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. 1997 Feb;179(4):1262–1267. doi: 10.1128/jb.179.4.1262-1267.1997

Identification and characterization of the dTDP-rhamnose biosynthesis and transfer genes of the lipopolysaccharide-related rfb locus in Leptospira interrogans serovar Copenhageni.

M Mitchison 1, D M Bulach 1, T Vinh 1, K Rajakumar 1, S Faine 1, B Adler 1
PMCID: PMC178824  PMID: 9023210

Abstract

Immunity to leptospirosis is principally humorally mediated and involves opsonization of leptospires for phagocytosis by macrophages and neutrophils. The only protective antigen identified to date is the leptospiral lipopolysaccharide (LPS), which biochemically resembles typical gram-negative LPS but has greatly reduced endotoxic activity. Little is known about the structure of leptospiral LPS. A 2.1-kb EcoRI fragment from the chromosome of serovar Copenhageni was cloned in pUC18 in Escherichia coli, after which flanking regions were cloned from a genomic library constructed in bacteriophage lambda GEM12. Sequence analysis identified four open reading frames which showed similarity to the rfbC, rfbD, rfbB, and rfbA genes, transcribed in that order, which encode the four enzymes involved in the biosynthesis of dTDP-rhamnose for the assembly of LPS in Salmonella enterica, E. coli, and Shigella flexneri. An additional open reading frame downstream of the rfbCDBA locus showed similarity with the rhamnosyltransferase genes of Shigella and Yersinia enterocolitica but not Salmonella. Comparison of deduced amino acid sequences showed up to 85% similarity of the leptospiral proteins with those of other gram-negative bacteria. Polyacrylamide gel electrophoresis of recombinant clones identified the putative RfbCDBA proteins, while reverse transcriptase-mediated PCR analysis indicated that the rfbCDBA gene cluster was expressed in Leptospira. Moreover, it could restore normal LPS phenotype to a defined rfbB::Tn5 mutant of S. flexneri which was deficient in all four genes, thereby confirming the functional identification of a part of the leptospiral rfb locus.

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Selected References

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  1. Adler B., Faine S. A pomona serogroup-specific, agglutinating antigen in Leptospira, identified by monoclonal antibodies. Pathology. 1983 Jul;15(3):247–250. doi: 10.3109/00313028309083501. [DOI] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Brahmbhatt H. N., Wyk P., Quigley N. B., Reeves P. R. Complete physical map of the rfb gene cluster encoding biosynthetic enzymes for the O antigen of Salmonella typhimurium LT2. J Bacteriol. 1988 Jan;170(1):98–102. doi: 10.1128/jb.170.1.98-102.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cinco M., Banfi E., Soranzo M. R. Studies on the interaction between macrophages and leptospires. J Gen Microbiol. 1981 Jun;124(2):409–413. doi: 10.1099/00221287-124-2-409. [DOI] [PubMed] [Google Scholar]
  5. Faine S., Adler B., Palit A. Chemical, serological and biological properties of a serotype-specific polysaccharide antigen in Leptospira. Aust J Exp Biol Med Sci. 1974 Apr;52(2):311–319. doi: 10.1038/icb.1974.29. [DOI] [PubMed] [Google Scholar]
  6. Farrelly H. E., Adler B., Faine S. Opsonic monoclonal antibodies against lipopolysaccharide antigens of Leptospira interrogans serovar hardjo. J Med Microbiol. 1987 Feb;23(1):1–7. doi: 10.1099/00222615-23-1-1. [DOI] [PubMed] [Google Scholar]
  7. Finco D. R., Low D. G. Endotoxin properties of Leptospira canicola. Am J Vet Res. 1967 Nov;28(127):1863–1872. [PubMed] [Google Scholar]
  8. Haake D. A., Champion C. I., Martinich C., Shang E. S., Blanco D. R., Miller J. N., Lovett M. A. Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp. J Bacteriol. 1993 Jul;175(13):4225–4234. doi: 10.1128/jb.175.13.4225-4234.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Isogai E., Isogai H., Kurebayashi Y., Ito N. Biological activities of leptospiral lipopolysaccharide. Zentralbl Bakteriol Mikrobiol Hyg A. 1986 Feb;261(1):53–64. doi: 10.1016/s0176-6724(86)80062-1. [DOI] [PubMed] [Google Scholar]
  11. Jiang X. M., Neal B., Santiago F., Lee S. J., Romana L. K., Reeves P. R. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol. 1991 Mar;5(3):695–713. doi: 10.1111/j.1365-2958.1991.tb00741.x. [DOI] [PubMed] [Google Scholar]
  12. Johnson R. C., Walby J., Henry R. A., Auran N. E. Cultivation of parasitic leptospires: effect of pyruvate. Appl Microbiol. 1973 Jul;26(1):118–119. doi: 10.1128/am.26.1.118-119.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jost B. H., Adler B., Faine S. Experimental immunisation of hamsters with lipopolysaccharide antigens of Leptospira interrogans. J Med Microbiol. 1989 Jun;29(2):115–120. doi: 10.1099/00222615-29-2-115. [DOI] [PubMed] [Google Scholar]
  14. Jost B. H., Adler B., Vinh T., Faine S. A monoclonal antibody reacting with a determinant on leptospiral lipopolysaccharide protects guinea pigs against leptospirosis. J Med Microbiol. 1986 Nov;22(3):269–275. doi: 10.1099/00222615-22-3-269. [DOI] [PubMed] [Google Scholar]
  15. Klena J. D., Schnaitman C. A. Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Mol Microbiol. 1993 Jul;9(2):393–402. doi: 10.1111/j.1365-2958.1993.tb01700.x. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Liu D., Reeves P. R. Escherichia coli K12 regains its O antigen. Microbiology. 1994 Jan;140(Pt 1):49–57. doi: 10.1099/13500872-140-1-49. [DOI] [PubMed] [Google Scholar]
  18. Macpherson D. F., Manning P. A., Morona R. Characterization of the dTDP-rhamnose biosynthetic genes encoded in the rfb locus of Shigella flexneri. Mol Microbiol. 1994 Jan;11(2):281–292. doi: 10.1111/j.1365-2958.1994.tb00308.x. [DOI] [PubMed] [Google Scholar]
  19. McGrath H., Adler B., Vinh T., Faine S. Phagocytosis of virulent and avirulent leptospires by guinea-pig and human polymorphonuclear leukocytes in vitro. Pathology. 1984 Jul;16(3):243–249. doi: 10.3109/00313028409068531. [DOI] [PubMed] [Google Scholar]
  20. Midwinter A., Faine S., Adler B. Vaccination of mice with lipopolysaccharide (LPS) and LPS-derived immuno-conjugates from Leptospira interrogans. J Med Microbiol. 1990 Nov;33(3):199–204. doi: 10.1099/00222615-33-3-199. [DOI] [PubMed] [Google Scholar]
  21. Midwinter A., Vinh T., Faine S., Adler B. Characterization of an antigenic oligosaccharide from Leptospira interrogans serovar pomona and its role in immunity. Infect Immun. 1994 Dec;62(12):5477–5482. doi: 10.1128/iai.62.12.5477-5482.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morona R., Macpherson D. F., Van Den Bosch L., Carlin N. I., Manning P. A. Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes. Mol Microbiol. 1995 Oct;18(2):209–223. doi: 10.1111/j.1365-2958.1995.mmi_18020209.x. [DOI] [PubMed] [Google Scholar]
  23. Morona R., Mavris M., Fallarino A., Manning P. A. Characterization of the rfc region of Shigella flexneri. J Bacteriol. 1994 Feb;176(3):733–747. doi: 10.1128/jb.176.3.733-747.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paster B. J., Dewhirst F. E., Weisburg W. G., Tordoff L. A., Fraser G. J., Hespell R. B., Stanton T. B., Zablen L., Mandelco L., Woese C. R. Phylogenetic analysis of the spirochetes. J Bacteriol. 1991 Oct;173(19):6101–6109. doi: 10.1128/jb.173.19.6101-6109.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rajakumar K., Jost B. H., Sasakawa C., Okada N., Yoshikawa M., Adler B. Nucleotide sequence of the rhamnose biosynthetic operon of Shigella flexneri 2a and role of lipopolysaccharide in virulence. J Bacteriol. 1994 Apr;176(8):2362–2373. doi: 10.1128/jb.176.8.2362-2373.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reeves P. Evolution of Salmonella O antigen variation by interspecific gene transfer on a large scale. Trends Genet. 1993 Jan;9(1):17–22. doi: 10.1016/0168-9525(93)90067-R. [DOI] [PubMed] [Google Scholar]
  28. Sasakawa C., Kamata K., Sakai T., Murayama S. Y., Makino S., Yoshikawa M. Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. Infect Immun. 1986 Feb;51(2):470–475. doi: 10.1128/iai.51.2.470-475.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schoone G. J., Everard C. O., Korver H., Carrington D. G., Inniss V. A., Baulu J., Terpstra W. J. An immunoprotective monoclonal antibody directed against Leptospira interrogans serovar copenhageni. J Gen Microbiol. 1989 Jan;135(1):73–78. doi: 10.1099/00221287-135-1-73. [DOI] [PubMed] [Google Scholar]
  30. Shang E. S., Exner M. M., Summers T. A., Martinich C., Champion C. I., Hancock R. E., Haake D. A. The rare outer membrane protein, OmpL1, of pathogenic Leptospira species is a heat-modifiable porin. Infect Immun. 1995 Aug;63(8):3174–3181. doi: 10.1128/iai.63.8.3174-3181.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  32. Sturm S., Jann B., Jann K., Fortnagel P., Timmis K. N. Genetic and biochemical analysis of Shigella dysenteriae 1 O antigen polysaccharide biosynthesis in Escherichia coli K-12: structure and functions of the rfb gene cluster. Microb Pathog. 1986 Jun;1(3):307–324. doi: 10.1016/0882-4010(86)90056-2. [DOI] [PubMed] [Google Scholar]
  33. Tu V., Adler B., Faine S. The role of macrophages in the protection of mice against leptospirosis: in vitro and in vivo studies. Pathology. 1982 Oct;14(4):463–468. doi: 10.3109/00313028209092128. [DOI] [PubMed] [Google Scholar]
  34. Vinh T. U., Shi M. H., Adler B., Faine S. Characterization and taxonomic significance of lipopolysaccharides of Leptospira interrogans serovar hardjo. J Gen Microbiol. 1989 Oct;135(10):2663–2673. doi: 10.1099/00221287-135-10-2663. [DOI] [PubMed] [Google Scholar]
  35. Vinh T., Adler B., Faine S. Ultrastructure and chemical composition of lipopolysaccharide extracted from Leptospira interrogans serovar copenhageni. J Gen Microbiol. 1986 Jan;132(1):103–109. doi: 10.1099/00221287-132-1-103. [DOI] [PubMed] [Google Scholar]
  36. Vinh T., Faine S., Handley C. J., Adler B. Immunochemical studies of opsonic epitopes of the lipopolysaccharide of Leptospira interrogans serovar hardjo. FEMS Immunol Med Microbiol. 1994 Feb;8(2):99–107. doi: 10.1111/j.1574-695X.1994.tb00431.x. [DOI] [PubMed] [Google Scholar]
  37. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  38. Zhang L., al-Hendy A., Toivanen P., Skurnik M. Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems. Mol Microbiol. 1993 Jul;9(2):309–321. doi: 10.1111/j.1365-2958.1993.tb01692.x. [DOI] [PubMed] [Google Scholar]

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