1887

Abstract

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

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2012-03-01
2024-12-03
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References

  1. Ahlgren J. A., Ordal G. W. ( 1983). Methyl esterification of glutamic acid residues of methyl-accepting chemotaxis proteins in Bacillus subtilis . Biochem J 213:759–763[PubMed]
    [Google Scholar]
  2. Artiushin S., Timoney J. F., Nally J., Verma A. ( 2004). Host-inducible immunogenic sphingomyelinase-like protein, Lk73.5, of Leptospira interrogans . Infect Immun 72:742–749 [View Article][PubMed]
    [Google Scholar]
  3. Bagos P. G., Liakopoulos T. D., Spyropoulos I. C., Hamodrakas S. J. ( 2004). PRED-TMBB: a web server for predicting the topology of β-barrel outer membrane proteins. Nucleic Acids Res 32:Web Server issueW400–W404 [View Article][PubMed]
    [Google Scholar]
  4. Beck M., Malmström J. A., Lange V., Schmidt A., Deutsch E. W., Aebersold R. ( 2009). Visual proteomics of the human pathogen Leptospira interrogans . Nat Methods 6:817–823 [View Article][PubMed]
    [Google Scholar]
  5. Bharti A. R., Nally J. E., Ricaldi J. N., Matthias M. A., Diaz M. M., Lovett M. A., Levett P. N., Gilman R. H., Willig M. R. & other authors ( 2003). Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3:757–771 [View Article][PubMed]
    [Google Scholar]
  6. Bourhy P., Louvel H., Saint Girons I., Picardeau M. ( 2005). Random insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis, using a mariner transposon. J Bacteriol 187:3255–3258 [View Article][PubMed]
    [Google Scholar]
  7. Cao X. J., Dai J., Xu H., Nie S., Chang X., Hu B. Y., Sheng Q. H., Wang L. S., Ning Z. B. & other authors ( 2010). High-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete Leptospira interrogans . Cell Res 20:197–210 [View Article][PubMed]
    [Google Scholar]
  8. Chao C. C., Wu S. L., Ching W. M. ( 2004). Using LC-MS with de novo software to fully characterize the multiple methylations of lysine residues in a recombinant fragment of an outer membrane protein from a virulent strain of Rickettsia prowazekii . Biochim Biophys Acta 1702:145–152[PubMed] [CrossRef]
    [Google Scholar]
  9. Chao C. C., Chelius D., Zhang T., Mutumanje E., Ching W. M. ( 2007). Insight into the virulence of Rickettsia prowazekii by proteomic analysis and comparison with an avirulent strain. Biochim Biophys Acta 1774:373–381[PubMed] [CrossRef]
    [Google Scholar]
  10. Chao C. C., Zhang Z., Wang H., Alkhalil A., Ching W. M. ( 2008). Serological reactivity and biochemical characterization of methylated and unmethylated forms of a recombinant protein fragment derived from outer membrane protein B of Rickettsia typhi . Clin Vaccine Immunol 15:684–690 [View Article][PubMed]
    [Google Scholar]
  11. Clarke S. ( 1993). Protein methylation. Curr Opin Cell Biol 5:977–983 [View Article][PubMed]
    [Google Scholar]
  12. Cullen P. A., Cordwell S. J., Bulach D. M., Haake D. A., Adler B. ( 2002). Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect Immun 70:2311–2318 [View Article][PubMed]
    [Google Scholar]
  13. Cullen P. A., Xu X., Matsunaga J., Sanchez Y., Ko A. I., Haake D. A., Adler B. ( 2005). Surfaceome of Leptospira spp. Infect Immun 73:4853–4863 [View Article][PubMed]
    [Google Scholar]
  14. Deitsch K. W., Lukehart S. A., Stringer J. R. ( 2009). Common strategies for antigenic variation by bacterial, fungal and protozoan pathogens. Nat Rev Microbiol 7:493–503 [View Article][PubMed]
    [Google Scholar]
  15. El-Manzalawy Y., Dobbs D., Honavar V. ( 2008). Predicting linear B-cell epitopes using string kernels. J Mol Recognit 21:243–255 [View Article][PubMed]
    [Google Scholar]
  16. Ellinghausen H. C. Jr, McCullough W. G. ( 1965). Nutrition of Leptospira pomona and growth of 13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80. Am J Vet Res 26:45–51[PubMed]
    [Google Scholar]
  17. Emanuelsson O., Brunak S., von Heijne G., Nielsen H. ( 2007). Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971 [View Article][PubMed]
    [Google Scholar]
  18. Eshghi A., Cullen P. A., Cowen L., Zuerner R. L., Cameron C. E. ( 2009). Global proteome analysis of Leptospira interrogans . J Proteome Res 8:4564–4578 [View Article][PubMed]
    [Google Scholar]
  19. Garbom S., Forsberg A., Wolf-Watz H., Kihlberg B. M. ( 2004). Identification of novel virulence-associated genes via genome analysis of hypothetical genes. Infect Immun 72:1333–1340 [View Article][PubMed]
    [Google Scholar]
  20. Garbom S., Olofsson M., Björnfot A.-C., Srivastava M. K., Robinson V. L., Oyston P. C. F., Titball R. W., Wolf-Watz H. ( 2007). Phenotypic characterization of a virulence-associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the type III secretion system. Microbiology 153:1464–1473 [View Article][PubMed]
    [Google Scholar]
  21. Giacomodonato M. N., Sarnacki S. H., Llana M. N., Cerquetti M. C. ( 2009). Dam and its role in pathogenicity of Salmonella enterica . J Infect Dev Ctries 3:484–490 [View Article][PubMed]
    [Google Scholar]
  22. Guerreiro H., Croda J., Flannery B., Mazel M., Matsunaga J., Galvão Reis M., Levett P. N., Ko A. I., Haake D. A. ( 2001). Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans. Infect Immun 69:4958–4968 [View Article][PubMed]
    [Google Scholar]
  23. Haake D. A., Matsunaga J. ( 2002). Characterization of the leptospiral outer membrane and description of three novel leptospiral membrane proteins. Infect Immun 70:4936–4945 [View Article][PubMed]
    [Google Scholar]
  24. Haake D. A., Chao G., Zuerner R. L., Barnett J. K., Barnett D., Mazel M., Matsunaga J., Levett P. N., Bolin C. A. ( 2000). The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect Immun 68:2276–2285 [View Article][PubMed]
    [Google Scholar]
  25. Hensel M., Shea J. E., Gleeson C., Jones M. D., Dalton E., Holden D. W. ( 1995). Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400–403 [View Article][PubMed]
    [Google Scholar]
  26. Heurgué-Hamard V., Champ S., Engström A., Ehrenberg M., Buckingham R. H. ( 2002). The hemK gene in Escherichia coli encodes the N 5-glutamine methyltransferase that modifies peptide release factors. EMBO J 21:769–778 [View Article][PubMed]
    [Google Scholar]
  27. Johnson R. C., Harris V. G. ( 1967). Differentiation of pathogenic and saprophytic letospires. I. Growth at low temperatures. J Bacteriol 94:27–31[PubMed]
    [Google Scholar]
  28. Kehry M. R., Doak T. G., Dahlquist F. W. ( 1984). Stimulus-induced changes in methylesterase activity during chemotaxis in Escherichia coli . J Biol Chem 259:11828–11835[PubMed]
    [Google Scholar]
  29. Kleene S. J., Toews M. L., Adler J. ( 1977). Isolation of glutamic acid methyl ester from an Escherichia coli membrane protein involved in chemotaxis. J Biol Chem 252:3214–3218[PubMed]
    [Google Scholar]
  30. Kloczkowski A., Ting K. L., Jernigan R. L., Garnier J. ( 2002). Combining the GOR V algorithm with evolutionary information for protein secondary structure prediction from amino acid sequence. Proteins 49:154–166 [View Article][PubMed]
    [Google Scholar]
  31. Ko A. I., Galvão Reis M., Ribeiro Dourado C. M., Johnson W. D. Jr, Riley L. W. Salvador Leptospirosis Study Group ( 1999). Urban epidemic of severe leptospirosis in Brazil. Lancet 354:820–825[PubMed] [CrossRef]
    [Google Scholar]
  32. Ko A. I., Goarant C., Picardeau M. ( 2009). Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 7:736–747 [View Article][PubMed]
    [Google Scholar]
  33. Krzywinska E., Bhatnagar S., Sweet L., Chatterjee D., Schorey J. S. ( 2005). Mycobacterium avium 104 deleted of the methyltransferase D gene by allelic replacement lacks serotype-specific glycopeptidolipids and shows attenuated virulence in mice. Mol Microbiol 56:1262–1273 [View Article][PubMed]
    [Google Scholar]
  34. Levett P. N. ( 2001). Leptospirosis. Clin Microbiol Rev 14:296–326 [View Article][PubMed]
    [Google Scholar]
  35. Liao S., Sun A., Ojcius D. M., Wu S., Zhao J., Yan J. ( 2009). Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai. BMC Microbiol 9:253–262 [View Article][PubMed]
    [Google Scholar]
  36. Lo M., Cordwell S. J., Bulach D. M., Adler B. ( 2009). Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature. PLoS Negl Trop Dis 3:e560 [View Article][PubMed]
    [Google Scholar]
  37. Luo Y., Liu Y., Sun D., Ojcius D. M., Zhao J., Lin X., Wu D., Zhang R., Chen M. & other authors ( 2011). InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals. J Biol Chem 286:36852–36863 [View Article][PubMed]
    [Google Scholar]
  38. Ma B., Zhang K., Hendrie C., Liang C., Li M., Doherty-Kirby A., Lajoie G. ( 2003). peaks: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun Mass Spectrom 17:2337–2342 [View Article][PubMed]
    [Google Scholar]
  39. Malmström J., Beck M., Schmidt A., Lange V., Deutsch E. W., Aebersold R. ( 2009). Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans . Nature 460:762–765 [View Article][PubMed]
    [Google Scholar]
  40. Matsunaga J., Werneid K., Zuerner R. L., Frank A., Haake D. A. ( 2006). LipL46 is a novel surface-exposed lipoprotein expressed during leptospiral dissemination in the mammalian host. Microbiology 152:3777–3786 [View Article][PubMed]
    [Google Scholar]
  41. Matsunaga J., Lo M., Bulach D. M., Zuerner R. L., Adler B., Haake D. A. ( 2007). Response of Leptospira interrogans to physiologic osmolarity: relevance in signaling the environment-to-host transition. Infect Immun 75:2864–2874 [View Article][PubMed]
    [Google Scholar]
  42. McBride A. J., Athanazio D. A., Reis M. G., Ko A. I. ( 2005). Leptospirosis. Curr Opin Infect Dis 18:376–386 [View Article][PubMed]
    [Google Scholar]
  43. Merien F., Truccolo J., Rougier Y., Baranton G., Perolat P. ( 1998). In vivo apoptosis of hepatocytes in guinea pigs infected with Leptospira interrogans serovar icterohaemorrhagiae. FEMS Microbiol Lett 169:95–102 [View Article][PubMed]
    [Google Scholar]
  44. Monahan A. M., Callanan J. J., Nally J. E. ( 2008). Proteomic analysis of Leptospira interrogans shed in urine of chronically infected hosts. Infect Immun 76:4952–4958 [View Article][PubMed]
    [Google Scholar]
  45. Murray G. L., Srikram A., Henry R., Puapairoj A., Sermswan R. W., Adler B. ( 2009). Leptospira interrogans requires heme oxygenase for disease pathogenesis. Microbes Infect 11:311–314 [View Article][PubMed]
    [Google Scholar]
  46. Murray G. L., Srikram A., Henry R., Hartskeerl R. A., Sermswan R. W., Adler B. ( 2010). Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence. Mol Microbiol 78:701–709 [View Article][PubMed]
    [Google Scholar]
  47. Nakahigashi K., Kubo N., Narita S.-i., Shimaoka T., Goto S., Oshima T., Mori H., Maeda M., Wada C., Inokuchi H. ( 2002). HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination. Proc Natl Acad Sci U S A 99:1473–1478 [View Article][PubMed]
    [Google Scholar]
  48. Nally J. E., Artiushin S., Timoney J. F. ( 2001a). Molecular characterization of thermoinduced immunogenic proteins Q1p42 and Hsp15 of Leptospira interrogans . Infect Immun 69:7616–7624 [View Article][PubMed]
    [Google Scholar]
  49. Nally J. E., Timoney J. F., Stevenson B. ( 2001b). Temperature-regulated protein synthesis by Leptospira interrogans . Infect Immun 69:400–404 [View Article][PubMed]
    [Google Scholar]
  50. Nally J. E., Whitelegge J. P., Bassilian S., Blanco D. R., Lovett M. A. ( 2007). Characterization of the outer membrane proteome of Leptospira interrogans expressed during acute lethal infection. Infect Immun 75:766–773 [View Article][PubMed]
    [Google Scholar]
  51. Nishiyama S. I., Umemura T., Nara T., Homma M., Kawagishi I. ( 1999). Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant. Mol Microbiol 32:357–365 [View Article][PubMed]
    [Google Scholar]
  52. Parra M., Pickett T., Delogu G., Dheenadhayalan V., Debrie A. S., Locht C., Brennan M. J. ( 2004). The mycobacterial heparin-binding hemagglutinin is a protective antigen in the mouse aerosol challenge model of tuberculosis. Infect Immun 72:6799–6805 [View Article][PubMed]
    [Google Scholar]
  53. Pinne M., Haake D. A. ( 2009). A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans . PLoS ONE 4:e6071 [View Article][PubMed]
    [Google Scholar]
  54. Pinne M., Haake D. ( 2011). Immuno-fluorescence assay of leptospiral surface-exposed proteins. J Vis Exp532805[PubMed]
    [Google Scholar]
  55. Rahman O., Cummings S., Harrington D., Sutcliffe I. ( 2008). Methods for the bioinformatic identification of bacterial lipoproteins encoded in the genomes of Gram-positive bacteria. World J Microb Biot 24:2377–2382 [View Article]
    [Google Scholar]
  56. Rice M. S., Dahlquist F. W. ( 1991). Sites of deamidation and methylation in Tsr, a bacterial chemotaxis sensory transducer. J Biol Chem 266:9746–9753[PubMed]
    [Google Scholar]
  57. Ristow P., Bourhy P., da Cruz McBride F. W., Figueira C. P., Huerre M., Ave P., Girons I. S., Ko A. I., Picardeau M. ( 2007). The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathog 3:e97 [View Article][PubMed]
    [Google Scholar]
  58. Sakolvaree Y., Maneewatch S., Jiemsup S., Klaysing B., Tongtawe P., Srimanote P., Saengjaruk P., Banyen S., Tapchaisri P. & other authors ( 2007). Proteome and immunome of pathogenic Leptospira spp. revealed by 2DE and 2DE-immunoblotting with immune serum. Asian Pac J Allergy Immunol 25:53–73[PubMed]
    [Google Scholar]
  59. Sassetti C. M., Boyd D. H., Rubin E. J. ( 2001). Comprehensive identification of conditionally essential genes in mycobacteria. Proc Natl Acad Sci U S A 98:12712–12717 [View Article][PubMed]
    [Google Scholar]
  60. Seguro A. C., Lomar A. V., Rocha A. S. ( 1990). Acute renal failure of leptospirosis: nonoliguric and hypokalemic forms. Nephron 55:146–151 [View Article][PubMed]
    [Google Scholar]
  61. Sen T. Z., Jernigan R. L., Garnier J., Kloczkowski A. ( 2005). GOR V server for protein secondary structure prediction. Bioinformatics 21:2787–2788 [View Article][PubMed]
    [Google Scholar]
  62. Setubal J. C., Reis M. G., Matsunaga J., Haake D. A. ( 2006). Lipoprotein computational prediction in spirochaetal genomes. Microbiology 152:113–121 [View Article][PubMed]
    [Google Scholar]
  63. Temmerman S., Pethe K., Parra M., Alonso S., Rouanet C., Pickett T., Drowart A., Debrie A. S., Delogu G. & other authors ( 2004). Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin. Nat Med 10:935–941 [View Article][PubMed]
    [Google Scholar]
  64. Thiermann A. B., McClellan R. D., Hill H. T. ( 1984). Improved techniques for the isolation of leptospires from swine abortion cases. American Assn of Veterinary Laboratory Diagnosticians 27:233–244
    [Google Scholar]
  65. Tsirigos K. D., Bagos P. G., Hamodrakas S. J. ( 2011). OMPdb: a database of β-barrel outer membrane proteins from Gram-negative bacteria. Nucleic Acids Res 39:Database issueD324–D331 [View Article][PubMed]
    [Google Scholar]
  66. Velineni S., Asuthkar S., Sritharan M. ( 2006). Iron limitation and expression of immunoreactive outer membrane proteins in Leptospira interrogans serovar icterohaemorrhagiae strain lai. Indian J Med Microbiol 24:339–342 [View Article][PubMed]
    [Google Scholar]
  67. Verma A., Rathinam S. R., Priya C. G., Muthukkaruppan V. R., Stevenson B., Timoney J. F. ( 2008). LruA and LruB antibodies in sera of humans with leptospiral uveitis. Clin Vaccine Immunol 15:1019–1023 [View Article][PubMed]
    [Google Scholar]
  68. Vieira M. L., Pimenta D. C., de Morais Z. M., Vasconcellos S. A., Nascimento A. L. ( 2009). Proteome analysis of Leptospira interrogans virulent strain. Open Microbiol J 3:69–74 [View Article][PubMed]
    [Google Scholar]
  69. Wadhams G. H., Armitage J. P. ( 2004). Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 5:1024–1037 [View Article][PubMed]
    [Google Scholar]
  70. WHO ( 1999). Leptospirosis worldwide, 1999. Wkly Epidemiol Rec 74:237–242[PubMed]
    [Google Scholar]
  71. Wilkins M. R., Gasteiger E., Gooley A. A., Herbert B. R., Molloy M. P., Binz P. A., Ou K., Sanchez J. C., Bairoch A. & other authors ( 1999). High-throughput mass spectrometric discovery of protein post-translational modifications. J Mol Biol 289:645–657 [View Article][PubMed]
    [Google Scholar]
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