellin and Flagellin Fragments
[0161] As shown above, full-length flagellin provides protection from both hematopoietic and gastrointestinal syndromes. The radioprotective potential of flagellin fragments was similarly tested after gamma-irradiation with 11 Gy (dose that induces hematopoietic syndrome-associated mortality in mice) or 14 Gy (dose that causes death from GI syndrome). Mice (10 animals per group) were injected subcutaneously with 5.0 μg/mouse (0.2 mg/kg) of flagellin or its fragments, AA' or BB', and gamma-irradiated 1 hour later.
[0162] The degree of radioprotection displayed by the AA' fragment is at least comparable to full-length flagellin (FIG. 16). Both the AN fragment and full-length flagellin showed 100% 30-day survival for mice irradiated with 11 Gy and 14 Gy. Meanwhile, 0% of mice injected with the BB' fragment survived to 30 days. This is expected since the BB' fragment is incapable of inducing NF-κB in vitro. These results show that significant reduction in the size of flagellin (about 40% removed) may be achieved without a decrease in the degree of radioprotection. In addition, the ability to predict radioprotective potential from results of in vitro NF-κB activation is confirmed.
Example 10
Identification of Cellular Targets of Flagellin-Mediated Radioprotection
[0163] Tissue samples of intestinal mucosa were taken 5 days after 14 Gy irradiation from mice pretreated with flagellin and control mice. Control animals were treated with 5.0 μg/mouse (0.2 mg/kg) bacterial RNA-polymerase. Pathomorphological analysis of the small intestine reveals reduction of the size of crypts and villi and a number of the cells with condensed apoptotic nuclei in control mouse and near-normal morphology in the treated mouse (data not shown). Tissue samples (small intestine and skin from the back) were also obtained from mice treated with the AA' fragment. The results shown in FIG. 17 are areas of typical morphology observed over a set of at least 3 mice. After treatment with flagellin and the AA' fragment, mice demonstrated near-normal intestinal morphology with preservation of the villi/crypt structure (FIG. 17A).
[0164] In addition to purely histological observation of cell death and survival, we performed more specialized tests of apoptotic cell death in intestinal tissue using a TUNEL assay, which detects apoptosis-associated DNA fragmentation. These experiments allowed us to define with a high degree of probability cellular populations that are depleted by radiation and rescued by flagellin fragment treatment. The earliest radiation-induced alterations detectable in the small intestine after treatment with IR is apoptosis occurring in vascular endothelial cells of villi, which is seen as early as 5 hours post treatment (FIG. 17B). This apoptosis, which is believed to be critical for radiosensitivity of the small intestine, was almost completely blocked in the mice pretreated with the AN fragment (FIG. 17B, bottom panel). Degeneration of villi and crypts, occurring within the next several days post treatment and greatly suppressed in AA' fragment-treated animals, comes as a consequence of injury of blood vessels. Effective protection of endothelial cells of the small intestine by the flagellin fragment may be due to expression of TLR5 in these cells.
[0165] Remarkably, the AA' fragment and flagellin also prevented the radiation-induced disappearance of sebaceous glands located at the base of skin hair follicles (FIG. 27C). These results further confirm the suitability of AA' fragment for radioprotection and for the prevention of radiation-induced hair loss.
Example 11
Protection from Supralethal Radiation
[0166] In order to explore the limits of radioprotection provided by the AA' fragment, we irradiated mice with 17 Gy and 20 Gy single doses of total body gamma-radiation. The experiment was performed as described above using inactive flagellin fragment (CB) as a negative control.
[0167] As expected, we observed a 100% mortality in both groups at 17 and 20 Gy (FIG. 18). However, death was significantly delayed in both cases by administration of the AA' fragment. Most remarkably, the kinetics of death at 17 Gy in control mice conform to GI syndrome (6-7 day mortality), while death of mice treated with the AA' fragment appear to be mediated by hematopoietic syndrome (10-15 day mortality). This shows that flagellin and flagellin fragments may protect against the GI syndrome at doses as high as 17 Gy. In addition, this shows that even further radioprotection may be obtained by flagellin and flagellin fragments combined with hematopoietic radioprotectors.
Example 12
Immunogenicity and Repetitive Administration Studies
[0168] Overall immunogenicity of a protein may determines its suitability for repeated use. Antibodies generated by immune system are capable of reducing the therapeutic activity of the protein and also may induce anaphylactic reaction upon second exposure if IgE antibodies are produced against the protein. Thus, any reduction in the amount and variety of antibodies compared to full-length flagellin is an improvement. Accordingly, after repeated introduction of flagellin or its fragments we monitored; a) efficiency of radioprotection afforded at second exposure; b) local and general allergic reactions; and c) antibody titer.
[0169] We tested the ability of AA' to protect mice that were exposed to it. A group of 20 NIH-Swiss mice were subcutaneously injected with 5 μg/mouse (0.2 mg/kg) of AA'. A second injection of a equal dose of AA' was administered after 21 (10 mice) and 28 days (another 10 mice), with the time elapsed being sufficient for formation of antibodies. The second injection of AA' was followed by 13 Gy of whole-body gamma irradiation (1 h post-injection). 100% 30-day survival was observed in both groups, as it was observed with mice that had no previous exposure to AA' (data not shown). These results show that activity of AA' is not diminished over long-term repeated administration and reaffirm its potential for multiple-use applications. Also, no local allergic reaction or anaphylaxis was observed either with flagellin or AA'.
[0170] We also performed an ELISA determination of antibody titers in order to quantify the effect that AA' has on the immune status of the organism. 96-well plates were coated with flagellin or AA', 20 mg/ml, 50 ml/well, and incubated overnight at +4° C. Blood serum samples collected from mice were added to the wells in several dilutions and incubated overnight followed by 6 hrs reaction with secondary goat anti-mouse IgG HPO-conjugate antibodies. Measurements were performed using a spectrophotometer with a 414 nm filter. The antibody titers determined for individual mice and average titers are shown in FIG. 19 and FIG. 20.
[0171] AA' induces far lower antibody levels in mice (FIG. 19), on the order of 0.8 mg/ml serum at 21 day and about 10% more at 28 days. Flagellin, on another hand (FIG. 20), induces a high titer of antibodies, around 20 mg/ml, at both 21 and 28 days. Overall, this shows that removal of the hypervariable domain sharply reduces the immunogenicity of AA' compared to the original protein (approximately 25×). FIG. 19 also shows that the majority of AA'-specific antibodies are capable of recognizing flagellin. This confirms that the rational design of AA' does not produce a sizable number of new immunogenic epitopes while removing >95% of the immunogenicity of the original protein.
Example 13
Acute Toxicity Studies of Flagellin and AA'
[0172] The lethal dose of Salmonella flagellin is between 1 mg/kg (systemic inflammation) and 10 mg/kg (100% lethality). We subcutaneously administered increasing doses of AA' to mice (4 mice per dose group) at 0.5, 1, 2, 4 and 8 mg/kg. Due to the lower (˜60%) molecular weight, 8 mg/kg of AA' correspond to a molar-equivalent dose of 13.3 mg/kg of flagellin. Several days after administration at all doses, no visible detrimental effects were observed, such as mortality, morbidity or signs of systemic inflammation such as reduced activity and fever. This shows that the pro-inflammatory effect of AA' is negligible compared to full-length flagellin, especially considering that AA' provides an efficient radioprotection at 0.2 mg/kg. The reduced toxicity may be sue to the absence of the central pro-inflammatory domain in the AA' fragment.
Example 14
Protection from Fractioned Irradiation by AA'
[0173] Repetitive irradiation within a short period of time may be common, for example, in space radiation events and in clinical radiotherapy regimens. We tested the ability of the AA' flagellin fragment to protect mice from sub-lethal (4 treatments of 3 Gy) and 100% lethal (4 treatments of 4 Gy) regimens of fractionated gamma-irradiation. Fragment AA' or saline buffer was given to NIH-Swiss female mice before every irradiation (once a day for 4 days). AA' was administered as described above for single-dose irradiation (5 μg/mouse, given subcutaneously 1 h before irradiation).
[0174] The results in FIG. 21 show that AA' provides significant protection against repetitive doses of radiation received within a short timeframe. The cumulative dose of fractionated radiation that is still compatible with 100% 30-day survival after AA' treatment is comparable to such obtained in single-dose irradiation scenarios.
Example 15
AA' Protects Normal Tissues without Compromising the Anti-Tumor Therapeutic Effect of Radiation
[0175] The ultimate test of a potential radioprotective agent in cancer treatment is tumor selectivity, its ability to protect normal tissues while providing no or little protection to the tumor. We injected 10 NIH-Swiss mice subcutaneously, in both flanks (20 tumors total), with 2×106 cells of syngeneic sarcoma cell line model (NIH3T3-derived and spontaneously transformed sarcoma with p53 inactivated by dominant negative inhibitor GSE56). When tumors reached the size of 5-7 mm in diameter (day 5), the mice were injected subcutaneously with 0.2 mg/kg of AA' or saline vehicle and irradiated 1 hours later with 4 Gy of total-body γ-irradiation (3×4.3 Gy=12.9 Gy total dose). Injections and irradiations were done at days 5, 6 and 7.
[0176] As the results show in FIG. 11, AA' enhanced the radiation-induced shrinkage of tumors. By day 18, all the irradiated tumor-bearing mice died from acute radiation toxicity whereas 100% of mice that obtained both radiotherapy and AA' were both cured and survived the treatment. Similar result was obtained with another syngeneic tumor model--B16 melanoma cells (FIG. 11, right panel). Surprisingly, even in unirradiated mice, AA' administration caused a decrease in the growth rate of tumors. This may be due to AN-induced immunostimulating, which is known to be caused by other ligands of Toll-like receptors. These results indicate that AA' increases the tolerance of mice to radiation with no effect on the radiosensitivity of two types of tumors, thus opening the possibility of combining radiotherapy with AA' to improve treatment outcome.
Example 16
Radioprotective Mechanisms of AA' and LPS are Different
[0177] Lipopolysaccharide of gram-negative bacteria (LPS) is a ligand of another Toll-like receptor, TLR4. LPS is a strong inducer of NF-κB and a subsequent cascade of cytokines LPS is known as a radioprotective compound, but its high toxicity makes its use unfeasible (radioprotective dose is very close to the lethal dose). One of the major mechanisms underlying the radioprotection by LPS is the activation of cyclooxygenase 2 (COX-2) that, in turn, drives the synthesis of GI-protective prostaglandins. The possibility that radioprotection by TLR5 also relies on COX-2 activity was tested by administering s.c. LPS (2 mg/kg), AA' (0.2 mg/kg') or vehicle 1 hr before irradiation of NIH-Swiss mice in combination with i.p injection of 1 mg/kg of NS398, a synthetic COX-2 inhibitor, or the corresponding vehicle. The mice were then treated with 13 Gy of total-body γ-irradiation. NS398 completely abolished LPS-mediated radioprotection but not the radioprotection of AA' (FIG. 12). This result shows that AN does not significantly rely on COX-2 for its activity and induces radioprotection by a mechanism different from the mechanism of LPS-mediated protection.
Example 17
AA' Protects Multiple Mouse Strains
[0178] We have extensively confirmed that AA' protects NIH-Swiss and ICR mice from radiation. To confirm that the radioprotection activity of AA' is not confined to a few mouse strains, several additional strains of mice with dissimilar origins were tested for protection by AA': 129/Sv, DBA/2 (relatively radioresistant), Balb/c (relatively radiosensitive) and Balb/c x DBA/2 F1 hybrid CD2F1. Experimental groups were injected with 0.2 mg/kg AA' 30 minutes before irradiation, while control groups were injected with vehicle (PBS).
[0179] All groups of mice (8-10 mice each, 8-12 week old females) were exposed to 10 Gy of single-dose, whole-body gamma-irradiation. Survival of mice at days 10 and 30 is shown. The results are shown in FIG. 27 as a cone graph. At 10 days, only Balb/c mice display mortality, which is drastically reduced by AA' administration (0% vs. 100% survival). At day 30, all tested strains display improved survival (0-25% vs. 50-100%) after AA' administration.
Example 18
Pharmacokinetics of AA'
[0180] Pharmacokinetic parameters (effective concentration and the duration of drug the presence in the organism) may be important for route, dose and time of drug administration. The pharmacokinetics of CBLB502 (AA') were thus tested for four common routes of injection: intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.) or intramuscular (i.m.). A radioprotective dose of CBLB502 (AA'), 0.2 mg/kg, was injected in 12-15 week old ICR mice and plasma samples were collected at the specified times after injection (at least 3 mice/point). The levels of CBLB502 in plasma were measured by sandwich ELISA using known concentrations of CBLB502 spiked in the control ICR plasma for calibration. The results are shown in FIG. 28 and FIG. 29.
[0181] The results show that the highest levels and longest persistence of CBLB502 in plasma are provided by intramuscular or intraperitoneal injection. After intramuscular injection, significant (>5 ng/ml) levels of CBLB502 are observed in mouse plasma >3 hours. Intravenous injection leads to a more rapid disappearance of CBLB502 from the bloodstream.
Example 19
Influence of AA' on Gamma-Irradiation Induced Cell Death and Growth Inhibition in A549 Cells
[0182] The A549 human lung adenocarcinoma cell line is reported to respond to flagellin by activation of NF-κB DNA-binding activity (Tallant T., et. al., BMC Microbiol. 2004 Aug. 23; 4:33). We decided to check whether this activation translates in the protection of cells from γ-IR in cell growth inhibition assay.
[0183] Tumor cells were seeded in wells of three 96-well plates in 3 different densities (0.5×104, 1×104 and 2×104 cells/well, producing single-cell, spare or semi-contact layer). After cells had attached to plastic, CBLB502 (2 μg/ml) was added to the wells of non-irradiated cells, or 15 min prior to 7 Gy or 10 Gy of gamma-irradiation. Control wells received equal volume of vehicle (PBS). All points were done in quadruplicate. 72 hours after irradiation, medium was replaced with methylene blue in 50%-methanol and the relative numbers of viable cells in wells were measured using spectrophotometer at 650 nm. The results are shown in FIG. 30. This experiment was also repeated with fixed dose of 1×104 A549 cells/well, CBLB502 was added 1 hour before 5, 10 or 15 Gy of gamma-irradiation (data not shown). We observed a similar effect of flagellin in all tested experiment conditions.
[0184] Gamma-irradiation induced a dose-dependent reduction in the number of A549 cells plated at all three densities (up to 60% as compared to non-irradiated control wells). CBLB502 had no or slight effect on cell numbers, with or without gamma-irradiation. This indicates that tumor cells are not significantly protected by CBLB502 (AA') from radiation. This effect may be due to tumor cells having constitutively active NF-κB pathway or some other mechanism.
Example 20
Influence of AA' on Gamma-Irradiation Induced Cell Death and Growth Inhibition in Multiple Cell Lines
[0185] Based on the results using A549 cells, several additional tumor cell lines (human melanoma Mel-7 and Mel-29, colon cancer HCT116, lung cancer HT1080), immortalized kidney epithelial cells (NKE) and normal mouse aortal endothelial cells (MAEC)) were tested in growth inhibition assay after 10 and 15 Gy of gamma-irradiation, as compared with intact control, with or without pretreatment with CBLB502. Cells were seeded in 96-well plates night before the treatments. CBLB502 (2 μg/ml) was added to the wells 4 hrs, 1 hr or 10 min before irradiation (all points were done in quadruplicate). 48 hours later, methylene blue staining was performed to determine the relative amount of the viable cells in the wells. All three time-points had shown the same effect (results for CBLB502 added 1 hr before irradiation are shown in FIG. 31). The percent of growth inhibition was calculated from the OD650 in control non-irradiated wells, taken as 0% inhibition.
[0186] Both human melanoma cell lines and MAEC cells were rather resistant to gamma-irradiation and showed only slight (<20%) growth inhibition after both 10 and 15 Gy comparing with intact cells (0 Gy). NKE, HT1080 and HCT116 cells showed up to 40% of growth inhibition after gamma-irradiation. Remarkably, CBLB502 had no or only a slight inhibitory effect on tumor cell growth, irradiated or not. The experiment was repeated twice. In addition, similar results were obtained on tested lung adenocarcinoma H1299 and prostate cancer CWR22 (data not shown). This indicates that there is no significant protection provided by CBLB502 to the tumor cell lines against radiation-induced cell death.
Example 21
Influence of Irradiation and AA' on BrdU Incorporation in Small Intestinal Crypts
[0187] Besides a direct inhibition of apoptosis, temporary halt of proliferation followed by repair may be an alternative mechanism of radioprotection, and has been described for other radioprotectors such as TGF-133 (Booth D., et. al., Int J Cancer. 2000 Apr. 1; 86(1):53-9). Accordingly, we decided to examine the possible influence of CBLB502 (AA') on the proliferative activity of the cells in small intestine (with and without irradiation) during the first hours after its administration (FIG. 32). CBLB502 or PBS was injected i.p. in mice, followed after 30 min by 15 Gy irradiation (if used). 2 hr after injection (1.5 hr after irradiation if it was applied), BrdU was injected intraperitoneally. Samples of small intestine were obtained after additional 1.5 hours.
[0188] Without irradiation, BrdU was incorporated at high levels in the nuclei of cells in the intestinal crypts of untreated NIH-Swiss mice (FIG. 32, top left), whereas DNA synthesis (as measured by BrdU incorporation) was nearly undetectable in the crypts of CBLB502-treated mice (FIG. 32, top right). In vehicle-treated irradiated mice, the incorporation of BrdU was lower than in control mice. Importantly, the level of BrdU incorporation was strongly reduced by CBLB502, possibly indicating quick (S phase) growth arrest, as opposed to later (G2 phase) irradiation-induced growth arrest. Therefore, cytostatic activity of CBLB502 or flagellin may be an additional mechanism of radioprotection of small intestine.
Example 22
Duration of AA'-Mediated Growth Arrest and Reduced BrdU Incorporation
[0189] We next determined the duration of the CBLB502-induced growth arrest in small intestine. CBLB502 or PBS was injected i.p. in mice, BrdU was injected 1 or 4 hours later and samples of small intestine were obtained after additional 1.5 hours from several mice (samples from three mice are shown) (FIG. 33).
[0190] Incorporation of BrdU in intestine was reduced as compared to control if BrdU was injected after 1 hour (as it was shown in the previous experiment where BrdU was injected after 2 hr). NIH-Swiss, ICR and Balb/c mice displayed a similar degree of CBLB502-mediated block of BrdU incorporation (Balb/c samples are shown in FIG. 33). If BrdU was injected 4 hr after injection of CBLB502, the levels of incorporation/DNA synthesis were even higher than in control. This indicates that inhibition of intestinal stem cell proliferation by CBLB502 may be temporary and may be quickly resolved (by 4 hours), followed by a period of increased proliferation (possibly due to the partial synchronization of cells).
Example 23
Influence of AA' on BrdU Incorporation in Colonic Crypts
[0191] The colon is much less radiosensitive than small intestine. To further examine the relationship between reduced proliferation in the small intestine and radioprotection, we determined the effect of CBLB502 on BrdU incorporation in the colon. CBLB502 or PBS was injected i.p. in mice, BrdU was injected 1 hour later and samples of small intestine were obtained after additional 1.5 hours (FIG. 34).
[0192] Unlike in the small intestine, CBLB502 has no effect on BrdU incorporation in colon. This is surprising since TLR5 is plentiful in both organs. The difference in effects may be due to the higher amount of symbiotic bacteria in the colon, which may mask the effect of additional TLR5 signaling induced by CBLB502.
Example 24
Comparison of Radioprotective Potential by Route of Administration
[0193] We next tested radioprotection provided by FliC flagellin administered via several routes: intravenous (i.v.), intraperitoneal (i.p.), intramuscular (i.m.), subcutaneous (s.c.) and gavage. For parenteral (non-gavage) routes, mice were injected with 0.2 mg/kg of FliC flagellin dissolved in PBS or vehicle, followed 1 hr later by 13 Gy irradiation. In gavage delivery experiment, 5 mice were given to swallow an increased dose (50 μg) of FliC in 50 μl of PBS 1 h before 13 Gy gamma-irradiation. Both experiments were done in 8-10 week old female NIH-Swiss mice, 5-10 mice/group.
[0194] All tested routes besides gavage afforded similar degree of protection, leading to 85-90% 30-day survival of mice (data not shown). No protection against radiation was provided by gavage delivery, which may be due to digestion of the protein by the gastrointestinal environment. In addition, flagellin receptor, TLR5, is absent on the luminal side of intestinal epithelium that is exposed to intestinal contents (Gewirtz A T., et. al., J Immunol. 2001 Aug. 15; 167(4):1882-5)
Example 25
Effect of AA' on the Morphology of Small Intestine
[0195] Flagellin (and CBLB502) may induce NF-κB activity via binding to TLR5. Accordingly, CBLB502-mediated radioprotection may be dependent on the presence and activity of TLR5. MOLF/Ei mice are a known natural model of TLR5 deficiency (Sebastiani G., et. al., Genomics. 2000 Mar. 15; 64(3):230-40). To verify that CBLB502-mediated radioprotection is indeed TLR5-dependent, we tested the protection of small intestine from radiation by CBLB502 in MOLF/Ei and NIH-Swiss mice (FIG. 35). Both strains of mice were given 0.2 mg/kg CBLB502 (AA') or PBS 0.5 hr before 15 Gy of gamma-irradiation. The samples of small intestine were obtained 4 days after irradiation, stained by hematoxylin-eosin and subjected to pathomorphological analysis.
[0196] In NIH-Swiss (TLR5 wild type) mice, CBLB502 pretreatment led to preservation of intestinal morphology (long villae, normal crypts) as compared to short villae and disappearance of normal crypt structure in PBS-treated mice. Meanwhile, in TLR5-deficient MOLF/Ei mice the administration of CBLB502 had no improving effect on intestinal morphology after 15 Gy of gamma-irradiation: short villae and destruction of the normal crypt structure was observed, with or without CBLB502. This indicates that the presence of TLR5 may be necessary for CBLB502-mediated radioprotection in the small intestine.
Example 26
Flagellin Derivatives
[0197] Additional flagellin variants were produced based on the domain structure shown in FIG. 36. The flagellin variants were then tested along with some of the variants discussed above for NF-κB stimulating activity (Table 3). A549 cells were left unstimulated or stimulated with TNF (10 ng/ml) as indicated or 1 μg/ml of purified flagellin or the various indicated flagellin derivatives for 45 min and whole cell extracts prepared as described in Example 7. EMSA assays were preformed and the NF-κB DNA-protein complex detected as described in Example 7.
TABLE-US-00003 TABLE 3 NF-κB Name N-terminal C-terminal DNA Protein Stimulation AA' 1-176 402-505 SEQ ID NO: 7 SEQ ID NO: 8 Yes AB' 1-176 402-450 SEQ ID NO: 9 SEQ ID NO: 10 Yes BA' 54-176 402-505 SEQ ID NO: 11 SEQ ID NO: 12 Yes BB' 54-176 402-450 SEQ ID NO: 13 SEQ ID NO: 14 No CA' 54-100 402-505 SEQ ID NO: 15 SEQ ID NO: 16 No CB' 54-100 402-450 SEQ ID NO: 17 SEQ ID NO: 18 No AA'n1-170 1-170 402-505 SEQ ID NO: 29 SEQ ID NO: 30 Yes AA'n54-170 54-170 402-505 SEQ ID NO: 31 SEQ ID NO: 32 Yes AB'n1-170 1-170 402-450 SEQ ID NO: 37 SEQ ID NO: 38 Yes AA'n1-163 1-163 402-505 SEQ ID NO: 33 SEQ ID NO: 34 Yes AA'n54-163 54-163 402-505 SEQ ID NO: 35 SEQ ID NO: 36 Yes AB'n1-163 1-163 402-450 SEQ ID NO: 39 SEQ ID NO: 40 Yes AA'n1-129 1-129 402-505 SEQ ID NO: 41 SEQ ID NO: 42 Yes AA'n54-129 54-129 402-505 SEQ ID NO: 43 SEQ ID NO: 44 Yes AB'n1-129 1-129 402-450 SEQ ID NO: 45 SEQ ID NO: 46 untested AB'n54-129 54-129 402-450 SEQ ID NO: 47 SEQ ID NO: 48 Untested AA'n1-100 1-100 402-505 SEQ ID NO: 49 SEQ ID NO: 50 untested AB'n1-100 1-100 402-450 SEQ ID NO: 51 SEQ ID NO: 52 untested AA'n1-70 1-70 402-505 SEQ ID NO: 53 SEQ ID NO: 54 No AB'n1-70 1-70 402-450 SEQ ID NO: 55 SEQ ID NO: 56 No A 1-176 SEQ ID NO: 19 SEQ ID NO: 20 No B 54-176 SEQ ID NO: 21 SEQ ID NO: 22 No C 54-100 SEQ ID NO: 23 SEQ ID NO: 24 No GST-A' 402-505 SEQ ID NO: 25 SEQ ID NO: 26 No GST-B' 402-450 SEQ ID NO: 27 SEQ ID NO: 28 No
[0198] The results in Table 3 indicate that flagellin variants with at least one polymerization domain (aa1-50 or aa 450-505) that linked to domains contained within the amino-terminal region (aa 1-176) and those of the carboxy terminus (aa 402-505) are capable of stimulating NF-κB and would thus be expected to be radioprotectors. Physical linkage of the recognition domains may be required for activity as domains supplied unlinked in trans fail to activate NF-κB. As an alternative to the linking of the domains in a single polypeptide, the domains may be linked using a linker, which is a molecule that is used to join two molecules. The linker may be capable of forming covalent bonds or high-affinity non-covalent bonds to both molecules. Suitable linkers are well known to those of ordinary skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. The linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine).
[0199] The region between amino acids 163 and 176 may be required for activity when the carboxyl polymerization domain (aa 450-505) is absent. Since this region is dispensable for activity when the carboxyl polymerization domain is present it may be involved in stabilizing the derivative. The region between amino acids 70 and 129 may be important for activation and may be involved in derivative recognition. The region between amino acids 402 and 450 may also be required for activity. The domains identified above are located within three large α-helices (located within amino acids 54-129 and 402-450) and, to produce an active derivative, may need to form a ring-like structure (with or without polymerization domain).
Example 27
Dynamic of Weight Loss and Viability of Mice Treated with Cisplatinum with or without Protectan CBLB502
[0200] This example demonstrates that pretreatment with CBLB502 strongly reduces chemotherapy toxicity when administered prior to chemotherapeutic treatment. Cisplatinum treatment was used as an example of chemotherapy-associated toxicity. When injected at toxic doses, cisplatinum is known to induce myelosuppression and renal toxicity in animals, which result in weight loss and, in the case of severe adverse effects, death. Less severe or delayed weight loss and mortality can be used as indicators of the efficacy of agents capable of reducing cisplatinum-mediated toxicity. The effects of CBLB502 were tested using this rationale.
[0201] The following experiments were performed using NIH Swiss mouse females that were 12-15 weeks in age (6-10 mice/group). CBLB502 was injected subcutaneously (s.c.) 30 min. prior to injecting cisplatinum at a dose of 1 μg/mouse (0.04 mg/kg), which are the optimal conditions for protecting mammals from total body ionizing radiation. Cisplatinum in PBS solution was injected interperitoneally (i.p.) at either 10 mg/kg (maximum tolerable dose, [MTD]) or 20 mg/kg (2×MTD). Mice were monitored daily, including for weight and behavioral abnormalities, for 30 d or until death. Mice that lost more than 25% of their weight were sacrificed. The experiment was repeated three times, each time yielding similar results.
[0202] The results of a representative experiment are shown in FIG. 39. FIG. 39A shows the dynamics of weight loss of mice that received 2×MTD cisplatinum (20 mg/kg), pretreated with either CBLB502 or PBS (as indicated). While the majority of mice in the control group died or experienced >25% weight loss by day 10, 100% of animals that received CBLB502 30 min before being injected with cisplatinum survived, and never lost more than 20% of their weight. FIG. 39B shows the dynamics of the deaths of animals that received 20 mg/kg cisplatinum, with or without CBLB502. FIG. 39C shows the results of experimental groups treated as described for FIG. 39C, but which received 10 mg/kg cisplatinum. Again, the CBLB502-treated group differed substantially from the control group by not losing weight in response to cisplatinum treatment. No deaths were observed among mice in these groups. FIG. 39D, however, shows that there were obvious behavioral changes exhibited in mice that received cisplatinum with PBS (limited mobility as a sign of morbidity) compared to animals that received a similar dose of cisplatinum, but were pretreated with a single injection of CBLB502 (active animals).
[0203] FIG. 40 shows the weight loss effects in NIH Swiss mice of cisplatinum alone at a dose of 10 mg/kg (n=11) compared with the same dose of cisplatinum when 0.04 mg/kg of CBLB502 is injected 30 min prior to the cisplatinum (n=11). While cisplatinum-only treated mice showed almost a mean 30% loss in body weight after two weeks, the mice treated with CBLB502 prior to cisplatinum treatment never exhibited a mean weight loss greater than about 15% on any given day. The CBLB502-treated mice significantly less weight than the cisplatinum-only treated mice on Days 7, 10 and 13.
[0204] Furthermore, FIG. 41 shows that while no mice out of 11 treated with cisplatinum alone survived past Day 13, only one mouse out of eleven treated with CBLB502 beforehand died (at Day 13). All of the remaining ten mice survived to at least Day 30. The difference in survival rate between groups was statistically significant from Day 10 to 30. This result indicates that CBLB502 significantly increases the survival rate of mice treated with cisplatinum.
[0205] FIG. 42 shows that CBLB502 can also mitigate cisplatinum-induced weight loss in FVB mice. As for the NIH Swiss mice, FVB mice were treated with 10 mg/kg, i.p. cisplatinum alone (n=3) or with CBLB502 at 0.04 mg/kg, s.c. 30 min prior to receiving cisplatinum (n=3). CBLB502-treated mice showed lower mean weight loss compared to mice treated with cisplatinum alone. Thus, the protective effect of CBLB502 against cisplatinum toxicity is not limited to only one mouse strain.
[0206] FIG. 43 shows that renal toxicity caused by cisplatinum is also reduced by CBLB502. Blood urea levels in mice treated with cisplatin (20 mg/kg, i.p.) (n=3) were compared with mice injected with CLBL502 (0.04 mg/kg, s.c.) (n=3) 30 min prior to cisplatinum treatment, to mice treated with CLBL502 alone (n=3), and to untreated mice (n=3). Urea was detected in mouse blood serum at Day 4 post-treatment using a QuantiChrom Urea Assay Kit (BioAssay Systems). There was very little difference in mean blood urea levels between CLBL502-only and untreated mice. Both groups of mice showed a mean OD 490 of less than 0.50. On the other hand, cisplatinum-only treated mice exhibited a mean OD 490 of over 1.00. Strikingly, treating mice with CLBL502 prior to cisplatinum treatment resulted in a mean OD 490 that was very similar to mean levels in CBLB502-only and untreated mice. These results indicate that renal toxicity of cisplatinum is completely suppressed by treating mice with CLBL502.
[0207] These experiments show that administering CBLB502 prior to cisplatinum treatment strongly reduces the toxicity of this chemotherapeutic drug, as obviated by the less severe weight loss, and reduced lethality and renal toxicity in CBLB502-treated mice. Although this result was achieved by using one representative of a large group of genotoxic chemotherapeutic drugs (including cyclophosphamide, doxorubicin, 5-fluorouracil, camptothecin, methotrexate, melphalan, taxanes, etc.), indicates that CBLB502 can be used more widely as an agent to reduce the adverse effects and toxicity of chemotherapeutic drugs.
Sequence CWU
1
1
761505PRTSalmonella dublin 1Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser
Leu Leu Thr Gln Asn 1 5 10
15 Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg Leu
20 25 30 Ser Ser
Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln 35
40 45 Ala Ile Ala Asn Arg Phe Thr
Ser Asn Ile Lys Gly Leu Thr Gln Ala 50 55
60 Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln
Thr Thr Glu Gly 65 70 75
80 Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ser
85 90 95 Val Gln Ala
Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser Ile 100
105 110 Gln Asp Glu Ile Gln Gln Arg Leu
Glu Glu Ile Asp Arg Val Ser Asn 115 120
125 Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp
Asn Gln Met 130 135 140
Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp Leu 145
150 155 160 Gln Lys Ile Asp
Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val Asn 165
170 175 Gly Pro Lys Glu Ala Thr Val Gly Asp
Leu Lys Ser Ser Phe Lys Asn 180 185
190 Val Thr Gly Tyr Asp Thr Tyr Ala Ala Gly Ala Asp Lys Tyr
Arg Val 195 200 205
Asp Ile Asn Ser Gly Ala Val Val Thr Asp Ala Ala Ala Pro Asp Lys 210
215 220 Val Tyr Val Asn Ala
Ala Asn Gly Gln Leu Thr Thr Asp Asp Ala Glu 225 230
235 240 Asn Asn Thr Ala Val Asp Leu Phe Lys Thr
Thr Lys Ser Thr Ala Gly 245 250
255 Thr Ala Glu Ala Lys Ala Ile Ala Gly Ala Ile Lys Gly Gly Lys
Glu 260 265 270 Gly
Asp Thr Phe Asp Tyr Lys Gly Val Thr Phe Thr Ile Asp Thr Lys 275
280 285 Thr Gly Asp Asp Gly Asn
Gly Lys Val Ser Thr Thr Ile Asn Gly Glu 290 295
300 Lys Val Thr Leu Thr Val Ala Asp Ile Ala Thr
Gly Ala Ala Asp Val 305 310 315
320 Asn Ala Ala Thr Leu Gln Ser Ser Lys Asn Val Tyr Thr Ser Val Val
325 330 335 Asn Gly
Gln Phe Thr Phe Asp Asp Lys Thr Lys Asn Glu Ser Ala Lys 340
345 350 Leu Ser Asp Leu Glu Ala Asn
Asn Ala Val Lys Gly Glu Ser Lys Ile 355 360
365 Thr Val Asn Gly Ala Glu Tyr Thr Ala Asn Ala Thr
Gly Asp Lys Ile 370 375 380
Thr Leu Ala Gly Lys Thr Met Phe Ile Asp Lys Thr Ala Ser Gly Val 385
390 395 400 Ser Thr Leu
Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala 405
410 415 Asn Pro Leu Ala Ser Ile Asp Ser
Ala Leu Ser Lys Val Asp Ala Val 420 425
430 Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser
Ala Ile Thr 435 440 445
Asn Leu Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile 450
455 460 Glu Asp Ala Asp
Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln 465 470
475 480 Ile Leu Gln Gln Ala Gly Thr Ser Val
Leu Ala Gln Ala Asn Gln Val 485 490
495 Pro Gln Asn Val Leu Ser Leu Leu Arg 500
505 21518DNASalmonella dublin 2atggcacaag tcattaatac
aaacagcctg tcgctgttga cccagaataa cctgaacaaa 60tctcagtcct cactgagttc
cgctattgag cgtctgtcct ctggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg
ccaggcgatt gctaaccgct tcacttctaa tatcaaaggc 180ctgactcagg cttcccgtaa
cgctaacgac ggcatttcta ttgcgcagac cactgaaggt 240gcgctgaatg aaatcaacaa
caacctgcag cgtgtgcgtg agttgtctgt tcaggccact 300aacgggacta actctgattc
cgatctgaaa tctatccagg atgaaattca gcaacgtctg 360gaagaaatcg atcgcgtttc
taatcagact caatttaacg gtgttaaagt cctctctcag 420gacaaccaga tgaaaatcca
ggttggtgct aacgatggtg aaaccattac catcgatctg 480caaaaaattg atgtgaaaag
ccttggcctt gatgggttca atgttaatgg gccaaaagaa 540gcgacagtgg gtgatctgaa
atccagcttc aagaatgtta cgggttacga cacctatgca 600gcgggtgccg ataaatatcg
tgtagatatt aattccggtg ctgtagtgac tgatgcagca 660gcaccggata aagtatatgt
aaatgcagca aacggtcagt taacaactga cgatgcggaa 720aataacactg cggttgatct
ctttaagacc actaaatcta ctgctggtac cgctgaagcc 780aaagcgatag ctggtgccat
taaaggtggt aaggaaggag atacctttga ttataaaggc 840gtgactttta ctattgatac
aaaaactggt gatgacggta atggtaaggt ttctactacc 900atcaatggtg aaaaagttac
gttaactgtc gctgatattg ccactggcgc ggcggatgtt 960aatgctgcta ccttacaatc
aagcaaaaat gtttatacat ctgtagtgaa cggtcagttt 1020acttttgatg ataaaaccaa
aaacgagagt gcgaaacttt ctgatttgga agcaaacaat 1080gctgttaagg gcgaaagtaa
aattacagta aatggggctg aatatactgc taacgccacg 1140ggtgataaga tcaccttagc
tggcaaaacc atgtttattg ataaaacagc ttctggcgta 1200agtacattaa tcaatgaaga
cgctgccgca gccaagaaaa gtaccgctaa cccactggct 1260tcaattgatt ctgcattgtc
aaaagtggac gcagttcgtt cttctctggg ggcaattcaa 1320aaccgttttg attcagccat
taccaacctt ggcaatacgg taaccaatct gaactccgcg 1380cgtagccgta tcgaagatgc
tgactatgca acggaagttt ctaatatgtc taaagcgcag 1440attctgcagc aggctggtac
ttccgttctg gcgcaggcta accaggttcc gcaaaacgtc 1500ctctctttac tgcgttaa
1518316PRTArtificial
SequencePeptide linker 3Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu
Asp Ser Met Gly 1 5 10
15 416PRTArtificial SequencePeptide linker 4Ile Pro Gly Ile Ser Gly
Gly Gly Gly Gly Ile Leu Asp Ser Met Gly 1 5
10 15 546DNAArtificial SequencePeptide linker
5tccccgggaa tttccggtgg tggtggtgga attctagact ccatgg
46646DNAArtificial SequencePeptide linker 6atcccgggaa tttccggtgg
tggtggtgga attctagact ccatgg 467600DNASalmonella
dublin 7atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
60atgggtcggg atctgtacga cgatgacgat aaggatccga tggcacaagt cattaataca
120aacagcctgt cgctgttgac ccagaataac ctgaacaaat ctcagtcctc actgagttcc
180gctattgagc gtctgtcctc tggtctgcgt atcaacagcg cgaaagacga tgcggcaggc
240caggcgattg ctaaccgctt cacttctaat atcaaaggcc tgactcaggc ttcccgtaac
300gctaacgacg gcatttctat tgcgcagacc actgaaggtg cgctgaatga aatcaacaac
360aacctgcagc gtgtgcgtga gttgtctgtt caggccacta acgggactaa ctctgattcc
420gatctgaaat ctatccagga tgaaattcag caacgtctgg aagaaatcga tcgcgtttct
480aatcagactc aatttaacgg tgttaaagtc ctctctcagg acaaccagat gaaaatccag
540gttggtgcta acgatggtga aaccattacc atcgatctgc aaaaaattga tgtgaaaagc
6008329PRTSalmonella dublin 8Met Arg Gly Ser His His His His His His Gly
Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30 Pro Met
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln 35
40 45 Asn Asn Leu Asn Lys Ser Gln
Ser Ser Leu Ser Ser Ala Ile Glu Arg 50 55
60 Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp
Asp Ala Ala Gly 65 70 75
80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
85 90 95 Ala Ser Arg
Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu 100
105 110 Gly Ala Leu Asn Glu Ile Asn Asn
Asn Leu Gln Arg Val Arg Glu Leu 115 120
125 Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp
Leu Lys Ser 130 135 140
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser 145
150 155 160 Asn Gln Thr Gln
Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln 165
170 175 Met Lys Ile Gln Val Gly Ala Asn Asp
Gly Glu Thr Ile Thr Ile Asp 180 185
190 Leu Gln Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly Phe
Asn Val 195 200 205
Asn Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met 210
215 220 Gly Thr Leu Ile Asn
Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala 225 230
235 240 Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu
Ser Lys Val Asp Ala Val 245 250
255 Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile
Thr 260 265 270 Asn
Leu Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile 275
280 285 Glu Asp Ala Asp Tyr Ala
Thr Glu Val Ser Asn Met Ser Lys Ala Gln 290 295
300 Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala
Gln Ala Asn Gln Val 305 310 315
320 Pro Gln Asn Val Leu Ser Leu Leu Arg 325
9825DNASalmonella dublin 9atgcggggtt ctcatcatca tcatcatcat
ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg atctgtacga cgatgacgat
aaggatccga tggcacaagt cattaataca 120aacagcctgt cgctgttgac ccagaataac
ctgaacaaat ctcagtcctc actgagttcc 180gctattgagc gtctgtcctc tggtctgcgt
atcaacagcg cgaaagacga tgcggcaggc 240caggcgattg ctaaccgctt cacttctaat
atcaaaggcc tgactcaggc ttcccgtaac 300gctaacgacg gcatttctat tgcgcagacc
actgaaggtg cgctgaatga aatcaacaac 360aacctgcagc gtgtgcgtga gttgtctgtt
caggccacta acgggactaa ctctgattcc 420gatctgaaat ctatccagga tgaaattcag
caacgtctgg aagaaatcga tcgcgtttct 480aatcagactc aatttaacgg tgttaaagtc
ctctctcagg acaaccagat gaaaatccag 540gttggtgcta acgatggtga aaccattacc
atcgatctgc aaaaaattga tgtgaaaagc 600cttggccttg atgggttcaa tgttaattcc
ccgggaattt ccggtggtgg tggtggaatt 660ctagactcca tgggtacatt aatcaatgaa
gacgctgccg cagccaagaa aagtaccgct 720aacccactgg cttcaattga ttctgcattg
tcaaaagtgg acgcagttcg ttcttctctg 780ggggcaattc aaaaccgttt tgattcagcc
attaccaacc tttag 82510274PRTSalmonella dublin 10Met
Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1
5 10 15 Gly Gly Gln Gln Met Gly
Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20
25 30 Pro Met Ala Gln Val Ile Asn Thr Asn Ser
Leu Ser Leu Leu Thr Gln 35 40
45 Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile
Glu Arg 50 55 60
Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly 65
70 75 80 Gln Ala Ile Ala Asn
Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln 85
90 95 Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser
Ile Ala Gln Thr Thr Glu 100 105
110 Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu
Leu 115 120 125 Ser
Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser 130
135 140 Ile Gln Asp Glu Ile Gln
Gln Arg Leu Glu Glu Ile Asp Arg Val Ser 145 150
155 160 Asn Gln Thr Gln Phe Asn Gly Val Lys Val Leu
Ser Gln Asp Asn Gln 165 170
175 Met Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp
180 185 190 Leu Gln
Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val 195
200 205 Asn Ser Pro Gly Ile Ser Gly
Gly Gly Gly Gly Ile Leu Asp Ser Met 210 215
220 Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys
Lys Ser Thr Ala 225 230 235
240 Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val
245 250 255 Arg Ser Ser
Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr 260
265 270 Asn Leu 11831DNASalmonella
dublin 11atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccgt tcacttctaa
tatcaaaggc 120ctgactcagg cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac
cactgaaggt 180gcgctgaatg aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt
tcaggccact 240aacgggacta actctgattc cgatctgaaa tctatccagg atgaaattca
gcaacgtctg 300gaagaaatcg atcgcgtttc taatcagact caatttaacg gtgttaaagt
cctctctcag 360gacaaccaga tgaaaatcca ggttggtgct aacgatggtg aaaccattac
catcgatctg 420caaaaaattg atgtgaaaag ccttggcctt gatgggttca atgttaattc
cccgggaatt 480tccggtggtg gtggtggaat tctagactcc atgggtacat taatcaatga
agacgctgcc 540gcagccaaga aaagtaccgc taacccactg gcttcaattg attctgcatt
gtcaaaagtg 600gacgcagttc gttcttctct gggggcaatt caaaaccgtt ttgattcagc
cattaccaac 660cttggcaata cggtaaccaa tctgaactcc gcgcgtagcc gtatcgaaga
tgctgactat 720gcaacggaag tttctaatat gtctaaagcg cagattctgc agcaggctgg
tacttccgtt 780ctggcgcagg ctaaccaggt tccgcaaaac gtcctctctt tactgcgtta g
83112276PRTSalmonella dublin 12Met Arg Gly Ser His His His
His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp
Asp Asp Asp Lys Asp 20 25
30 Pro Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn
Ala 35 40 45 Asn
Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu 50
55 60 Ile Asn Asn Asn Leu Gln
Arg Val Arg Glu Leu Ser Val Gln Ala Thr 65 70
75 80 Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser
Ile Gln Asp Glu Ile 85 90
95 Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser Asn Gln Thr Gln Phe
100 105 110 Asn Gly
Val Lys Val Leu Ser Gln Asp Asn Gln Met Lys Ile Gln Val 115
120 125 Gly Ala Asn Asp Gly Glu Thr
Ile Thr Ile Asp Leu Gln Lys Ile Asp 130 135
140 Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val Asn
Ser Pro Gly Ile 145 150 155
160 Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met Gly Thr Leu Ile Asn
165 170 175 Glu Asp Ala
Ala Ala Ala Lys Lys Ser Thr Ala Asn Pro Leu Ala Ser 180
185 190 Ile Asp Ser Ala Leu Ser Lys Val
Asp Ala Val Arg Ser Ser Leu Gly 195 200
205 Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu
Gly Asn Thr 210 215 220
Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile Glu Asp Ala Asp Tyr 225
230 235 240 Ala Thr Glu Val
Ser Asn Met Ser Lys Ala Gln Ile Leu Gln Gln Ala 245
250 255 Gly Thr Ser Val Leu Ala Gln Ala Asn
Gln Val Pro Gln Asn Val Leu 260 265
270 Ser Leu Leu Arg 275 13666DNASalmonella
dublin 13atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccgt tcacttctaa
tatcaaaggc 120ctgactcagg cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac
cactgaaggt 180gcgctgaatg aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt
tcaggccact 240aacgggacta actctgattc cgatctgaaa tctatccagg atgaaattca
gcaacgtctg 300gaagaaatcg atcgcgtttc taatcagact caatttaacg gtgttaaagt
cctctctcag 360gacaaccaga tgaaaatcca ggttggtgct aacgatggtg aaaccattac
catcgatctg 420caaaaaattg atgtgaaaag ccttggcctt gatgggttca atgttaattc
cccgggaatt 480tccggtggtg gtggtggaat tctagactcc atgggtacat taatcaatga
agacgctgcc 540gcagccaaga aaagtaccgc taacccactg gcttcaattg attctgcatt
gtcaaaagtg 600gacgcagttc gttcttctct gggggcaatt caaaaccgtt ttgattcagc
cattaccaac 660ctttag
66614221PRTSalmonella dublin 14Met Arg Gly Ser His His His
His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp
Asp Asp Asp Lys Asp 20 25
30 Pro Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn
Ala 35 40 45 Asn
Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu 50
55 60 Ile Asn Asn Asn Leu Gln
Arg Val Arg Glu Leu Ser Val Gln Ala Thr 65 70
75 80 Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser
Ile Gln Asp Glu Ile 85 90
95 Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser Asn Gln Thr Gln Phe
100 105 110 Asn Gly
Val Lys Val Leu Ser Gln Asp Asn Gln Met Lys Ile Gln Val 115
120 125 Gly Ala Asn Asp Gly Glu Thr
Ile Thr Ile Asp Leu Gln Lys Ile Asp 130 135
140 Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val Asn
Ser Pro Gly Ile 145 150 155
160 Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met Gly Thr Leu Ile Asn
165 170 175 Glu Asp Ala
Ala Ala Ala Lys Lys Ser Thr Ala Asn Pro Leu Ala Ser 180
185 190 Ile Asp Ser Ala Leu Ser Lys Val
Asp Ala Val Arg Ser Ser Leu Gly 195 200
205 Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu
210 215 220 15603DNASalmonella
dublin 15atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccgt tcacttctaa
tatcaaaggc 120ctgactcagg cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac
cactgaaggt 180gcgctgaatg aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt
tcaggccact 240tccccgggaa tttccggtgg tggtggtgga attctagact ccatgggtac
attaatcaat 300gaagacgctg ccgcagccaa gaaaagtacc gctaacccac tggcttcaat
tgattctgca 360ttgtcaaaag tggacgcagt tcgttcttct ctgggggcaa ttcaaaaccg
ttttgattca 420gccattacca accttggcaa tacggtaacc aatctgaact ccgcgcgtag
ccgtatcgaa 480gatgctgact atgcaacgga agtttctaat atgtctaaag cgcagattct
gcagcaggct 540ggtacttccg ttctggcgca ggctaaccag gttccgcaaa acgtcctctc
tttactgcgt 600tag
60316200PRTSalmonella dublin 16Met Arg Gly Ser His His His
His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp
Asp Asp Asp Lys Asp 20 25
30 Pro Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn
Ala 35 40 45 Asn
Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu 50
55 60 Ile Asn Asn Asn Leu Gln
Arg Val Arg Glu Leu Ser Val Gln Ala Thr 65 70
75 80 Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile
Leu Asp Ser Met Gly 85 90
95 Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala Asn
100 105 110 Pro Leu
Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val Arg 115
120 125 Ser Ser Leu Gly Ala Ile Gln
Asn Arg Phe Asp Ser Ala Ile Thr Asn 130 135
140 Leu Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg
Ser Arg Ile Glu 145 150 155
160 Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln Ile
165 170 175 Leu Gln Gln
Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val Pro 180
185 190 Gln Asn Val Leu Ser Leu Leu Arg
195 200 17438DNASalmonella dublin 17atgcggggtt
ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg
atctgtacga cgatgacgat aaggatccgt tcacttctaa tatcaaaggc 120ctgactcagg
cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac cactgaaggt 180gcgctgaatg
aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt tcaggccact 240tccccgggaa
tttccggtgg tggtggtgga attctagact ccatgggtac attaatcaat 300gaagacgctg
ccgcagccaa gaaaagtacc gctaacccac tggcttcaat tgattctgca 360ttgtcaaaag
tggacgcagt tcgttcttct ctgggggcaa ttcaaaaccg ttttgattca 420gccattacca
acctttag
43818145PRTSalmonella dublin 18Met Arg Gly Ser His His His His His His
Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
Asp 20 25 30 Pro
Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn Ala 35
40 45 Asn Asp Gly Ile Ser Ile
Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu 50 55
60 Ile Asn Asn Asn Leu Gln Arg Val Arg Glu
Leu Ser Val Gln Ala Thr 65 70 75
80 Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met
Gly 85 90 95 Thr
Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala Asn
100 105 110 Pro Leu Ala Ser Ile
Asp Ser Ala Leu Ser Lys Val Asp Ala Val Arg 115
120 125 Ser Ser Leu Gly Ala Ile Gln Asn Arg
Phe Asp Ser Ala Ile Thr Asn 130 135
140 Leu 145 19639DNASalmonella dublin 19atgcggggtt
ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg
atctgtacga cgatgacgat aaggatccga tggcacaagt cattaataca 120aacagcctgt
cgctgttgac ccagaataac ctgaacaaat ctcagtcctc actgagttcc 180gctattgagc
gtctgtcctc tggtctgcgt atcaacagcg cgaaagacga tgcggcaggc 240caggcgattg
ctaaccgctt cacttctaat atcaaaggtc tgactcaggc ttcccgtaac 300gctaacgacg
gcatttctat tgcgcagacc actgaaggtg cgctgaatga aatcaacaac 360aacctgcagc
gtgtgcgtga gttgtctgtt caggccacta acgggactaa ctctgattcc 420gatctgaaat
ctatccagga tgaaattcag caacgtctgg aagaaatcga tcgcgtttct 480aatcagactc
aatttaacgg tgttaaagtc ctgtctcagg acaaccagat gaaaatccag 540gttggtgcta
acgatggtga aaccattacc atcgatctgc aaaaaattga tgtgaaaagc 600cttggccttg
atgggttcaa tgttaattcc ccgggatga
63920212PRTSalmonella dublin 20Met Arg Gly Ser His His His His His His
Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
Asp 20 25 30 Pro
Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln 35
40 45 Asn Asn Leu Asn Lys Ser
Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg 50 55
60 Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys
Asp Asp Ala Ala Gly 65 70 75
80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
85 90 95 Ala Ser
Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu 100
105 110 Gly Ala Leu Asn Glu Ile Asn
Asn Asn Leu Gln Arg Val Arg Glu Leu 115 120
125 Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser
Asp Leu Lys Ser 130 135 140
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser 145
150 155 160 Asn Gln Thr
Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln 165
170 175 Met Lys Ile Gln Val Gly Ala Asn
Asp Gly Glu Thr Ile Thr Ile Asp 180 185
190 Leu Gln Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly
Phe Asn Val 195 200 205
Asn Ser Pro Gly 210 21480DNASalmonella dublin 21atgcggggtt
ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg
atctgtacga cgatgacgat aaggatccgt tcacttctaa tatcaaaggt 120ctgactcagg
cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac cactgaaggt 180gcgctgaatg
aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt tcaggccact 240aacgggacta
actctgattc cgatctgaaa tctatccagg atgaaattca gcaacgtctg 300gaagaaatcg
atcgcgtttc taatcagact caatttaacg gtgttaaagt cctgtctcag 360gacaaccaga
tgaaaatcca ggttggtgct aacgatggtg aaaccattac catcgatctg 420caaaaaattg
atgtgaaaag ccttggcctt gatgggttca atgttaattc cccgggatga
48022159PRTSalmonella dublin 22Met Arg Gly Ser His His His His His His
Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
Asp 20 25 30 Pro
Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn Ala 35
40 45 Asn Asp Gly Ile Ser Ile
Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu 50 55
60 Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu
Ser Val Gln Ala Thr 65 70 75
80 Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser Ile Gln Asp Glu Ile
85 90 95 Gln Gln
Arg Leu Glu Glu Ile Asp Arg Val Ser Asn Gln Thr Gln Phe 100
105 110 Asn Gly Val Lys Val Leu Ser
Gln Asp Asn Gln Met Lys Ile Gln Val 115 120
125 Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp Leu
Gln Lys Ile Asp 130 135 140
Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val Asn Ser Pro Gly 145
150 155 23252DNASalmonella
dublin 23atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccgt tcacttctaa
tatcaaaggt 120ctgactcagg cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac
cactgaaggt 180gcgctgaatg aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt
tcaggccact 240tccccgggat ga
2522483PRTSalmonella dublin 24Met Arg Gly Ser His His His His
His His Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp
Asp Lys Asp 20 25 30
Pro Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn Ala
35 40 45 Asn Asp Gly Ile
Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu 50
55 60 Ile Asn Asn Asn Leu Gln Arg Val
Arg Glu Leu Ser Val Gln Ala Thr 65 70
75 80 Ser Pro Gly 251038DNASalmonella dublin
25atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
60ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
120tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
180ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
240atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
300gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
360gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
420acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
480gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
540aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
600tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
660ctggttccgc gtggatcccc gggaatttcc ggtggtggtg gtggaattct agactccatg
720ggtacattaa tcaatgaaga cgctgccgca gccaagaaaa gtaccgctaa cccactggct
780tcaattgatt ctgcattgtc aaaagtggac gcagttcgtt cttctctggg ggcaattcaa
840aaccgttttg attcagccat taccaacctt ggcaatacgg taaccaatct gaactccgcg
900cgtagccgta tcgaagatgc tgactatgca acggaagttt ctaatatgtc taaagcgcag
960attctgcagc aggctggtac ttccgttctg gcgcaggcta accaggttcc gcaaaacgtc
1020ctctctttac tgcgttag
103826345PRTSalmonella dublin 26Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile
Lys Gly Leu Val Gln Pro 1 5 10
15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His
Leu 20 25 30 Tyr
Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35
40 45 Gly Leu Glu Phe Pro Asn
Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55
60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile
Ala Asp Lys His Asn 65 70 75
80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95 Gly Ala
Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100
105 110 Lys Asp Phe Glu Thr Leu Lys
Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120
125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys
Thr Tyr Leu Asn 130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145
150 155 160 Val Val Leu
Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165
170 175 Val Cys Phe Lys Lys Arg Ile Glu
Ala Ile Pro Gln Ile Asp Lys Tyr 180 185
190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly
Trp Gln Ala 195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg 210
215 220 Gly Ser Pro Gly
Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met 225 230
235 240 Gly Thr Leu Ile Asn Glu Asp Ala Ala
Ala Ala Lys Lys Ser Thr Ala 245 250
255 Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp
Ala Val 260 265 270
Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr
275 280 285 Asn Leu Gly Asn
Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile 290
295 300 Glu Asp Ala Asp Tyr Ala Thr Glu
Val Ser Asn Met Ser Lys Ala Gln 305 310
315 320 Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln
Ala Asn Gln Val 325 330
335 Pro Gln Asn Val Leu Ser Leu Leu Arg 340
345 27873DNASalmonella dublin 27atgtccccta tactaggtta ttggaaaatt
aagggccttg tgcaacccac tcgacttctt 60ttggaatatc ttgaagaaaa atatgaagag
catttgtatg agcgcgatga aggtgataaa 120tggcgaaaca aaaagtttga attgggtttg
gagtttccca atcttcctta ttatattgat 180ggtgatgtta aattaacaca gtctatggcc
atcatacgtt atatagctga caagcacaac 240atgttgggtg gttgtccaaa agagcgtgca
gagatttcaa tgcttgaagg agcggttttg 300gatattagat acggtgtttc gagaattgca
tatagtaaag actttgaaac tctcaaagtt 360gattttctta gcaagctacc tgaaatgctg
aaaatgttcg aagatcgttt atgtcataaa 420acatatttaa atggtgatca tgtaacccat
cctgacttca tgttgtatga cgctcttgat 480gttgttttat acatggaccc aatgtgcctg
gatgcgttcc caaaattagt ttgttttaaa 540aaacgtattg aagctatccc acaaattgat
aagtacttga aatccagcaa gtatatagca 600tggcctttgc agggctggca agccacgttt
ggtggtggcg accatcctcc aaaatcggat 660ctggttccgc gtggatcccc gggaatttcc
ggtggtggtg gtggaattct agactccatg 720ggtacattaa tcaatgaaga cgctgccgca
gccaagaaaa gtaccgctaa cccactggct 780tcaattgatt ctgcattgtc aaaagtggac
gcagttcgtt cttctctggg ggcaattcaa 840aaccgttttg attcagccat taccaacctt
tag 87328290PRTSalmonella dublin 28Met
Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1
5 10 15 Thr Arg Leu Leu Leu Glu
Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20
25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg
Asn Lys Lys Phe Glu Leu 35 40
45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp
Val Lys 50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65
70 75 80 Met Leu Gly Gly Cys
Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85
90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val
Ser Arg Ile Ala Tyr Ser 100 105
110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro
Glu 115 120 125 Met
Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130
135 140 Gly Asp His Val Thr His
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150
155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp
Ala Phe Pro Lys Leu 165 170
175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190 Leu Lys
Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195
200 205 Thr Phe Gly Gly Gly Asp His
Pro Pro Lys Ser Asp Leu Val Pro Arg 210 215
220 Gly Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile
Leu Asp Ser Met 225 230 235
240 Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala
245 250 255 Asn Pro Leu
Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val 260
265 270 Arg Ser Ser Leu Gly Ala Ile Gln
Asn Arg Phe Asp Ser Ala Ile Thr 275 280
285 Asn Leu 290 29972DNASalmonella dublin
29atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
60atgggtcggg atctgtacga cgatgacgat aaggatccga tggcacaagt cattaataca
120aacagcctgt cgctgttgac ccagaataac ctgaacaaat ctcagtcctc actgagttcc
180gctattgagc gtctgtcctc tggtctgcgt atcaacagcg cgaaagacga tgcggcaggc
240caggcgattg ctaaccgctt cacttctaat atcaaaggcc tgactcaggc ttcccgtaac
300gctaacgacg gcatttctat tgcgcagacc actgaaggtg cgctgaatga aatcaacaac
360aacctgcagc gtgtgcgtga gttgtctgtt caggccacta acgggactaa ctctgattcc
420gatctgaaat ctatccagga tgaaattcag caacgtctgg aagaaatcga tcgcgtttct
480aatcagactc aatttaacgg tgttaaagtc ctctctcagg acaaccagat gaaaatccag
540gttggtgcta acgatggtga aaccattacc atcgatctgc aaaaaattga tgtgaaaagc
600cttggcctta tcccgggaat ttccggtggt ggtggtggaa ttctagactc catgggtaca
660ttaatcaatg aagacgctgc cgcagccaag aaaagtaccg ctaacccact ggcttcaatt
720gattctgcat tgtcaaaagt ggacgcagtt cgttcttctc tgggggcaat tcaaaaccgt
780tttgattcag ccattaccaa ccttggcaat acggtaacca atctgaactc cgcgcgtagc
840cgtatcgaag atgctgacta tgcaacggaa gtttctaata tgtctaaagc gcagattctg
900cagcaggctg gtacttccgt tctggcgcag gctaaccagg ttccgcaaaa cgtcctctct
960ttactgcgtt ag
97230323PRTSalmonella dublin 30Met Arg Gly Ser His His His His His His
Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
Asp 20 25 30 Pro
Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln 35
40 45 Asn Asn Leu Asn Lys Ser
Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg 50 55
60 Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys
Asp Asp Ala Ala Gly 65 70 75
80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
85 90 95 Ala Ser
Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu 100
105 110 Gly Ala Leu Asn Glu Ile Asn
Asn Asn Leu Gln Arg Val Arg Glu Leu 115 120
125 Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser
Asp Leu Lys Ser 130 135 140
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser 145
150 155 160 Asn Gln Thr
Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln 165
170 175 Met Lys Ile Gln Val Gly Ala Asn
Asp Gly Glu Thr Ile Thr Ile Asp 180 185
190 Leu Gln Lys Ile Asp Val Lys Ser Leu Gly Leu Ile Pro
Gly Ile Ser 195 200 205
Gly Gly Gly Gly Gly Ile Leu Asp Ser Met Gly Thr Leu Ile Asn Glu 210
215 220 Asp Ala Ala Ala
Ala Lys Lys Ser Thr Ala Asn Pro Leu Ala Ser Ile 225 230
235 240 Asp Ser Ala Leu Ser Lys Val Asp Ala
Val Arg Ser Ser Leu Gly Ala 245 250
255 Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu Gly Asn
Thr Val 260 265 270
Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile Glu Asp Ala Asp Tyr Ala
275 280 285 Thr Glu Val Ser
Asn Met Ser Lys Ala Gln Ile Leu Gln Gln Ala Gly 290
295 300 Thr Ser Val Leu Ala Gln Ala Asn
Gln Val Pro Gln Asn Val Leu Ser 305 310
315 320 Leu Leu Arg 31813DNASalmonella dublin
31atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
60atgggtcggg atctgtacga cgatgacgat aaggatccgt tcacttctaa tatcaaaggc
120ctgactcagg cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac cactgaaggt
180gcgctgaatg aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt tcaggccact
240aacgggacta actctgattc cgatctgaaa tctatccagg atgaaattca gcaacgtctg
300gaagaaatcg atcgcgtttc taatcagact caatttaacg gtgttaaagt cctctctcag
360gacaaccaga tgaaaatcca ggttggtgct aacgatggtg aaaccattac catcgatctg
420caaaaaattg atgtgaaaag ccttggcctt atcccgggaa tttccggtgg tggtggtgga
480attctagact ccatgggtac attaatcaat gaagacgctg ccgcagccaa gaaaagtacc
540gctaacccac tggcttcaat tgattctgca ttgtcaaaag tggacgcagt tcgttcttct
600ctgggggcaa ttcaaaaccg ttttgattca gccattacca accttggcaa tacggtaacc
660aatctgaact ccgcgcgtag ccgtatcgaa gatgctgact atgcaacgga agtttctaat
720atgtctaaag cgcagattct gcagcaggct ggtacttccg ttctggcgca ggctaaccag
780gttccgcaaa acgtcctctc tttactgcgt tag
81332270PRTSalmonella dublin 32Met Arg Gly Ser His His His His His His
Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
Asp 20 25 30 Pro
Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn Ala 35
40 45 Asn Asp Gly Ile Ser Ile
Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu 50 55
60 Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu
Ser Val Gln Ala Thr 65 70 75
80 Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser Ile Gln Asp Glu Ile
85 90 95 Gln Gln
Arg Leu Glu Glu Ile Asp Arg Val Ser Asn Gln Thr Gln Phe 100
105 110 Asn Gly Val Lys Val Leu Ser
Gln Asp Asn Gln Met Lys Ile Gln Val 115 120
125 Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp Leu
Gln Lys Ile Asp 130 135 140
Val Lys Ser Leu Gly Leu Ile Pro Gly Ile Ser Gly Gly Gly Gly Gly 145
150 155 160 Ile Leu Asp
Ser Met Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala 165
170 175 Lys Lys Ser Thr Ala Asn Pro Leu
Ala Ser Ile Asp Ser Ala Leu Ser 180 185
190 Lys Val Asp Ala Val Arg Ser Ser Leu Gly Ala Ile Gln
Asn Arg Phe 195 200 205
Asp Ser Ala Ile Thr Asn Leu Gly Asn Thr Val Thr Asn Leu Asn Ser 210
215 220 Ala Arg Ser Arg
Ile Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn 225 230
235 240 Met Ser Lys Ala Gln Ile Leu Gln Gln
Ala Gly Thr Ser Val Leu Ala 245 250
255 Gln Ala Asn Gln Val Pro Gln Asn Val Leu Ser Leu Leu Arg
260 265 270
33951DNASalmonella dublin 33atgcggggtt ctcatcatca tcatcatcat ggtatggcta
gcatgactgg tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccga
tggcacaagt cattaataca 120aacagcctgt cgctgttgac ccagaataac ctgaacaaat
ctcagtcctc actgagttcc 180gctattgagc gtctgtcctc tggtctgcgt atcaacagcg
cgaaagacga tgcggcaggc 240caggcgattg ctaaccgctt cacttctaat atcaaaggcc
tgactcaggc ttcccgtaac 300gctaacgacg gcatttctat tgcgcagacc actgaaggtg
cgctgaatga aatcaacaac 360aacctgcagc gtgtgcgtga gttgtctgtt caggccacta
acgggactaa ctctgattcc 420gatctgaaat ctatccagga tgaaattcag caacgtctgg
aagaaatcga tcgcgtttct 480aatcagactc aatttaacgg tgttaaagtc ctctctcagg
acaaccagat gaaaatccag 540gttggtgcta acgatggtga aaccattacc atcgatctgc
aaaaaattat cccgggaatt 600tccggtggtg gtggtggaat tctagactcc atgggtacat
taatcaatga agacgctgcc 660gcagccaaga aaagtaccgc taacccactg gcttcaattg
attctgcatt gtcaaaagtg 720gacgcagttc gttcttctct gggggcaatt caaaaccgtt
ttgattcagc cattaccaac 780cttggcaata cggtaaccaa tctgaactcc gcgcgtagcc
gtatcgaaga tgctgactat 840gcaacggaag tttctaatat gtctaaagcg cagattctgc
agcaggctgg tacttccgtt 900ctggcgcagg ctaaccaggt tccgcaaaac gtcctctctt
tactgcgtta g 95134316PRTSalmonella dublin 34Met Arg Gly Ser
His His His His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu
Tyr Asp Asp Asp Asp Lys Asp 20 25
30 Pro Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu
Thr Gln 35 40 45
Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg 50
55 60 Leu Ser Ser Gly Leu
Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly 65 70
75 80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn
Ile Lys Gly Leu Thr Gln 85 90
95 Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr
Glu 100 105 110 Gly
Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu 115
120 125 Ser Val Gln Ala Thr Asn
Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser 130 135
140 Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu
Ile Asp Arg Val Ser 145 150 155
160 Asn Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln
165 170 175 Met Lys
Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp 180
185 190 Leu Gln Lys Ile Ile Pro Gly
Ile Ser Gly Gly Gly Gly Gly Ile Leu 195 200
205 Asp Ser Met Gly Thr Leu Ile Asn Glu Asp Ala Ala
Ala Ala Lys Lys 210 215 220
Ser Thr Ala Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val 225
230 235 240 Asp Ala Val
Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser 245
250 255 Ala Ile Thr Asn Leu Gly Asn Thr
Val Thr Asn Leu Asn Ser Ala Arg 260 265
270 Ser Arg Ile Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser
Asn Met Ser 275 280 285
Lys Ala Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala 290
295 300 Asn Gln Val Pro
Gln Asn Val Leu Ser Leu Leu Arg 305 310
315 35792DNASalmonella dublin 35atgcggggtt ctcatcatca tcatcatcat
ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg atctgtacga cgatgacgat
aaggatccgt tcacttctaa tatcaaaggc 120ctgactcagg cttcccgtaa cgctaacgac
ggcatttcta ttgcgcagac cactgaaggt 180gcgctgaatg aaatcaacaa caacctgcag
cgtgtgcgtg agttgtctgt tcaggccact 240aacgggacta actctgattc cgatctgaaa
tctatccagg atgaaattca gcaacgtctg 300gaagaaatcg atcgcgtttc taatcagact
caatttaacg gtgttaaagt cctctctcag 360gacaaccaga tgaaaatcca ggttggtgct
aacgatggtg aaaccattac catcgatctg 420caaaaaatta tcccgggaat ttccggtggt
ggtggtggaa ttctagactc catgggtaca 480ttaatcaatg aagacgctgc cgcagccaag
aaaagtaccg ctaacccact ggcttcaatt 540gattctgcat tgtcaaaagt ggacgcagtt
cgttcttctc tgggggcaat tcaaaaccgt 600tttgattcag ccattaccaa ccttggcaat
acggtaacca atctgaactc cgcgcgtagc 660cgtatcgaag atgctgacta tgcaacggaa
gtttctaata tgtctaaagc gcagattctg 720cagcaggctg gtacttccgt tctggcgcag
gctaaccagg ttccgcaaaa cgtcctctct 780ttactgcgtt ag
79236263PRTSalmonella dublin 36Met Arg
Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg
Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25
30 Pro Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala
Ser Arg Asn Ala 35 40 45
Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu
50 55 60 Ile Asn Asn
Asn Leu Gln Arg Val Arg Glu Leu Ser Val Gln Ala Thr 65
70 75 80 Asn Gly Thr Asn Ser Asp Ser
Asp Leu Lys Ser Ile Gln Asp Glu Ile 85
90 95 Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser
Asn Gln Thr Gln Phe 100 105
110 Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln Met Lys Ile Gln
Val 115 120 125 Gly
Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp Leu Gln Lys Ile Ile 130
135 140 Pro Gly Ile Ser Gly Gly
Gly Gly Gly Ile Leu Asp Ser Met Gly Thr 145 150
155 160 Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys
Ser Thr Ala Asn Pro 165 170
175 Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val Arg Ser
180 185 190 Ser Leu
Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu 195
200 205 Gly Asn Thr Val Thr Asn Leu
Asn Ser Ala Arg Ser Arg Ile Glu Asp 210 215
220 Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys
Ala Gln Ile Leu 225 230 235
240 Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val Pro Gln
245 250 255 Asn Val Leu
Ser Leu Leu Arg 260 37807DNASalmonella dublin
37atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
60atgggtcggg atctgtacga cgatgacgat aaggatccga tggcacaagt cattaataca
120aacagcctgt cgctgttgac ccagaataac ctgaacaaat ctcagtcctc actgagttcc
180gctattgagc gtctgtcctc tggtctgcgt atcaacagcg cgaaagacga tgcggcaggc
240caggcgattg ctaaccgctt cacttctaat atcaaaggcc tgactcaggc ttcccgtaac
300gctaacgacg gcatttctat tgcgcagacc actgaaggtg cgctgaatga aatcaacaac
360aacctgcagc gtgtgcgtga gttgtctgtt caggccacta acgggactaa ctctgattcc
420gatctgaaat ctatccagga tgaaattcag caacgtctgg aagaaatcga tcgcgtttct
480aatcagactc aatttaacgg tgttaaagtc ctctctcagg acaaccagat gaaaatccag
540gttggtgcta acgatggtga aaccattacc atcgatctgc aaaaaattga tgtgaaaagc
600cttggcctta tcccgggaat ttccggtggt ggtggtggaa ttctagactc catgggtaca
660ttaatcaatg aagacgctgc cgcagccaag aaaagtaccg ctaacccact ggcttcaatt
720gattctgcat tgtcaaaagt ggacgcagtt cgttcttctc tgggggcaat tcaaaaccgt
780tttgattcag ccattaccaa cctttag
80738268PRTSalmonella dublin 38Met Arg Gly Ser His His His His His His
Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
Asp 20 25 30 Pro
Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln 35
40 45 Asn Asn Leu Asn Lys Ser
Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg 50 55
60 Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys
Asp Asp Ala Ala Gly 65 70 75
80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
85 90 95 Ala Ser
Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu 100
105 110 Gly Ala Leu Asn Glu Ile Asn
Asn Asn Leu Gln Arg Val Arg Glu Leu 115 120
125 Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser
Asp Leu Lys Ser 130 135 140
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser 145
150 155 160 Asn Gln Thr
Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln 165
170 175 Met Lys Ile Gln Val Gly Ala Asn
Asp Gly Glu Thr Ile Thr Ile Asp 180 185
190 Leu Gln Lys Ile Asp Val Lys Ser Leu Gly Leu Ile Pro
Gly Ile Ser 195 200 205
Gly Gly Gly Gly Gly Ile Leu Asp Ser Met Gly Thr Leu Ile Asn Glu 210
215 220 Asp Ala Ala Ala
Ala Lys Lys Ser Thr Ala Asn Pro Leu Ala Ser Ile 225 230
235 240 Asp Ser Ala Leu Ser Lys Val Asp Ala
Val Arg Ser Ser Leu Gly Ala 245 250
255 Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu
260 265 39786DNASalmonella dublin
39atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
60atgggtcggg atctgtacga cgatgacgat aaggatccga tggcacaagt cattaataca
120aacagcctgt cgctgttgac ccagaataac ctgaacaaat ctcagtcctc actgagttcc
180gctattgagc gtctgtcctc tggtctgcgt atcaacagcg cgaaagacga tgcggcaggc
240caggcgattg ctaaccgctt cacttctaat atcaaaggcc tgactcaggc ttcccgtaac
300gctaacgacg gcatttctat tgcgcagacc actgaaggtg cgctgaatga aatcaacaac
360aacctgcagc gtgtgcgtga gttgtctgtt caggccacta acgggactaa ctctgattcc
420gatctgaaat ctatccagga tgaaattcag caacgtctgg aagaaatcga tcgcgtttct
480aatcagactc aatttaacgg tgttaaagtc ctctctcagg acaaccagat gaaaatccag
540gttggtgcta acgatggtga aaccattacc atcgatctgc aaaaaattat cccgggaatt
600tccggtggtg gtggtggaat tctagactcc atgggtacat taatcaatga agacgctgcc
660gcagccaaga aaagtaccgc taacccactg gcttcaattg attctgcatt gtcaaaagtg
720gacgcagttc gttcttctct gggggcaatt caaaaccgtt ttgattcagc cattaccaac
780ctttag
78640261PRTSalmonella dublin 40Met Arg Gly Ser His His His His His His
Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
Asp 20 25 30 Pro
Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln 35
40 45 Asn Asn Leu Asn Lys Ser
Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg 50 55
60 Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys
Asp Asp Ala Ala Gly 65 70 75
80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
85 90 95 Ala Ser
Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu 100
105 110 Gly Ala Leu Asn Glu Ile Asn
Asn Asn Leu Gln Arg Val Arg Glu Leu 115 120
125 Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser
Asp Leu Lys Ser 130 135 140
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser 145
150 155 160 Asn Gln Thr
Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln 165
170 175 Met Lys Ile Gln Val Gly Ala Asn
Asp Gly Glu Thr Ile Thr Ile Asp 180 185
190 Leu Gln Lys Ile Ile Pro Gly Ile Ser Gly Gly Gly Gly
Gly Ile Leu 195 200 205
Asp Ser Met Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys 210
215 220 Ser Thr Ala Asn
Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val 225 230
235 240 Asp Ala Val Arg Ser Ser Leu Gly Ala
Ile Gln Asn Arg Phe Asp Ser 245 250
255 Ala Ile Thr Asn Leu 260
41849DNASalmonella dublin 41atgcggggtt ctcatcatca tcatcatcat ggtatggcta
gcatgactgg tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccga
tggcacaagt cattaataca 120aacagcctgt cgctgttgac ccagaataac ctgaacaaat
ctcagtcctc actgagttcc 180gctattgagc gtctgtcctc tggtctgcgt atcaacagcg
cgaaagacga tgcggcaggc 240caggcgattg ctaaccgctt cacttctaat atcaaaggcc
tgactcaggc ttcccgtaac 300gctaacgacg gcatttctat tgcgcagacc actgaaggtg
cgctgaatga aatcaacaac 360aacctgcagc gtgtgcgtga gttgtctgtt caggccacta
acgggactaa ctctgattcc 420gatctgaaat ctatccagga tgaaattcag caacgtctgg
aagaaatcga tcgcgtttct 480aatcagatcc cgggaatttc cggtggtggt ggtggaattc
tagactccat gggtacatta 540atcaatgaag acgctgccgc agccaagaaa agtaccgcta
acccactggc ttcaattgat 600tctgcattgt caaaagtgga cgcagttcgt tcttctctgg
gggcaattca aaaccgtttt 660gattcagcca ttaccaacct tggcaatacg gtaaccaatc
tgaactccgc gcgtagccgt 720atcgaagatg ctgactatgc aacggaagtt tctaatatgt
ctaaagcgca gattctgcag 780caggctggta cttccgttct ggcgcaggct aaccaggttc
cgcaaaacgt cctctcttta 840ctgcgttag
84942282PRTSalmonella dublin 42Met Arg Gly Ser His
His His His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr
Asp Asp Asp Asp Lys Asp 20 25
30 Pro Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr
Gln 35 40 45 Asn
Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg 50
55 60 Leu Ser Ser Gly Leu Arg
Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly 65 70
75 80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile
Lys Gly Leu Thr Gln 85 90
95 Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu
100 105 110 Gly Ala
Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu 115
120 125 Ser Val Gln Ala Thr Asn Gly
Thr Asn Ser Asp Ser Asp Leu Lys Ser 130 135
140 Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile
Asp Arg Val Ser 145 150 155
160 Asn Gln Ile Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser
165 170 175 Met Gly Thr
Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr 180
185 190 Ala Asn Pro Leu Ala Ser Ile Asp
Ser Ala Leu Ser Lys Val Asp Ala 195 200
205 Val Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp
Ser Ala Ile 210 215 220
Thr Asn Leu Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg 225
230 235 240 Ile Glu Asp Ala
Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala 245
250 255 Gln Ile Leu Gln Gln Ala Gly Thr Ser
Val Leu Ala Gln Ala Asn Gln 260 265
270 Val Pro Gln Asn Val Leu Ser Leu Leu Arg 275
280 43690DNASalmonella dublin 43atgcggggtt ctcatcatca
tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg atctgtacga
cgatgacgat aaggatccgt tcacttctaa tatcaaaggc 120ctgactcagg cttcccgtaa
cgctaacgac ggcatttcta ttgcgcagac cactgaaggt 180gcgctgaatg aaatcaacaa
caacctgcag cgtgtgcgtg agttgtctgt tcaggccact 240aacgggacta actctgattc
cgatctgaaa tctatccagg atgaaattca gcaacgtctg 300gaagaaatcg atcgcgtttc
taatcagatc ccgggaattt ccggtggtgg tggtggaatt 360ctagactcca tgggtacatt
aatcaatgaa gacgctgccg cagccaagaa aagtaccgct 420aacccactgg cttcaattga
ttctgcattg tcaaaagtgg acgcagttcg ttcttctctg 480ggggcaattc aaaaccgttt
tgattcagcc attaccaacc ttggcaatac ggtaaccaat 540ctgaactccg cgcgtagccg
tatcgaagat gctgactatg caacggaagt ttctaatatg 600tctaaagcgc agattctgca
gcaggctggt acttccgttc tggcgcaggc taaccaggtt 660ccgcaaaacg tcctctcttt
actgcgttag 69044229PRTSalmonella
dublin 44Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15 Gly Gly
Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20
25 30 Pro Phe Thr Ser Asn Ile Lys
Gly Leu Thr Gln Ala Ser Arg Asn Ala 35 40
45 Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly
Ala Leu Asn Glu 50 55 60
Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ser Val Gln Ala Thr 65
70 75 80 Asn Gly Thr
Asn Ser Asp Ser Asp Leu Lys Ser Ile Gln Asp Glu Ile 85
90 95 Gln Gln Arg Leu Glu Glu Ile Asp
Arg Val Ser Asn Gln Ile Pro Gly 100 105
110 Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met Gly
Thr Leu Ile 115 120 125
Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala Asn Pro Leu Ala 130
135 140 Ser Ile Asp Ser
Ala Leu Ser Lys Val Asp Ala Val Arg Ser Ser Leu 145 150
155 160 Gly Ala Ile Gln Asn Arg Phe Asp Ser
Ala Ile Thr Asn Leu Gly Asn 165 170
175 Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile Glu Asp
Ala Asp 180 185 190
Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln Ile Leu Gln Gln
195 200 205 Ala Gly Thr Ser
Val Leu Ala Gln Ala Asn Gln Val Pro Gln Asn Val 210
215 220 Leu Ser Leu Leu Arg 225
45684DNASalmonella dublin 45atgcggggtt ctcatcatca tcatcatcat
ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg atctgtacga cgatgacgat
aaggatccga tggcacaagt cattaataca 120aacagcctgt cgctgttgac ccagaataac
ctgaacaaat ctcagtcctc actgagttcc 180gctattgagc gtctgtcctc tggtctgcgt
atcaacagcg cgaaagacga tgcggcaggc 240caggcgattg ctaaccgctt cacttctaat
atcaaaggcc tgactcaggc ttcccgtaac 300gctaacgacg gcatttctat tgcgcagacc
actgaaggtg cgctgaatga aatcaacaac 360aacctgcagc gtgtgcgtga gttgtctgtt
caggccacta acgggactaa ctctgattcc 420gatctgaaat ctatccagga tgaaattcag
caacgtctgg aagaaatcga tcgcgtttct 480aatcagatcc cgggaatttc cggtggtggt
ggtggaattc tagactccat gggtacatta 540atcaatgaag acgctgccgc agccaagaaa
agtaccgcta acccactggc ttcaattgat 600tctgcattgt caaaagtgga cgcagttcgt
tcttctctgg gggcaattca aaaccgtttt 660gattcagcca ttaccaacct ttag
68446227PRTSalmonella dublin 46Met Arg
Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg
Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25
30 Pro Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser
Leu Leu Thr Gln 35 40 45
Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg
50 55 60 Leu Ser Ser
Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly 65
70 75 80 Gln Ala Ile Ala Asn Arg Phe
Thr Ser Asn Ile Lys Gly Leu Thr Gln 85
90 95 Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile
Ala Gln Thr Thr Glu 100 105
110 Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu
Leu 115 120 125 Ser
Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser 130
135 140 Ile Gln Asp Glu Ile Gln
Gln Arg Leu Glu Glu Ile Asp Arg Val Ser 145 150
155 160 Asn Gln Ile Pro Gly Ile Ser Gly Gly Gly Gly
Gly Ile Leu Asp Ser 165 170
175 Met Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr
180 185 190 Ala Asn
Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala 195
200 205 Val Arg Ser Ser Leu Gly Ala
Ile Gln Asn Arg Phe Asp Ser Ala Ile 210 215
220 Thr Asn Leu 225 47525DNASalmonella
dublin 47atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccgt tcacttctaa
tatcaaaggc 120ctgactcagg cttcccgtaa cgctaacgac ggcatttcta ttgcgcagac
cactgaaggt 180gcgctgaatg aaatcaacaa caacctgcag cgtgtgcgtg agttgtctgt
tcaggccact 240aacgggacta actctgattc cgatctgaaa tctatccagg atgaaattca
gcaacgtctg 300gaagaaatcg atcgcgtttc taatcagatc ccgggaattt ccggtggtgg
tggtggaatt 360ctagactcca tgggtacatt aatcaatgaa gacgctgccg cagccaagaa
aagtaccgct 420aacccactgg cttcaattga ttctgcattg tcaaaagtgg acgcagttcg
ttcttctctg 480ggggcaattc aaaaccgttt tgattcagcc attaccaacc tttag
52548174PRTSalmonella dublin 48Met Arg Gly Ser His His His
His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp
Asp Asp Asp Lys Asp 20 25
30 Pro Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn
Ala 35 40 45 Asn
Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu 50
55 60 Ile Asn Asn Asn Leu Gln
Arg Val Arg Glu Leu Ser Val Gln Ala Thr 65 70
75 80 Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser
Ile Gln Asp Glu Ile 85 90
95 Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser Asn Gln Ile Pro Gly
100 105 110 Ile Ser
Gly Gly Gly Gly Gly Ile Leu Asp Ser Met Gly Thr Leu Ile 115
120 125 Asn Glu Asp Ala Ala Ala Ala
Lys Lys Ser Thr Ala Asn Pro Leu Ala 130 135
140 Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val
Arg Ser Ser Leu 145 150 155
160 Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu
165 170 49762DNASalmonella dublin
49atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
60atgggtcggg atctgtacga cgatgacgat aaggatccga tggcacaagt cattaataca
120aacagcctgt cgctgttgac ccagaataac ctgaacaaat ctcagtcctc actgagttcc
180gctattgagc gtctgtcctc tggtctgcgt atcaacagcg cgaaagacga tgcggcaggc
240caggcgattg ctaaccgctt cacttctaat atcaaaggcc tgactcaggc ttcccgtaac
300gctaacgacg gcatttctat tgcgcagacc actgaaggtg cgctgaatga aatcaacaac
360aacctgcagc gtgtgcgtga gttgtctgtt caggccacta tcccgggaat ttccggtggt
420ggtggtggaa ttctagactc catgggtaca ttaatcaatg aagacgctgc cgcagccaag
480aaaagtaccg ctaacccact ggcttcaatt gattctgcat tgtcaaaagt ggacgcagtt
540cgttcttctc tgggggcaat tcaaaaccgt tttgattcag ccattaccaa ccttggcaat
600acggtaacca atctgaactc cgcgcgtagc cgtatcgaag atgctgacta tgcaacggaa
660gtttctaata tgtctaaagc gcagattctg cagcaggctg gtacttccgt tctggcgcag
720gctaaccagg ttccgcaaaa cgtcctctct ttactgcgtt ag
76250253PRTSalmonella dublin 50Met Arg Gly Ser His His His His His His
Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys
Asp 20 25 30 Pro
Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln 35
40 45 Asn Asn Leu Asn Lys Ser
Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg 50 55
60 Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys
Asp Asp Ala Ala Gly 65 70 75
80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
85 90 95 Ala Ser
Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu 100
105 110 Gly Ala Leu Asn Glu Ile Asn
Asn Asn Leu Gln Arg Val Arg Glu Leu 115 120
125 Ser Val Gln Ala Thr Ile Pro Gly Ile Ser Gly Gly
Gly Gly Gly Ile 130 135 140
Leu Asp Ser Met Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys 145
150 155 160 Lys Ser Thr
Ala Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys 165
170 175 Val Asp Ala Val Arg Ser Ser Leu
Gly Ala Ile Gln Asn Arg Phe Asp 180 185
190 Ser Ala Ile Thr Asn Leu Gly Asn Thr Val Thr Asn Leu
Asn Ser Ala 195 200 205
Arg Ser Arg Ile Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met 210
215 220 Ser Lys Ala Gln
Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln 225 230
235 240 Ala Asn Gln Val Pro Gln Asn Val Leu
Ser Leu Leu Arg 245 250
51597DNASalmonella dublin 51atgcggggtt ctcatcatca tcatcatcat ggtatggcta
gcatgactgg tggacagcaa 60atgggtcggg atctgtacga cgatgacgat aaggatccga
tggcacaagt cattaataca 120aacagcctgt cgctgttgac ccagaataac ctgaacaaat
ctcagtcctc actgagttcc 180gctattgagc gtctgtcctc tggtctgcgt atcaacagcg
cgaaagacga tgcggcaggc 240caggcgattg ctaaccgctt cacttctaat atcaaaggcc
tgactcaggc ttcccgtaac 300gctaacgacg gcatttctat tgcgcagacc actgaaggtg
cgctgaatga aatcaacaac 360aacctgcagc gtgtgcgtga gttgtctgtt caggccacta
tcccgggaat ttccggtggt 420ggtggtggaa ttctagactc catgggtaca ttaatcaatg
aagacgctgc cgcagccaag 480aaaagtaccg ctaacccact ggcttcaatt gattctgcat
tgtcaaaagt ggacgcagtt 540cgttcttctc tgggggcaat tcaaaaccgt tttgattcag
ccattaccaa cctttag 59752198PRTSalmonella dublin 52Met Arg Gly Ser
His His His His His His Gly Met Ala Ser Met Thr 1 5
10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu
Tyr Asp Asp Asp Asp Lys Asp 20 25
30 Pro Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu
Thr Gln 35 40 45
Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg 50
55 60 Leu Ser Ser Gly Leu
Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly 65 70
75 80 Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn
Ile Lys Gly Leu Thr Gln 85 90
95 Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr
Glu 100 105 110 Gly
Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu 115
120 125 Ser Val Gln Ala Thr Ile
Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile 130 135
140 Leu Asp Ser Met Gly Thr Leu Ile Asn Glu Asp
Ala Ala Ala Ala Lys 145 150 155
160 Lys Ser Thr Ala Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys
165 170 175 Val Asp
Ala Val Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp 180
185 190 Ser Ala Ile Thr Asn Leu
195 53672DNASalmonella dublin 53atgcggggtt ctcatcatca
tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg atctgtacga
cgatgacgat aaggatccga tggcacaagt cattaataca 120aacagcctgt cgctgttgac
ccagaataac ctgaacaaat ctcagtcctc actgagttcc 180gctattgagc gtctgtcctc
tggtctgcgt atcaacagcg cgaaagacga tgcggcaggc 240caggcgattg ctaaccgctt
cacttctaat atcaaaggcc tgactcaggc ttcccgtaac 300gctaacgaca tcccgggaat
ttccggtggt ggtggtggaa ttctagactc catgggtaca 360ttaatcaatg aagacgctgc
cgcagccaag aaaagtaccg ctaacccact ggcttcaatt 420gattctgcat tgtcaaaagt
ggacgcagtt cgttcttctc tgggggcaat tcaaaaccgt 480tttgattcag ccattaccaa
ccttggcaat acggtaacca atctgaactc cgcgcgtagc 540cgtatcgaag atgctgacta
tgcaacggaa gtttctaata tgtctaaagc gcagattctg 600cagcaggctg gtacttccgt
tctggcgcag gctaaccagg ttccgcaaaa cgtcctctct 660ttactgcgtt ag
67254223PRTSalmonella dublin
54Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1
5 10 15 Gly Gly Gln Gln
Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20
25 30 Pro Met Ala Gln Val Ile Asn Thr Asn
Ser Leu Ser Leu Leu Thr Gln 35 40
45 Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile
Glu Arg 50 55 60
Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly 65
70 75 80 Gln Ala Ile Ala Asn
Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln 85
90 95 Ala Ser Arg Asn Ala Asn Asp Ile Pro Gly
Ile Ser Gly Gly Gly Gly 100 105
110 Gly Ile Leu Asp Ser Met Gly Thr Leu Ile Asn Glu Asp Ala Ala
Ala 115 120 125 Ala
Lys Lys Ser Thr Ala Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu 130
135 140 Ser Lys Val Asp Ala Val
Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg 145 150
155 160 Phe Asp Ser Ala Ile Thr Asn Leu Gly Asn Thr
Val Thr Asn Leu Asn 165 170
175 Ser Ala Arg Ser Arg Ile Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser
180 185 190 Asn Met
Ser Lys Ala Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu 195
200 205 Ala Gln Ala Asn Gln Val Pro
Gln Asn Val Leu Ser Leu Leu Arg 210 215
220 55507DNASalmonella dublin 55atgcggggtt ctcatcatca
tcatcatcat ggtatggcta gcatgactgg tggacagcaa 60atgggtcggg atctgtacga
cgatgacgat aaggatccga tggcacaagt cattaataca 120aacagcctgt cgctgttgac
ccagaataac ctgaacaaat ctcagtcctc actgagttcc 180gctattgagc gtctgtcctc
tggtctgcgt atcaacagcg cgaaagacga tgcggcaggc 240caggcgattg ctaaccgctt
cacttctaat atcaaaggcc tgactcaggc ttcccgtaac 300gctaacgaca tcccgggaat
ttccggtggt ggtggtggaa ttctagactc catgggtaca 360ttaatcaatg aagacgctgc
cgcagccaag aaaagtaccg ctaacccact ggcttcaatt 420gattctgcat tgtcaaaagt
ggacgcagtt cgttcttctc tgggggcaat tcaaaaccgt 480tttgattcag ccattaccaa
cctttag 50756168PRTSalmonella
dublin 56Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15 Gly Gly
Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20
25 30 Pro Met Ala Gln Val Ile Asn
Thr Asn Ser Leu Ser Leu Leu Thr Gln 35 40
45 Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser
Ser Ala Ile Glu Arg 50 55 60
Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly
65 70 75 80 Gln Ala
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
85 90 95 Ala Ser Arg Asn Ala Asn
Asp Ile Pro Gly Ile Ser Gly Gly Gly Gly 100
105 110 Gly Ile Leu Asp Ser Met Gly Thr Leu Ile
Asn Glu Asp Ala Ala Ala 115 120
125 Ala Lys Lys Ser Thr Ala Asn Pro Leu Ala Ser Ile Asp Ser
Ala Leu 130 135 140
Ser Lys Val Asp Ala Val Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg 145
150 155 160 Phe Asp Ser Ala Ile
Thr Asn Leu 165 57488PRTPseudomonas
aeruginosa 57Met Ala Leu Thr Val Asn Thr Asn Ile Ala Ser Leu Asn Thr Gln
Arg 1 5 10 15 Asn
Leu Asn Ala Ser Ser Asn Asp Leu Asn Thr Ser Leu Gln Arg Leu
20 25 30 Thr Thr Gly Tyr Arg
Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35
40 45 Gln Ile Ser Asn Arg Leu Ser Asn Gln
Ile Ser Gly Leu Asn Val Ala 50 55
60 Thr Arg Asn Ala Asn Asp Gly Ile Ser Leu Ala Gln Thr
Ala Glu Gly 65 70 75
80 Ala Leu Gln Gln Ser Thr Asn Ile Leu Gln Arg Ile Arg Asp Leu Ala
85 90 95 Leu Gln Ser Ala
Asn Gly Ser Asn Ser Asp Ala Asp Arg Ala Ala Leu 100
105 110 Gln Lys Glu Val Ala Ala Gln Gln Ala
Glu Leu Thr Arg Ile Ser Asp 115 120
125 Thr Thr Thr Phe Gly Gly Arg Lys Leu Leu Asp Gly Ser Phe
Gly Thr 130 135 140
Thr Ser Phe Gln Val Gly Ser Asn Ala Tyr Glu Thr Ile Asp Ile Ser 145
150 155 160 Leu Gln Asn Ala Ser
Ala Ser Ala Ile Gly Ser Tyr Gln Val Gly Ser 165
170 175 Asn Gly Ala Gly Thr Val Ala Ser Val Ala
Gly Thr Ala Thr Ala Ser 180 185
190 Gly Ile Ala Ser Gly Thr Val Asn Leu Val Gly Gly Gly Gln Val
Lys 195 200 205 Asn
Ile Ala Ile Ala Ala Gly Asp Ser Ala Lys Ala Ile Ala Glu Lys 210
215 220 Met Asp Gly Ala Ile Pro
Asn Leu Ser Ala Arg Ala Arg Thr Val Phe 225 230
235 240 Thr Ala Asp Val Ser Gly Val Thr Gly Gly Ser
Leu Asn Phe Asp Val 245 250
255 Thr Val Gly Ser Asn Thr Val Ser Leu Ala Gly Val Thr Ser Thr Gln
260 265 270 Asp Leu
Ala Asp Gln Leu Asn Ser Asn Ser Ser Lys Leu Gly Ile Thr 275
280 285 Ala Ser Ile Asn Asp Lys Gly
Val Leu Thr Ile Thr Ser Ala Thr Gly 290 295
300 Glu Asn Val Lys Phe Gly Ala Gln Thr Gly Thr Ala
Thr Ala Gly Gln 305 310 315
320 Val Ala Val Lys Val Gln Gly Ser Asp Gly Lys Phe Glu Ala Ala Ala
325 330 335 Lys Asn Val
Val Ala Ala Gly Thr Ala Ala Thr Thr Thr Ile Val Thr 340
345 350 Gly Tyr Val Gln Leu Asn Ser Pro
Thr Ala Tyr Ser Val Ser Gly Thr 355 360
365 Gly Thr Gln Ala Ser Gln Val Phe Gly Asn Ala Ser Ala
Ala Gln Lys 370 375 380
Ser Ser Val Ala Ser Val Asp Ile Ser Thr Ala Asp Gly Ala Gln Asn 385
390 395 400 Ala Ile Ala Val
Val Asp Asn Ala Leu Ala Ala Ile Asp Ala Gln Arg 405
410 415 Ala Asp Leu Gly Ala Val Gln Asn Arg
Phe Lys Asn Thr Ile Asp Asn 420 425
430 Leu Thr Asn Ile Ser Glu Asn Ala Thr Asn Ala Arg Ser Arg
Ile Lys 435 440 445
Asp Thr Asp Phe Ala Ala Glu Thr Ala Ala Leu Ser Lys Asn Gln Val 450
455 460 Leu Gln Gln Ala Gly
Thr Ala Ile Leu Ala Gln Ala Asn Gln Leu Pro 465 470
475 480 Gln Ala Val Leu Ser Leu Leu Arg
485 58475PRTLegionella pneumophila 58Met Ala Gln Val
Ile Asn Thr Asn Val Ala Ser Leu Thr Ala Gln Arg 1 5
10 15 Asn Leu Gly Val Ser Gly Asn Met Met
Gln Thr Ser Ile Gln Arg Leu 20 25
30 Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala
Gly Leu 35 40 45
Ala Ile Ser Gln Arg Met Thr Ala Gln Ile Arg Gly Met Asn Gln Ala 50
55 60 Val Arg Asn Ala Asn
Asp Gly Ile Ser Leu Ala Gln Val Ala Glu Gly 65 70
75 80 Ala Met Gln Glu Thr Thr Asn Ile Leu Gln
Arg Met Arg Glu Leu Ser 85 90
95 Val Gln Ala Ala Asn Ser Thr Asn Asn Ser Ser Asp Arg Ala Ser
Ile 100 105 110 Gln
Ser Glu Ile Ser Gln Leu Lys Ser Glu Leu Glu Arg Ile Ala Gln 115
120 125 Asn Thr Glu Phe Asn Gly
Gln Arg Ile Leu Asp Gly Ser Phe Ser Gly 130 135
140 Ala Ser Phe Gln Val Gly Ala Asn Ser Asn Gln
Thr Ile Asn Phe Ser 145 150 155
160 Ile Gly Ser Ile Lys Ala Ser Ser Ile Gly Gly Ile Ala Thr Ala Thr
165 170 175 Gly Thr
Glu Val Ala Gly Ala Ala Ala Thr Asp Ile Thr Ile Ala Ile 180
185 190 Gly Gly Gly Ala Ala Thr Ser
Ile Asn Ser Ser Ala Asn Phe Thr Gly 195 200
205 Ala Leu Asn Gly Gln Asp Ala Thr Ser Ala Tyr Ala
Lys Ala Ala Ala 210 215 220
Ile Asn Asp Ala Gly Ile Gly Gly Leu Ser Val Thr Ala Ser Thr Ser 225
230 235 240 Gly Thr Gln
Ala Val Gly Ala Ile Gly Gly Thr Ala Gly Asp Thr Tyr 245
250 255 Asn Leu Thr Ile Asn Gly Val Ala
Ile Tyr Thr Asn Leu Asn Val Ala 260 265
270 Thr Ala Leu Thr Asn Ser Asp Leu Arg Asp Ala Ile Asn
Gly Val Ser 275 280 285
Asn Gln Thr Gly Val Val Ala Ser Leu Asn Gly Gly Asn Met Thr Leu 290
295 300 Thr Ala Ala Asp
Gly Arg Asn Ile Thr Val Thr Glu Ser Gly Thr Gly 305 310
315 320 Phe Thr Ala Gly Thr Asp Gly Leu Thr
Val Thr Gly Gly Ala Phe Asp 325 330
335 Gly Ala Leu Arg Gly Thr Leu Ser Ile Ser Ala Val Asp Thr
Ile Ala 340 345 350
Ile Gly Gly Thr Val Ala Asn Val Gly Leu Ser Ala Asn Ile Ala Lys
355 360 365 Asp Thr Val Gly
Val Asp Ser Leu Asp Val Ser Thr Ala Ala Gly Ala 370
375 380 Gln Thr Ala Ile Lys Arg Ile Asp
Ala Ala Leu Asn Ser Val Asn Ser 385 390
395 400 Asn Arg Ala Asn Met Gly Ala Leu Gln Asn Arg Phe
Glu Ser Thr Ile 405 410
415 Ala Asn Leu Gln Asn Val Ser Asp Asn Leu Ser Ala Ala Arg Ser Arg
420 425 430 Ile Gln Asp
Ala Asp Tyr Ala Ala Glu Met Ala Ser Leu Thr Lys Asn 435
440 445 Gln Ile Leu Gln Gln Ala Gly Thr
Ala Met Leu Ala Gln Ala Asn Ser 450 455
460 Leu Pro Gln Ser Val Leu Ser Leu Leu Gly Arg 465
470 475 59488PRTEscherichia coli 59Met Ala
Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn 1 5
10 15 Asn Leu Asn Lys Ser Gln Ser
Ser Leu Ser Ser Ala Ile Glu Arg Leu 20 25
30 Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp
Ala Ala Gly Gln 35 40 45
Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala
50 55 60 Ser Arg Asn
Ala Asn Asp Gly Ile Ser Val Ala Gln Thr Thr Glu Gly 65
70 75 80 Ala Leu Asn Glu Ile Asn Asn
Asn Leu Gln Arg Val Arg Glu Leu Thr 85
90 95 Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser
Asp Leu Ser Ser Ile 100 105
110 Gln Ala Glu Ile Thr Gln Arg Leu Glu Glu Ile Asp Arg Val Ser
Glu 115 120 125 Gln
Thr Gln Phe Asn Gly Val Lys Val Leu Ala Glu Asn Asn Glu Met 130
135 140 Lys Ile Gln Val Gly Ala
Asn Asp Gly Glu Thr Ile Thr Ile Asn Leu 145 150
155 160 Ala Lys Ile Asp Ala Lys Thr Leu Gly Leu Asp
Gly Phe Asn Ile Asp 165 170
175 Gly Ala Gln Lys Ala Thr Gly Ser Asp Leu Ile Ser Lys Phe Lys Ala
180 185 190 Thr Gly
Thr Asp Asn Tyr Asp Val Gly Gly Asp Ala Tyr Thr Val Asn 195
200 205 Val Asp Ser Gly Ala Val Lys
Asp Thr Thr Gly Asn Asp Ile Phe Val 210 215
220 Ser Ala Ala Asp Gly Ser Leu Thr Thr Lys Ser Asp
Thr Asn Ile Ala 225 230 235
240 Gly Thr Gly Ile Asp Ala Thr Ala Leu Ala Ala Ala Ala Lys Asn Lys
245 250 255 Ala Gln Asn
Asp Lys Phe Thr Phe Asn Gly Val Glu Phe Thr Thr Thr 260
265 270 Thr Ala Ala Asp Gly Asn Gly Asn
Gly Val Tyr Ser Ala Glu Ile Asp 275 280
285 Gly Lys Ser Val Thr Phe Thr Val Thr Asp Ala Asp Lys
Lys Ala Ser 290 295 300
Leu Ile Thr Ser Glu Thr Val Tyr Lys Asn Ser Ala Gly Leu Tyr Thr 305
310 315 320 Thr Thr Lys Val
Asp Asn Lys Ala Ala Thr Leu Ser Asp Leu Asp Leu 325
330 335 Asn Ala Ala Lys Lys Thr Gly Ser Thr
Leu Val Val Asn Gly Ala Thr 340 345
350 Tyr Asp Val Ser Ala Asp Gly Lys Thr Ile Thr Glu Thr Ala
Ser Gly 355 360 365
Asn Asn Lys Val Met Tyr Leu Ser Lys Ser Glu Gly Gly Ser Pro Ile 370
375 380 Leu Val Asn Glu Asp
Ala Ala Lys Ser Leu Gln Ser Thr Thr Asn Pro 385 390
395 400 Leu Glu Thr Ile Asp Lys Ala Leu Ala Lys
Val Asp Asn Leu Arg Ser 405 410
415 Asp Leu Gly Ala Val Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn
Leu 420 425 430 Gly
Asn Thr Val Asn Asn Leu Ser Ser Ala Arg Ser Arg Ile Glu Asp 435
440 445 Ala Asp Tyr Ala Thr Glu
Val Ser Asn Met Ser Arg Ala Gln Ile Leu 450 455
460 Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala
Asn Gln Thr Thr Gln 465 470 475
480 Asn Val Leu Ser Leu Leu Gln Gly 485
60351PRTSerratia marcescens 60Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser
Leu Met Ala Gln Asn 1 5 10
15 Asn Leu Asn Lys Ser Gln Ser Ser Leu Gly Thr Ala Ile Glu Arg Leu
20 25 30 Ser Ser
Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln 35
40 45 Ala Ile Ser Asn Arg Phe Thr
Ala Asn Ile Lys Gly Leu Thr Gln Ala 50 55
60 Ser Arg Asn Ala Asn Asp Gly Ile Ser Leu Ala Gln
Thr Thr Glu Gly 65 70 75
80 Ala Leu Asn Glu Val Asn Asp Asn Leu Gln Asn Ile Arg Arg Leu Thr
85 90 95 Val Gln Ala
Gln Asn Gly Ser Asn Ser Thr Ser Asp Leu Lys Ser Ile 100
105 110 Gln Asp Glu Ile Thr Gln Arg Leu
Ser Glu Ile Asn Arg Ile Ser Glu 115 120
125 Gln Thr Asp Phe Asn Gly Val Lys Val Leu Ser Ser Asp
Gln Lys Leu 130 135 140
Thr Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Thr Asp Ile Asp Leu 145
150 155 160 Lys Lys Ile Asp
Ala Lys Gln Leu Gly Met Asp Thr Phe Asp Val Thr 165
170 175 Thr Lys Ser Ala Lys Ala Gly Ala Glu
Ile Ala Thr Gly Thr Lys Ile 180 185
190 Thr Val Asp Ser Asp Ala Thr Lys Gln Ala Asp Ala Asp Val
Thr Gly 195 200 205
Leu Ala Lys Gly Gln Thr Leu Val Ser Gly Thr Asp Ala Asp Gly Lys 210
215 220 Ser Ala Tyr Phe Ile
Ala Thr Lys Asp Asp Ala Thr Gly Asp Val Ala 225 230
235 240 Tyr Thr Lys Ala Lys Val Ala Asp Asp Gly
Lys Val Thr Asp Ser Gly 245 250
255 Thr Asp Ala Gly Val Lys Asn Pro Leu Ala Thr Leu Asp Lys Ala
Leu 260 265 270 Ala
Gln Val Asp Gly Leu Arg Ser Ser Leu Gly Ala Val Gln Asn Arg 275
280 285 Phe Asp Ser Val Ile Asn
Asn Leu Asn Ser Thr Val Asn Asn Leu Ser 290 295
300 Ala Ser Gln Ser Arg Ile Gln Asp Ala Asp Tyr
Ala Thr Glu Val Ser 305 310 315
320 Asn Met Ser Arg Ala Asn Ile Leu Gln Gln Ala Gly Thr Ser Val Leu
325 330 335 Ala Gln
Ala Asn Gln Ser Thr Gln Asn Val Leu Ser Leu Leu Arg 340
345 350 61333PRTBacillus subtilis 61Met Arg
Ile Asn His Asn Ile Ala Ala Leu Asn Thr Ser Arg Gln Leu 1 5
10 15 Asn Ala Gly Ser Asn Ser Ala
Ala Lys Asn Met Glu Lys Leu Ser Ser 20 25
30 Gly Leu Arg Ile Asn Arg Ala Gly Asp Asp Ala Ala
Gly Leu Ala Ile 35 40 45
Ser Glu Lys Met Arg Ser Gln Ile Arg Gly Leu Asp Met Ala Ser Lys
50 55 60 Asn Ala Gln
Asp Gly Ile Ser Leu Ile Gln Thr Ser Glu Gly Ala Leu 65
70 75 80 Asn Glu Thr His Ser Ile Leu
Gln Arg Met Ser Glu Leu Ala Thr Gln 85
90 95 Ala Ala Asn Asp Thr Asn Thr Asp Ser Asp Arg
Ser Glu Leu Gln Lys 100 105
110 Glu Met Asp Gln Leu Ala Ser Glu Val Thr Arg Ile Ser Thr Asp
Thr 115 120 125 Glu
Phe Asn Thr Lys Lys Leu Leu Asp Gly Thr Ala Gln Asn Leu Thr 130
135 140 Phe Gln Ile Gly Ala Asn
Glu Gly Gln Thr Met Ser Leu Ser Ile Asn 145 150
155 160 Lys Met Asp Ser Glu Ser Leu Lys Val Gly Thr
Thr Tyr Thr Val Ser 165 170
175 Asn Asp Gln Asn Thr Leu Thr Ser Thr Asp Gly Lys Ser Thr Ala Thr
180 185 190 Trp Ala
Asp Glu Val Thr Asp Pro Asp Gly Lys Val Thr Lys Ala Ala 195
200 205 Gly Tyr Tyr Asp Thr Asp Gly
Lys Val Ile Ala Ser Glu Lys Leu Ala 210 215
220 Ala Asp Ser Lys Val Thr Lys Gly Ile Asp Ile Ser
Ser Ser Ala Lys 225 230 235
240 Ala Ala Ser Ser Ala Leu Thr Thr Ile Lys Thr Ala Ile Asp Thr Val
245 250 255 Ser Ser Glu
Arg Ala Lys Leu Gly Ala Val Gln Asn Arg Leu Glu His 260
265 270 Thr Ile Asn Asn Leu Gly Thr Ser
Ser Glu Asn Leu Thr Ser Ala Glu 275 280
285 Ser Arg Ile Arg Asp Val Asp Met Ala Ser Glu Met Met
Glu Tyr Thr 290 295 300
Lys Asn Asn Ile Leu Thr Gln Ala Ser Gln Ala Met Leu Ala Gln Ala 305
310 315 320 Asn Gln Gln Pro
Gln Gln Val Leu Gln Leu Leu Lys Gly 325
330 62287PRTListeria monocytogenes 62Met Lys Val Asn Thr Asn
Ile Ile Ser Leu Lys Thr Gln Glu Tyr Leu 1 5
10 15 Arg Lys Asn Asn Glu Gly Met Thr Gln Ala Gln
Glu Arg Leu Ala Ser 20 25
30 Gly Lys Arg Ile Asn Ser Ser Leu Asp Asp Ala Ala Gly Leu Ala
Val 35 40 45 Val
Thr Arg Met Asn Val Lys Ser Thr Gly Leu Asp Ala Ala Ser Lys 50
55 60 Asn Ser Ser Met Gly Ile
Asp Leu Leu Gln Thr Ala Asp Ser Ala Leu 65 70
75 80 Ser Ser Met Ser Ser Ile Leu Gln Arg Met Arg
Gln Leu Ala Val Gln 85 90
95 Ser Ser Asn Gly Ser Phe Ser Asp Glu Asp Arg Lys Gln Tyr Thr Ala
100 105 110 Glu Phe
Gly Ser Leu Ile Lys Glu Leu Asp His Val Ala Asp Thr Thr 115
120 125 Asn Tyr Asn Asn Ile Lys Leu
Leu Asp Gln Thr Ala Thr Gly Ala Ala 130 135
140 Thr Gln Val Ser Ile Gln Ala Ser Asp Lys Ala Asn
Asp Leu Ile Asn 145 150 155
160 Ile Asp Leu Phe Asn Ala Lys Gly Leu Ser Ala Gly Thr Ile Thr Leu
165 170 175 Gly Ser Gly
Ser Thr Val Ala Gly Tyr Ser Ala Leu Ser Val Ala Asp 180
185 190 Ala Asp Ser Ser Gln Gln Ala Thr
Glu Ala Ile Asp Glu Leu Ile Asn 195 200
205 Asn Ile Ser Asn Gly Arg Ala Leu Leu Gly Ala Gly Met
Ser Arg Leu 210 215 220
Ser Tyr Asn Val Ser Asn Val Asn Asn Gln Ser Ile Ala Thr Lys Ala 225
230 235 240 Ser Ala Ser Ser
Ile Glu Asp Ala Asp Met Ala Ala Glu Met Ser Glu 245
250 255 Met Thr Lys Tyr Lys Ile Leu Thr Gln
Thr Ser Ile Ser Met Leu Ser 260 265
270 Gln Ala Asn Gln Thr Pro Gln Met Leu Thr Gln Leu Ile Asn
Ser 275 280 285
63524PRTShigella sonnei 63Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu
Leu Thr Gln Asn 1 5 10
15 Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg Leu
20 25 30 Ser Ser Gly
Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln 35
40 45 Ala Ile Ala Asn Arg Phe Thr Ala
Asn Ile Lys Gly Leu Thr Gln Ala 50 55
60 Ser Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Gln Thr
Thr Glu Gly 65 70 75
80 Ala Leu Ser Glu Ile Asn Asn Asn Leu Gln Arg Ile Arg Glu Leu Ser
85 90 95 Val Gln Ala Thr
Asn Gly Thr Asn Ser Asp Ser Asp Leu Asn Ser Ile 100
105 110 Gln Asp Glu Ile Thr Gln Arg Leu Ser
Glu Ile Asp Arg Val Ser Asn 115 120
125 Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ala Ser Asp Gln
Thr Met 130 135 140
Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Glu Ile Ala Leu 145
150 155 160 Asp Lys Ile Asp Ala
Lys Thr Leu Gly Leu Asp Asn Phe Ser Val Ala 165
170 175 Leu Gly Lys Val Pro Met Ser Ser Ala Val
Ala Leu Lys Ser Glu Ala 180 185
190 Ala Pro Asp Leu Thr Lys Val Asn Ala Thr Asp Gly Ser Val Gly
Gly 195 200 205 Ala
Lys Ala Phe Gly Ser Asn Tyr Lys Asn Ala Asp Val Glu Thr Tyr 210
215 220 Phe Gly Thr Gly Asn Val
Gln Asp Thr Lys Asp Thr Thr Asp Ala Thr 225 230
235 240 Gly Thr Ala Gly Thr Lys Val Tyr Gln Val Gln
Val Glu Gly Gln Thr 245 250
255 Tyr Phe Val Gly Gln Asp Asn Asn Thr Asn Thr Asn Gly Phe Thr Leu
260 265 270 Leu Lys
Gln Asn Ser Thr Gly Tyr Glu Lys Val Gln Val Gly Gly Lys 275
280 285 Asp Val Gln Leu Ala Asn Phe
Gly Gly Arg Val Thr Ala Phe Val Glu 290 295
300 Asp Asn Gly Ser Ala Thr Ser Val Asp Leu Ala Ala
Gly Lys Met Gly 305 310 315
320 Lys Ala Leu Ala Tyr Asn Asp Ala Pro Met Ser Val Tyr Phe Gly Gly
325 330 335 Lys Asn Leu
Asp Val His Gln Val Gln Asp Thr Gln Gly Asn Pro Val 340
345 350 Pro Asn Ser Phe Ala Ala Lys Thr
Ser Asp Gly Thr Tyr Ile Ala Val 355 360
365 Asn Val Asp Ala Ala Thr Gly Asn Thr Ser Val Ile Thr
Asp Pro Asn 370 375 380
Gly Lys Ala Val Glu Trp Ala Val Lys Asn Asp Gly Ser Ala Gln Ala 385
390 395 400 Ile Met Arg Glu
Asp Asp Lys Val Tyr Thr Ala Asn Ile Thr Asn Lys 405
410 415 Thr Ala Thr Lys Gly Ala Glu Leu Ser
Ala Ser Asp Leu Lys Ala Leu 420 425
430 Ala Thr Thr Asn Pro Leu Ser Lys Leu Asp Glu Ala Leu Ala
Lys Val 435 440 445
Asp Lys Leu Arg Ser Ser Leu Gly Ala Val Gln Asn Arg Phe Asp Ser 450
455 460 Ala Ile Thr Asn Leu
Gly Asn Thr Val Asn Asp Leu Ser Ser Ala Arg 465 470
475 480 Ser Arg Ile Glu Asp Ala Asp Tyr Ala Thr
Glu Val Ser Asn Met Ser 485 490
495 Arg Ala Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln
Ala 500 505 510 Asn
Gln Thr Thr Gln Asn Val Leu Ser Leu Leu Arg 515
520 64416PRTEdwardsiella tarda 64Met Ala Gln Val Ile Asn
Thr Asn Ser Leu Ser Leu Met Ala Gln Asn 1 5
10 15 Asn Leu Asn Lys Ser Gln Ser Ala Leu Gly Thr
Ala Ile Glu Arg Leu 20 25
30 Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly
Gln 35 40 45 Ala
Ile Ser Asn Arg Phe Thr Ala Asn Ile Asn Gly Leu Thr Gln Ala 50
55 60 Ser Arg Asn Ala Asn Asp
Gly Ile Ser Leu Ala Gln Thr Thr Glu Gly 65 70
75 80 Ala Leu Asn Glu Val Asn Asp Asn Leu Gln Asn
Ile Arg Arg Leu Thr 85 90
95 Val Gln Ala Gln Asn Gly Ser Asn Ser Ser Ser Asp Leu Gln Ser Ile
100 105 110 Gln Asp
Glu Ile Thr Gln Arg Leu Ser Glu Ile Asp Arg Ile Ser Gln 115
120 125 Gln Thr Asp Phe Asn Gly Val
Lys Val Leu Ser Lys Asp Gln Lys Leu 130 135
140 Thr Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile
Asp Ile Asp Leu 145 150 155
160 Lys Asn Ile Asn Ala Gln Ser Leu Gly Leu Asp Lys Phe Asn Val Ala
165 170 175 Asp Ser Val
Asp Thr Thr Lys Val Ala Ala Ala Ala Pro Ala Lys Val 180
185 190 Glu Thr Asn Ile Asp Val Ala Ile
Asn Asn Asp Ala Thr Leu Lys Ala 195 200
205 Asp Ser Lys Asp Ile Thr Gly Tyr Glu Gln Lys Gly Ala
Asp Leu Tyr 210 215 220
Ala Lys Asn Thr Asp Gly Lys Leu Phe Lys Val Thr Thr Ile Asp Asn 225
230 235 240 Thr Thr Gly Lys
Val Thr Gly Val Asp Thr Thr Glu Tyr Thr Gly Gly 245
250 255 Gly Thr Thr Ala Val Thr Ser Ile Lys
Lys Glu Val Ala Pro Thr Gly 260 265
270 Pro Asp Ala Ala Asn Leu Arg Ala Tyr Ser Gly Thr Glu Lys
Gly Ala 275 280 285
Ser Ala Tyr Val Ile Gln Glu Gly Thr Gly Ala Asp Ala Lys Tyr Phe 290
295 300 Lys Ala Ser Val Ala
Asp Asp Gly Thr Val Thr Lys Gly Ser Ala Leu 305 310
315 320 Ser Thr Thr Val Lys Thr Ala Asp Pro Leu
Ala Thr Leu Asp Lys Ala 325 330
335 Leu Ser Gln Val Asp Asp Leu Arg Ser Gly Leu Gly Ala Val Gln
Asn 340 345 350 Arg
Phe Asp Ser Val Ile Asn Asn Leu Asn Ser Thr Val Asn Asn Leu 355
360 365 Ser Ala Ser Arg Ser Arg
Ile Gln Asp Ala Asp Tyr Ala Thr Glu Val 370 375
380 Ser Asn Met Ser Arg Ala Gln Ile Leu Gln Gln
Ala Gly Thr Ser Val 385 390 395
400 Leu Ala Gln Ala Asn Gln Ser Thr Gln Asn Val Leu Ser Leu Leu Arg
405 410 415
65492PRTAcidovorax avenae 65 Met Ala Ser Thr Ile Asn Thr Asn Val Ser Ser
Leu Thr Ala Gln Arg 1 5 10
15 Asn Leu Ser Leu Ser Gln Ser Ser Leu Asn Thr Ser Ile Gln Arg Leu
20 25 30 Ser Ser
Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35
40 45 Ala Ile Ser Glu Arg Phe Thr
Ser Gln Ile Arg Gly Leu Asn Gln Ala 50 55
60 Val Arg Asn Ala Asn Asp Gly Ile Ser Leu Ala Gln
Thr Ala Glu Gly 65 70 75
80 Ala Leu Lys Ser Thr Gly Asp Ile Leu Gln Arg Val Arg Glu Leu Ala
85 90 95 Val Gln Ser
Ala Asn Ala Thr Asn Ser Ser Gly Asp Arg Lys Ala Ile 100
105 110 Gln Ala Glu Val Gly Gln Leu Leu
Ser Glu Met Asp Arg Ile Ala Gly 115 120
125 Asn Thr Glu Phe Asn Gly Gln Lys Leu Leu Asp Gly Ser
Phe Gly Ser 130 135 140
Ala Thr Phe Gln Val Gly Ala Asn Ala Asn Gln Thr Ile Thr Ala Thr 145
150 155 160 Thr Gly Asn Phe
Arg Thr Asn Asn Tyr Gly Ala Gln Leu Thr Ala Ser 165
170 175 Ala Ser Gly Ala Ala Thr Ser Gly Ala
Ser Ala Gly Ser Ala Gly Ala 180 185
190 Ala Ala Gly Thr Val Val Ile Ala Gly Leu Gln Thr Lys Thr
Val Asn 195 200 205
Val Ala Ala Ser Gly Thr Ala Ala Asp Ile Ala Ser Ala Val Asn Ala 210
215 220 Val Ala Asp Ser Thr
Gly Val Thr Ala Ser Ala Arg Asn Val Ser Glu 225 230
235 240 Leu Lys Phe Ser Gly Thr Gly Ser Phe Thr
Leu Ala Val Lys Gly Asp 245 250
255 Asn Ser Thr Ala Ala Asn Val Thr Phe Asn Val Ser Ala Thr Ser
Thr 260 265 270 Ala
Ala Gly Leu Ala Glu Ala Val Lys Ala Phe Asn Asp Val Ser Ser 275
280 285 Gln Thr Gly Val Thr Ala
Lys Leu Asn Ser Asp Ser Ser Gly Leu Ile 290 295
300 Leu Thr Asn Glu Ser Gly Asn Asp Ile Asn Val
Ala Asn Gly Ala Ser 305 310 315
320 Ser Ala Ala Gly Ile Thr Leu Ala Ser Gln Asp Ala Ala Thr Thr Gln
325 330 335 Ser Ser
Gly Thr Leu Thr Phe Thr Thr Ala Thr Ala Ala Gly Thr Gly 340
345 350 Ala Thr Val Ala Ser Arg Gly
Thr Val Glu Tyr Lys Ser Asp Lys Gly 355 360
365 Tyr Thr Val Ser Gly Thr Gly Gly Thr Met Thr Thr
Thr Ala Ala Thr 370 375 380
Ser Ser Thr Leu Thr Lys Val Ser Asp Ile Asp Val Ser Thr Val Asp 385
390 395 400 Gly Ser Thr
Lys Ala Leu Lys Ile Ile Asp Ala Ala Leu Ser Ala Val 405
410 415 Asn Gly Gln Arg Ala Ser Phe Gly
Ala Leu Gln Ser Arg Phe Glu Thr 420 425
430 Thr Val Asn Asn Leu Gln Ser Thr Ser Glu Asn Met Ser
Ala Ser Arg 435 440 445
Ser Arg Ile Gln Asp Ala Asp Phe Ala Ala Glu Thr Ala Asn Leu Ser 450
455 460 Arg Ser Gln Ile
Leu Gln Gln Ala Gly Thr Ala Met Val Ala Gln Ala 465 470
475 480 Asn Gln Leu Pro Gln Gly Val Leu Ser
Leu Leu Lys 485 490
66369PRTYersinia pestis 66Met Ala Val Ile Asn Thr Asn Ser Leu Ser Leu Leu
Thr Gln Asn Asn 1 5 10
15 Leu Asn Lys Ser Gln Ser Ser Leu Gly Thr Ala Ile Glu Arg Leu Ser
20 25 30 Ser Gly Leu
Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala 35
40 45 Ile Ala Asn Arg Phe Thr Ser Asn
Ile Lys Gly Leu Thr Gln Ala Ala 50 55
60 Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr
Glu Gly Ser 65 70 75
80 Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Thr Val
85 90 95 Gln Ala Gln Asn
Gly Ser Asn Ser Ser Ser Asp Leu Asp Ser Ile Gln 100
105 110 Asp Glu Ile Ser Leu Arg Leu Ala Glu
Ile Asp Arg Val Ser Asp Gln 115 120
125 Thr Gln Phe Asn Gly Lys Lys Val Leu Ala Glu Asn Thr Thr
Met Ser 130 135 140
Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Asp Ile Asn Leu Gln 145
150 155 160 Lys Ile Asp Ser Lys
Ser Leu Gly Leu Gly Ser Tyr Ser Val Ser Gly 165
170 175 Val Ser Gly Ala Leu Thr Ser Leu Thr Asp
Thr Ser Val Thr Gly Val 180 185
190 Thr Thr Thr Thr Ala Leu Asp Phe Ser Asp Ile Ser Thr Phe Ala
Lys 195 200 205 Gly
Ala Thr Val His Gly Ile Gly Asp Val Gly Thr Asp Gly Ala Tyr 210
215 220 Ala Asp Gly Tyr Val Ile
Arg Thr Thr Asp Gly Lys Gln Tyr Lys Gly 225 230
235 240 Glu Val Asp Ala Thr Asn Gly Lys Val Thr Phe
Ala Asp Asp Ala Asn 245 250
255 Gly Asp Pro Ile Asp Asp Ala Thr Lys Leu Glu Ala Ala Ala Gln Phe
260 265 270 Ser Pro
Ala Gly Lys Ala Thr Ala Ser Pro Leu Glu Thr Leu Asp Asp 275
280 285 Ala Ile Lys Gln Val Asp Gly
Leu Arg Ser Ser Leu Gly Ala Val Gln 290 295
300 Asn Arg Phe Glu Ser Ala Val Thr Asn Leu Asn Asn
Thr Val Thr Asn 305 310 315
320 Leu Thr Ser Ala Arg Ser Arg Ile Glu Asp Ala Asp Tyr Ala Thr Glu
325 330 335 Val Ser Asn
Met Ser Arg Ala Gln Ile Leu Gln Gln Ala Gly Thr Ser 340
345 350 Val Leu Ser Gln Ala Asn Gln Val
Pro Gln Thr Val Leu Ser Leu Leu 355 360
365 Asn 67355PRTPhotorhabdus luminescens 67Met Ala Gln
Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn 1 5
10 15 Asn Leu Asn Arg Ser Gln Gly Thr
Leu Gly Ser Ala Ile Glu Arg Leu 20 25
30 Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala
Ala Gly Gln 35 40 45
Ala Ile Ala Asn Arg Phe Thr Ala Asn Val Arg Gly Leu Thr Gln Ala 50
55 60 Ala Arg Asn Ala
Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly 65 70
75 80 Ala Leu Asn Glu Ile Asn Thr Asn Leu
Gln Arg Ile Arg Glu Leu Thr 85 90
95 Val Gln Ser Gln Asn Gly Ser Asn Ser Glu Ser Asp Ile Lys
Ser Ile 100 105 110
Gln Glu Glu Val Thr Gln Arg Leu Lys Glu Ile Asp Arg Ile Ser Glu
115 120 125 Gln Thr Gln Phe
Asn Gly Val Arg Val Leu Arg Glu Asp Ser Lys Met 130
135 140 Thr Ile Gln Val Gly Ala Asn Asp
Asn Glu Val Ile Asp Ile Asp Leu 145 150
155 160 Lys Lys Ile Asp Lys Glu Ala Leu Asn Leu Gly Lys
Phe Thr Ile Ser 165 170
175 Ala Glu Val Pro His Gly Glu Val Val Lys Gln Val Asp Asp Gly Ala
180 185 190 Gly Gly Lys
Lys Asn Ile Asp His Thr Ala Asp Ala Lys Val Lys Gly 195
200 205 Leu Lys Ser Val Glu Val Tyr Glu
Ala Leu Lys Asp Asp Gly Ser Val 210 215
220 Asn Pro Gly Lys Tyr Val Val Lys Gly Thr Asp Asp Thr
Asp Gly Lys 225 230 235
240 Val Lys Tyr Phe Glu Ala Thr Ile Thr Lys Glu Gly Lys Leu Thr Ala
245 250 255 Asp Ala Asp Ile
Thr Ala Lys Ala Ser Glu Lys Pro Leu Glu Thr Leu 260
265 270 Asp Ser Ala Leu Ala Gln Val Asp Ser
Leu Arg Ser Ser Leu Gly Ala 275 280
285 Ile Gln Asn Arg Leu Glu Ser Thr Val Asn Asn Leu Asn Asn
Thr Val 290 295 300
Asn Asn Leu Ser Ala Ala Arg Ser Arg Ile Glu Asp Ala Asp Tyr Ala 305
310 315 320 Thr Glu Val Ser Asn
Met Ser Arg Gly Gln Ile Leu Gln Gln Ala Gly 325
330 335 Thr Ala Val Leu Ala Gln Ala Asn Gln Val
Pro Gln Asn Val Met Ser 340 345
350 Leu Leu Arg 355 68493PRTRhodobacter sphaeroides
68Met Thr Thr Ile Asn Thr Asn Ile Gly Ala Ile Ala Ala Gln Ala Asn 1
5 10 15 Met Thr Lys Val
Asn Asp Gln Phe Asn Thr Ala Met Thr Arg Leu Ser 20
25 30 Thr Gly Leu Arg Ile Asn Ala Ala Lys
Asp Asp Ala Ala Gly Met Ala 35 40
45 Ile Gly Glu Lys Met Thr Ala Gln Val Met Gly Leu Asn Gln
Ala Ile 50 55 60
Arg Asn Ala Gln Asp Gly Lys Asn Leu Val Asp Thr Thr Glu Gly Ala 65
70 75 80 His Val Glu Val Ser
Ser Met Leu Gln Arg Leu Arg Glu Leu Ala Val 85
90 95 Gln Ser Ser Asn Asp Thr Asn Thr Ala Ala
Asp Arg Gly Ser Leu Ala 100 105
110 Ala Glu Gly Lys Gln Leu Ile Ala Glu Ile Asn Arg Val Ala Glu
Ser 115 120 125 Thr
Thr Phe Asn Gly Met Lys Val Leu Asp Gly Ser Phe Thr Gly Lys 130
135 140 Gln Leu Gln Ile Gly Ala
Asp Ser Gly Gln Thr Met Ala Ile Asn Val 145 150
155 160 Asp Ser Ala Ala Ala Thr Asp Ile Gly Ala His
Lys Ile Ser Ser Ala 165 170
175 Ser Thr Val Val Ala Asp Ala Ala Leu Thr Asp Thr Thr Ile Ala Ala
180 185 190 Ser Thr
Asp Ile Thr Ile Thr Gly Phe Ala Gly Ser Asp Lys Ile Thr 195
200 205 Thr Ala Ala Gly Asp Ser Ala
Arg Thr Leu Ala Glu Ser Ile Asn Lys 210 215
220 Lys Thr Ser Ser Thr Gly Val Glu Ala Thr Ala Thr
Thr Lys Ala Gln 225 230 235
240 Leu Ser Gly Phe Thr Lys Gly Asp Thr Val Ser Phe Lys Ile Gly Thr
245 250 255 Ala Asp Gly
Asn Glu Val Ser Ile Gly Asp Val Ser Ile Thr Asp Ala 260
265 270 Ser Asp Val Arg Gly Leu Arg Asp
Ala Ile Asn Ala Val Ser Gly Gln 275 280
285 Thr Gly Ile Thr Ala Ala Met Ala Lys Asp Asp Asn Ser
Lys Ile Val 290 295 300
Leu Thr Asp Ala Asn Gly Asp Asp Ile Met Leu Thr Ser Val Ser Ser 305
310 315 320 Thr Thr Ala Asp
Phe Lys Val Thr Ala Leu Lys Ser Asp Gly Thr Ala 325
330 335 Thr Ala Thr Asn Val Asp Ile Gly Phe
Gly Thr Asn Lys Ser Ala Gly 340 345
350 Val Thr Gly Gln Val Asp Leu Val Ser Thr Lys Ser Phe Ser
Val Ala 355 360 365
Ala Ser Val Ser Gly Ser Ala Thr Ala His Phe Ala Asn Ala Asn Glu 370
375 380 Gly Ser Glu Leu Ser
Ser Val Ala Glu Ile Asp Leu Ser Thr Ala Glu 385 390
395 400 Gly Ala Ser Ala Ala Ile Gly Val Ile Asp
Val Ala Leu Ser Lys Ile 405 410
415 Ser Gln Ser Arg Ser Glu Leu Gly Ala Val Ser Asn Arg Leu Asp
Ser 420 425 430 Thr
Ile Ser Asn Leu Thr Asn Ile Ser Thr Ser Val Gln Ala Ala Lys 435
440 445 Ser Gln Val Met Asp Ala
Asp Phe Ala Ala Glu Ser Thr Asn Leu Ala 450 455
460 Arg Ser Gln Ile Leu Ser Gln Ala Ser Thr Ala
Met Leu Ala Gln Ala 465 470 475
480 Asn Ser Ser Lys Gln Asn Val Leu Ser Leu Leu Arg Gly
485 490 69313PRTXenorhabdus nematophila
69Met Ala Ser Val Ile Asn Thr Asn Asp Ser Ala Leu Leu Ala Gln Asn 1
5 10 15 Asn Leu Thr Lys
Ser Lys Gly Ile Leu Gly Ser Ala Ile Glu Arg Leu 20
25 30 Ser Ser Gly Leu Arg Ile Asn Ser Ala
Lys Asp Asp Ala Ala Gly Gln 35 40
45 Ala Ile Ala Asn Arg Phe Thr Ala Asn Val Lys Gly Leu Thr
Gln Ala 50 55 60
Ala Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly 65
70 75 80 Ala Leu Asn Glu Ile
Asn Asn Asn Leu Gln Arg Ile Arg Glu Leu Thr 85
90 95 Val Gln Ser Glu Asn Gly Ser Asn Ser Lys
Ser Asp Leu Asp Ser Ile 100 105
110 Gln Lys Glu Val Thr Gln Arg Leu Glu Glu Ile Asp Arg Ile Ser
Thr 115 120 125 Gln
Thr Gln Phe Asn Gly Ile Lys Val Leu Asn Gly Asp Val Thr Glu 130
135 140 Met Lys Ile Gln Val Gly
Ala Asn Asp Asn Glu Thr Ile Gly Ile Lys 145 150
155 160 Leu Gly Lys Ile Asn Ser Glu Lys Leu Asn Leu
Lys Glu Phe Ser Val 165 170
175 Val Glu Lys Glu Ala Val Ala Ala Lys Pro Ala Val Pro Ala Gln Pro
180 185 190 Ala Val
Pro Ala Asp Pro Lys Asn Gly Val Ala Ala Lys Pro Ala Val 195
200 205 Pro Ala Gln Pro Glu Val Lys
Ala Gln Glu Ala Val Lys Lys Thr Asp 210 215
220 Asn Pro Leu Asp Thr Leu Asp Lys Ala Leu Ala Gln
Val Asp Asp Met 225 230 235
240 Arg Ser Ser Leu Gly Ala Val Gln Asn Arg Leu Glu Ser Thr Val Asn
245 250 255 Asn Leu Asn
Asn Thr Val Asn Asn Leu Ser Ala Ala Arg Ser Arg Ile 260
265 270 Glu Asp Ala Asp Tyr Ala Val Glu
Val Ser Asn Met Ser Arg Gly Gln 275 280
285 Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala
Asn Gln Val 290 295 300
Pro Gln Thr Val Leu Ser Leu Leu Arg 305 310
70367PRTProteus mirabilis 70Met Ala Gln Val Ile Asn Thr Asn Tyr Leu Ser
Leu Val Thr Gln Asn 1 5 10
15 Asn Leu Asn Arg Ser Gln Ser Ala Leu Gly Asn Ala Ile Glu Arg Leu
20 25 30 Ser Ser
Gly Met Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln 35
40 45 Ala Ile Ala Asn Arg Phe Thr
Ser Asn Ile Asn Gly Leu Thr Gln Ala 50 55
60 Ser Arg Asn Ala Asn Asp Gly Ile Ser Val Ser Gln
Thr Thr Glu Gly 65 70 75
80 Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Ile Arg Glu Leu Thr
85 90 95 Val Gln Ala
Lys Asn Gly Thr Asn Ser Asn Ser Asp Ile Asn Ser Ile 100
105 110 Gln Asn Glu Val Asn Gln Arg Leu
Asp Glu Ile Asn Arg Val Ser Glu 115 120
125 Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gly Glu
Lys Ser Lys 130 135 140
Met Thr Ile Gln Val Gly Thr Asn Asp Asn Glu Val Ile Glu Phe Asn 145
150 155 160 Leu Asp Lys Ile
Asp Asn Asp Thr Leu Gly Val Ala Ser Asp Lys Leu 165
170 175 Phe Asp Ala Lys Thr Glu Lys Lys Gly
Val Thr Ala Ala Gly Asp Ala 180 185
190 Ile Asp Ala Asn Ala Leu Gly Ile Ser Gly Ser Lys Lys Tyr
Val Thr 195 200 205
Gly Ile Ser Val Lys Glu Tyr Lys Val Asp Gly Lys Val Ser Ser Asp 210
215 220 Lys Val Val Leu Asn
Asp Gly Ser Asp Asp Tyr Ile Val Ser Lys Ser 225 230
235 240 Asp Phe Thr Leu Lys Ser Gly Thr Thr Thr
Gly Glu Val Glu Phe Thr 245 250
255 Gly Ser Lys Thr Thr Lys Phe Thr Ala Asp Ala Gly Lys Asp Val
Lys 260 265 270 Val
Leu Asn Val Lys Asp Asp Ala Leu Ala Thr Leu Asp Asn Ala Ile 275
280 285 Ser Lys Val Asp Glu Ser
Arg Ser Lys Leu Gly Ala Ile Gln Asn Arg 290 295
300 Phe Gln Ser Thr Ile Asn Asn Leu Asn Asn Thr
Val Asn Asn Leu Ser 305 310 315
320 Ala Ser Arg Ser Arg Ile Leu Asp Ala Asp Tyr Ala Thr Glu Val Ser
325 330 335 Asn Met
Ser Lys Asn Gln Ile Leu Gln Gln Ala Gly Thr Ala Val Leu 340
345 350 Ala Gln Ala Asn Gln Val Pro
Gln Thr Val Leu Ser Leu Leu Arg 355 360
365 71472PRTButyrivibrio fibrisolvens 71Met Val Val Gln His
Asn Met Gln Ala Ala Asn Ala Ser Arg Met Leu 1 5
10 15 Gly Ile Thr Thr Gly Asp Gln Ser Lys Ser
Thr Glu Lys Leu Ser Ser 20 25
30 Gly Phe Lys Ile Asn Arg Ala Ala Asp Asp Ala Ala Gly Leu Ser
Ile 35 40 45 Ser
Glu Lys Met Arg Lys Gln Ile Arg Gly Leu Asp Gln Ala Ser Thr 50
55 60 Asn Ala Ser Asp Gly Ile
Ser Ala Val Gln Thr Ala Glu Gly Ala Leu 65 70
75 80 Thr Glu Val His Ser Met Leu Gln Arg Met Asn
Glu Leu Ala Val Gln 85 90
95 Ala Ala Asn Gly Thr Asn Ser Glu Ser Asp Arg Ser Ser Ile Gln Asp
100 105 110 Glu Ile
Asn Gln Leu Thr Thr Glu Ile Asp Arg Val Ala Glu Thr Thr 115
120 125 Lys Phe Asn Glu Thr Tyr Leu
Leu Lys Gly Gly Asn Gly Asp Arg Thr 130 135
140 Val Arg Val Tyr Ala His Asp Ala Gly Leu Val Gly
Ser Leu Ser Gln 145 150 155
160 Asn Thr Thr Lys Ala Thr Phe Gln Met Arg Lys Leu Glu Ile Gly Asp
165 170 175 Ser Tyr Thr
Ile Gly Gly Thr Thr Tyr Lys Ile Gly Ala Glu Thr Val 180
185 190 Lys Glu Ala Met Thr Ala Leu Lys
Ile Thr Ser Ala Ala Ser Ser Ser 195 200
205 Asp Asp Lys Ile Thr Ala Leu Lys Thr Gly Asp Val Ile
Lys Ile Asp 210 215 220
Gly Lys Ser Phe Thr Ile Asp Glu Asn Glu Thr Asp Met Glu Gln Ser 225
230 235 240 Arg Ile Lys Ala
Thr Met Ile Asn Lys Leu Ala Gly Ala Gly Ser Thr 245
250 255 Leu Glu Phe Asn Gly Arg Val Thr Val
Phe Ser Val Asp Val Gly Thr 260 265
270 Gly Val Asn Gly Val Asp Pro Asn Asn Lys Thr Leu Ile Thr
Ala Thr 275 280 285
Ala Ala Tyr Phe Arg Val Lys Thr Glu Leu Ile Ala Ala Ser Ser Val 290
295 300 Gly Ala Thr Asn Thr
Arg Ala Ala Ile Ile Gly Asp Pro Asp Ile Gly 305 310
315 320 Ala Thr Tyr Val Asn Phe Thr Ile Thr Arg
Gly Asp Val Ile Ser Lys 325 330
335 Asn Thr Leu Val Phe Thr Leu His Val Gly Ser Asp Ala Asp Met
Thr 340 345 350 Asn
Lys Ile Gly Val Glu Ile Asp Thr Met Asp Ala Lys Ser Leu Gly 355
360 365 Ile His Gly Met Asn Val
Ser Asp Asp Thr Gly Lys Ala Ala Thr Tyr 370 375
380 Ala Ile Asp Ala Ile Ser Asp Ala Leu Ala Lys
Val Ser Ala Gln Arg 385 390 395
400 Ser Ala Leu Gly Ser Ile Gln Asn Arg Leu Glu His Ser Ile Ala Asn
405 410 415 Leu Asp
Asn Val Val Glu Asn Thr Asn Ala Ala Glu Ser Arg Ile Arg 420
425 430 Asp Thr Asp Met Ala Asp Glu
Met Val Thr Tyr Ser Lys Asn Asn Ile 435 440
445 Leu Met Gln Ala Gly Gln Ser Met Leu Ala Gln Ala
Asn Gln Ala Thr 450 455 460
Gln Gly Val Leu Ser Ile Leu Gln 465 470
72391PRTBordetella pertussis 72Met Ala Ala Val Ile Asn Thr Asn Tyr Leu
Ser Leu Val Ala Gln Asn 1 5 10
15 Asn Leu Asn Lys Ser Gln Ser Ala Leu Gly Ser Ala Ile Glu Arg
Leu 20 25 30 Ser
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln 35
40 45 Ala Ile Ala Asn Arg Phe
Thr Ala Asn Val Lys Gly Leu Thr Gln Ala 50 55
60 Ala Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala
Gln Thr Thr Glu Gly 65 70 75
80 Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Ile Arg Glu Leu Thr
85 90 95 Val Gln
Ala Ser Asn Gly Thr Asn Ser Ala Ser Asp Ile Asp Ser Ile 100
105 110 Gln Gln Glu Val Asn Gln Arg
Leu Glu Glu Ile Asn Arg Ile Ala Glu 115 120
125 Gln Thr Asp Phe Asn Gly Ile Lys Val Leu Lys Ser
Asn Ala Thr Asp 130 135 140
Met Thr Leu Ser Ile Gln Val Gly Ala Lys Asp Asn Glu Thr Ile Asp 145
150 155 160 Ile Lys Ile
Asp Arg Asn Ser Asn Trp Asn Leu Tyr Asp Ala Val Gly 165
170 175 Thr Val Pro Gly Gly Thr Val Asn
Gly Glu Ala Arg Thr Val Asn Ala 180 185
190 Leu Gly Phe Asp Val Leu Ser Ala Val Thr Thr Thr Ile
Ala Ser Asp 195 200 205
Thr Val Thr Phe Asp Ala Ala Val Ala Ala Ala Glu Gln Ala Ala Gly 210
215 220 Ala Ala Ala Gly
Asp Gly Ser Val Val Ser Tyr Gly Asp Ala Ala Asn 225 230
235 240 Pro Gln Tyr Ala Val Val Val Asp Asn
Ala Gly Thr Leu Thr Ser Tyr 245 250
255 Ala Leu Thr Phe Asp Lys Asp Gly Lys Ala Ala Leu Gly Asp
Gln Leu 260 265 270
Gly Ala Val Ala Ser Gln Ala Ala Glu Ala Ala Val Gly Val Asn Asp
275 280 285 Val Ala Ser Gly
Asp Asn Val Thr Val Ser Gly Gly Asn Ala Asn Ala 290
295 300 Leu Ser Lys Leu Asp Asp Ala Met
Lys Ala Val Asp Glu Gln Arg Ser 305 310
315 320 Ser Leu Gly Ala Ile Gln Asn Arg Phe Glu Ser Thr
Val Ala Asn Leu 325 330
335 Asn Asn Thr Ile Thr Asn Leu Ser Ala Ala Arg Ser Arg Ile Glu Asp
340 345 350 Ser Asp Tyr
Ala Thr Glu Val Ser Asn Met Thr Lys Asn Gln Ile Leu 355
360 365 Gln Gln Ala Gly Thr Ser Val Leu
Ala Gln Ala Asn Gln Val Pro Gln 370 375
380 Asn Val Leu Ser Leu Leu Arg 385 390
73413PRTClostridium chauvoei 73Met Ile Ile Asn His Asn Met Asn Ala
Leu Asn Ala His Arg Asn Met 1 5 10
15 Met Gly Asn Ile Ala Thr Ala Gly Lys Ser Met Glu Lys Leu
Ser Ser 20 25 30
Gly Leu Arg Ile Asn Arg Ala Gly Asp Asp Ala Ala Gly Leu Ala Ile
35 40 45 Ser Glu Lys Met
Arg Gly Gln Ile Arg Gly Leu Asp Gln Ala Ser Arg 50
55 60 Asn Ala Gln Asp Gly Ile Ser Leu
Ile Gln Thr Ala Glu Gly Ala Leu 65 70
75 80 Ala Glu Thr His Ser Ile Leu Gln Arg Met Arg Glu
Leu Ser Val Gln 85 90
95 Ser Ala Asn Asp Thr Asn Val Ala Val Asp Arg Thr Ala Ile Gln Asp
100 105 110 Glu Ile Asn
Ser Leu Thr Glu Glu Ile Asn Arg Ile Ser Gly Asp Thr 115
120 125 Glu Phe Asn Thr Gln Lys Leu Leu
Asp Gly Gly Phe Lys Gly Glu Phe 130 135
140 Gln Ile Gly Ala Asn Ser Asn Gln Thr Val Lys Leu Asp
Ile Gly Asn 145 150 155
160 Met Ser Ala Ala Ser Leu Gly Leu Thr Thr Thr Asn Ser Leu Glu Ser
165 170 175 Lys Ala Leu Thr
Lys Asp Ser Asn Leu Ala Asp Gly Thr Tyr Lys Ile 180
185 190 Ser Gly Lys Asn Leu Val Asp Thr Asn
Gly Asn Ser Val Gly Thr Phe 195 200
205 Asp Ala Ala Ser Lys Lys Ile Thr Val Asn Gly Lys Asp Thr
Val Phe 210 215 220
Asp Lys Ala Ala Leu Ala Glu Asn Ala Val Leu Thr Val Lys Ser Gly 225
230 235 240 Thr Ala Glu Ile Lys
Asn Thr Met Thr Gly Ala Ala Thr Lys Leu Ser 245
250 255 Ser Gly Asn Tyr Glu Ile Lys Gly Thr Asn
Val Ile Lys Asp Gly Lys 260 265
270 Leu Ala Gly Thr Phe Asp Ala Ala Lys Lys Lys Leu Thr Ile Asp
Gly 275 280 285 Val
Gly Asp Val Ser Glu Ala Glu Leu Gly Phe Gln Thr Ser Lys Met 290
295 300 Leu Asp Lys Val Ser Phe
Thr Ile Asn Gly Ser Asp Val Ser Thr Arg 305 310
315 320 Glu Leu Ala Ser Gly Ser Ile Lys Thr Ile Asn
Ser Ala Ile Glu Gln 325 330
335 Val Ser Thr Gln Arg Ser Lys Leu Gly Ala Val Gln Asn Arg Leu Glu
340 345 350 His Thr
Ile Asn Asn Leu Asn Thr Ser Ser Glu Asn Leu Thr Ala Ala 355
360 365 Glu Ser Arg Val Arg Asp Val
Asp Met Ala Lys Glu Met Met Ala Phe 370 375
380 Ser Lys Asn Asn Ile Leu Ser Gln Ala Ala Gln Ala
Met Leu Gly Gln 385 390 395
400 Ala Asn Gln Gln Pro Gln Gly Val Leu Gln Leu Leu Arg
405 410 74399PRTXanthomonas campestris 74Met
Ala Gln Val Ile Asn Thr Asn Val Met Ser Leu Asn Ala Gln Arg 1
5 10 15 Asn Leu Asn Thr Asn Ser
Ser Ser Met Ala Leu Ser Ile Gln Gln Leu 20
25 30 Ser Ser Gly Lys Arg Ile Thr Ser Ala Ser
Val Asp Ala Ala Gly Leu 35 40
45 Ala Ile Ser Glu Arg Phe Thr Thr Gln Ile Arg Gly Leu Asp
Val Ala 50 55 60
Ser Arg Asn Ala Asn Asp Gly Ile Ser Leu Ala Gln Thr Ala Glu Gly 65
70 75 80 Ala Met Val Glu Ile
Gly Asn Asn Leu Gln Arg Ile Arg Glu Leu Ser 85
90 95 Val Gln Ser Ala Asn Ala Thr Asn Ser Ala
Thr Asp Arg Glu Ala Leu 100 105
110 Asn Ser Glu Val Lys Gln Leu Thr Ser Glu Ile Asp Arg Val Ala
Asn 115 120 125 Gln
Thr Ser Phe Asn Gly Thr Lys Leu Leu Asn Gly Asp Phe Ser Gly 130
135 140 Ala Leu Phe Gln Val Gly
Ala Asp Ala Gly Gln Thr Ile Gly Ile Asn 145 150
155 160 Ser Ile Val Asp Ala Asn Val Asp Ser Leu Gly
Lys Ala Asn Phe Ala 165 170
175 Ala Ser Val Ser Gly Ala Gly Val Thr Gly Ala Ala Thr Ala Ser Gly
180 185 190 Ser Leu
Ser Gly Ile Thr Leu Ala Phe Lys Asp Ala Ser Gly Ala Ala 195
200 205 Lys Ser Val Ala Val Ala Asp
Ile Lys Ile Ala Ser Gly Asp Thr Ala 210 215
220 Ala Asp Ile Asn Lys Lys Val Ala Ser Ala Ile Asn
Asp Lys Leu Asp 225 230 235
240 Gln Thr Gly Met Tyr Ala Ser Ile Asp Thr Ser Gly Asn Val Lys Leu
245 250 255 Glu Ser Leu
Lys Ala Gly Gln Asp Phe Thr Ser Leu Ser Gly Gly Thr 260
265 270 Ser Gly Ala Ala Gly Ile Thr Ala
Gly Ala Gly Ile Gln Thr Ala Ser 275 280
285 Ala Ala Ser Gly Ser Thr Ala Ser Thr Leu Ser Ser Leu
Asp Ile Ser 290 295 300
Thr Phe Ser Gly Ala Gln Lys Ala Leu Glu Ile Val Asp Lys Ala Leu 305
310 315 320 Thr Ser Val Asn
Ser Ser Arg Ala Asp Met Gly Ala Val Gln Asn Arg 325
330 335 Phe Thr Ser Thr Ile Ala Asn Leu Ala
Ala Thr Ser Glu Asn Leu Thr 340 345
350 Ala Ser Arg Ser Arg Ile Ala Asp Thr Asp Tyr Ala Lys Thr
Thr Ala 355 360 365
Glu Leu Thr Arg Thr Gln Ile Leu Gln Gln Ala Gly Thr Ala Met Leu 370
375 380 Ala Gln Ala Lys Ser
Val Pro Gln Asn Val Leu Ser Leu Leu Gln 385 390
395 75275PRTNitrosomonas europaea 75Met Pro Gln Val
Ile Asn Thr Asn Ile Ala Ser Leu Asn Ala Gln Arg 1 5
10 15 Asn Leu Asn Val Ser Gln Asn Ser Leu
Ser Thr Ala Leu Gln Arg Leu 20 25
30 Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala
Gly Leu 35 40 45
Ala Ile Ser Glu Arg Met Thr Ser Gln Ile Arg Gly Met Asn Gln Ala 50
55 60 Ala Arg Asn Ala Asn
Asp Gly Ile Ser Leu Ala Gln Thr Ala Glu Gly 65 70
75 80 Ala Leu Val Glu Ile Gly Asn Asn Leu Gln
Arg Ile Arg Glu Leu Ala 85 90
95 Val Gln Ser Ala Asn Ala Thr Asn Ser Glu Asp Asp Arg Glu Ala
Leu 100 105 110 Gln
Lys Glu Val Thr Gln Leu Ile Asp Glu Ile Gln Arg Val Gly Glu 115
120 125 Gln Thr Ser Phe Asn Gly
Thr Lys Leu Leu Asp Gly Ser Phe Ala Ser 130 135
140 Gln Ile Phe Gln Val Gly Ala Asn Glu Gly Glu
Thr Ile Asp Phe Thr 145 150 155
160 Asp Ile Ala Asp Val Thr Ala Ser Gly Leu Ser Val Asp Ser Val Asp
165 170 175 Ile Thr
Gly Thr Asp Gly Thr Ala Ala Ala Ser Val Ile Thr Thr Ile 180
185 190 Asp Asp Ala Leu Lys Ile Val
Asn Ser Thr Arg Ala Asp Leu Gly Ala 195 200
205 Ile Gln Asn Arg Phe Ser Ser Ala Ile Ala Asn Leu
Gln Thr Ser Ala 210 215 220
Glu Asn Leu Ser Ala Ser Arg Ser Arg Ile Gln Asp Ala Asp Phe Ala 225
230 235 240 Ala Glu Thr
Ala Ala Leu Thr Arg Ala Gln Ile Leu Gln Gln Ala Gly 245
250 255 Val Ala Met Leu Ser Gln Ala Asn
Ala Leu Pro Asn Asn Val Leu Ser 260 265
270 Leu Leu Arg 275 76490PRTCampylobacter lari
76Gly Phe Arg Ile Asn Thr Asn Gly Ala Ser Leu Asn Ala Gln Val Asn 1
5 10 15 Ala Gly Leu Asn
Ser Arg Asn Leu Asp Ser Ser Leu Ala Arg Leu Ser 20
25 30 Ser Gly Leu Arg Ile Asn Ser Ala Ala
Asp Asp Ala Ser Gly Leu Ala 35 40
45 Ile Ala Asp Ser Leu Lys Thr Gln Ala Asn Ser Leu Gly Gln
Ala Ile 50 55 60
Asn Asn Ala Asn Asp Ala Asn Ser Met Leu Gln Ile Ala Asp Lys Ala 65
70 75 80 Met Asp Glu Gln Leu
Lys Ile Leu Asp Thr Ile Lys Val Lys Ala Thr 85
90 95 Gln Ala Ala Gln Asp Gly Gln Thr Ala Lys
Thr Arg Ala Met Ile Gln 100 105
110 Gly Glu Ile Asn Lys Leu Met Glu Glu Leu Asp Asn Ile Ala Asn
Thr 115 120 125 Thr
Thr Tyr Asn Gly Lys Gln Leu Leu Ser Gly Ser Phe Ser Asn Ala 130
135 140 Gln Phe Gln Ile Gly Asp
Lys Ala Asn Gln Thr Val Asn Ala Thr Ile 145 150
155 160 Gly Ser Thr Asn Ser Ala Lys Val Gly Gln Thr
Arg Phe Glu Thr Gly 165 170
175 Ala Val Ile Thr Ala Ala Val Ser Asn Gly Phe Thr Ile Lys Ser Tyr
180 185 190 Asp Gly
Ile Asn Asp Tyr Lys Ile Asp Ser Val Ala Ile Ser Tyr Ser 195
200 205 Val Gly Thr Gly Leu Gly Ala
Leu Ala Ala Glu Ile Asn Lys Ala Ser 210 215
220 Asp Lys Thr Gly Val Arg Ala Thr Ala Thr Val Gln
Thr Ile Ser Ser 225 230 235
240 Gly Ser Leu Ala Ala Gly Ser Thr Gly Gln Thr Phe Ala Ile Asn Gly
245 250 255 Val Val Ile
Gly Lys Val Val Tyr Glu Ala Gly Asp Lys Asn Gly Ala 260
265 270 Leu Val Ser Ala Ile Asn Ala Lys
Lys Asp Thr Thr Gly Val Glu Ala 275 280
285 Ser Ile Val Asp Gly Lys Leu Val Leu Asn Ser Ala Asp
Gly Arg Gly 290 295 300
Ile Lys Leu Ser Gly Ser Ile Gly Ala Leu Gly Asp Gln Met Leu Glu 305
310 315 320 Glu Asn Tyr Gly
Arg Leu Ser Leu Val Lys Asn Asp Gly Ser Asn Ile 325
330 335 Phe Ile Ser Gly Thr Thr Val Ser Asn
Ile Gly Leu Gly Thr Ala Gln 340 345
350 Met Ala Glu Ala Thr Val Asn Leu Glu Ser Ile Lys Gly Gln
Ile Thr 355 360 365
Ala Asp Ile Ala Ser Ala Met Gly Phe Asn Ala Met Ser Thr Ala Asp 370
375 380 Thr Ala Gly Lys Lys
Gln Ser Ala Gly Val Thr Thr Leu Gln Gly Ala 385 390
395 400 Met Ala Val Met Asp Ile Ala Asp Thr Ala
Ile Ala Asn Leu Asp Thr 405 410
415 Ile Arg Ala Asn Ile Gly Ala Thr Gln Asn Gln Ile Thr Ser Thr
Ile 420 425 430 Asn
Asn Ile Ser Val Thr Gln Val Asn Val Lys Ala Ala Glu Ser Gln 435
440 445 Ile Arg Asp Val Asp Phe
Ala Ser Glu Ser Ala Asn Tyr Ser Lys Ala 450 455
460 Asn Ile Leu Ala Gln Ser Gly Ser Tyr Ala Met
Ala Gln Ala Asn Ala 465 470 475
480 Ala Ser Gln Asn Val Leu Arg Leu Leu Gln 485
490
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