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Abstract

Members of the bacterial genus utilize chitin both as a metabolic substrate and a signal to activate natural competence. is a bacterial enteric pathogen, sub-lineages of which can cause pandemic cholera. However, the chitin metabolic pathway in has been dissected using only a limited number of laboratory strains of this species. Here, we survey the complement of key chitin metabolism genes amongst 195 diverse . We show that the gene encoding GbpA, known to be an important colonization and virulence factor in pandemic isolates, is not ubiquitous amongst . We also identify a putatively novel chitinase, and present experimental evidence in support of its functionality. Our data indicate that the chitin metabolic pathway within is more complex than previously thought, and emphasize the importance of considering genes and functions in the context of a species in its entirety, rather than simply relying on traditional reference strains.

Keyword(s): ChiA , chitin , chitinase , cholera , GbpA and Vibrio cholerae
Funding
This study was supported by the:
  • Wellcome Trust (Award 206194)
    • Principle Award Recipient: MatthewJ. Dorman
  • Wellcome Trust (Award 206194)
    • Principle Award Recipient: NicholasR. Thomson
  • European Bioinformatics Institute (Award EBI-Sanger Postdoctoral (ESPOD) Fellowship)
    • Principle Award Recipient: GraceA. Blackwell
  • Churchill College, University of Cambridge (Award Junior Research Fellowship)
    • Principle Award Recipient: MatthewJ. Dorman
  • Amgen Foundation (Award Amgen Foundation Scholarship)
    • Principle Award Recipient: NicholasR. Thomson
  • Amgen Foundation (Award Amgen Foundation Scholarship)
    • Principle Award Recipient: TheaG. Fennell
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2021-06-08
2024-03-29
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References

  1. Morris JG, Black RE. Cholera and other vibrioses in the United States. N Engl J Med 1985; 312:343–350 [View Article][PubMed]
    [Google Scholar]
  2. Pollitzer R, Swaroop S, Burrows W. Cholera World Health Organization; 1959
    [Google Scholar]
  3. Kaper JB, Morris JG, Levine MM. Cholera. Clin Microbiol Rev 1995; 8:48–86 [View Article][PubMed]
    [Google Scholar]
  4. Devault AM, Golding GB, Waglechner N, Enk JM, Kuch M et al. Second-pandemic strain of Vibrio cholerae from the Philadelphia cholera outbreak of 1849. N Engl J Med 2014; 370:334–340 [View Article][PubMed]
    [Google Scholar]
  5. Cvjetanovic B, Barua D. The seventh pandemic of cholera. Nature 1972; 239:137–138 [View Article][PubMed]
    [Google Scholar]
  6. Furniss AL, Lee JV, Donovan TJ. The Vibrios London: His Majesty’s Stationery Office (H.M.S.O); 1978
    [Google Scholar]
  7. Weill F-. X, Domman D, Njamkepo E, Tarr C, Rauzier J et al. Genomic history of the seventh pandemic of cholera in Africa. Science 2017; 358:785–789 [View Article][PubMed]
    [Google Scholar]
  8. Domman D, Quilici ML, Dorman MJ, Njamkepo E, Mutreja A et al. Integrated view of Vibrio cholerae in the Americas. Science 2017; 358:789–793 [View Article][PubMed]
    [Google Scholar]
  9. Oprea M, Njamkepo E, Cristea D, Zhukova A, Clark CG et al. The seventh pandemic of cholera in Europe revisited by microbial genomics. Nat Commun 2020; 11:5347 [View Article][PubMed]
    [Google Scholar]
  10. Mutreja A, Kim DW, Thomson NR, Connor TR, Lee JH et al. Evidence for several waves of global transmission in the seventh cholera pandemic. Nature 2011; 477:462–465 [View Article][PubMed]
    [Google Scholar]
  11. Karlsson SL, Thomson N, Mutreja A, Connor T, Sur D et al. Retrospective analysis of serotype switching of Vibrio cholerae O1 in a cholera endemic region shows it is a non-random process. PLoS Negl Trop Dis 2016; 10:e0005044 [View Article][PubMed]
    [Google Scholar]
  12. Chun J, Grim CJ, Hasan NA, Lee JH, Choi SY et al. Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae . Proc Natl Acad Sci U S A 2009; 106:15442–15447 [View Article][PubMed]
    [Google Scholar]
  13. Clemens JD, Nair GB, Ahmed T, Qadri F, Holmgren J. Cholera. Lancet 2017; 390:1539–1549 [View Article]
    [Google Scholar]
  14. Jones MK, Oliver JD. Vibrio vulnificus: Disease and pathogenesis. Infect Immun 2009; 77:1723–1733 [View Article][PubMed]
    [Google Scholar]
  15. Daniels NA, MacKinnon L, Bishop R, Altekruse S, Ray B et al. Vibrio parahaemolyticus infections in the United States, 1973-1998. J Infect Dis 2000; 181:1661–1666 [View Article][PubMed]
    [Google Scholar]
  16. Goarant C, Reynaud Y, Ansquer D, de Decker S, Saulnier D et al. Molecular epidemiology of Vibrio nigripulchritudo, a pathogen of cultured penaeid shrimp (Litopenaeus stylirostris) in New Caledonia. Syst Appl Microbiol 2006; 29:570–580 [View Article]
    [Google Scholar]
  17. Goarant C, Ansquer D, Herlin J, Domalain D, Imbert F et al. “Summer Syndrome” in Litopenaeus stylirostris in New Caledonia: Pathology and epidemiology of the etiological agent, Vibrio nigripulchritudo . Aquaculture 2006; 253:105–113 [View Article]
    [Google Scholar]
  18. Frans I, Michiels CW, Bossier P, Willems KA, Lievens B et al. Vibrio anguillarum as a fish pathogen: Virulence factors, diagnosis and prevention. J Fish Dis 2011; 34:643–661 [View Article][PubMed]
    [Google Scholar]
  19. Zhang X-. H, He X, Austin B. Vibrio harveyi: A serious pathogen of fish and invertebrates in mariculture. Mar Life Sci Technol 2020; 2:231–245 [View Article]
    [Google Scholar]
  20. Hunt DE, Gevers D, Vahora NM, Polz MF. Conservation of the chitin utilization pathway in the Vibrionaceae . Appl Environ Microbiol 2008; 74:44–51 [View Article][PubMed]
    [Google Scholar]
  21. Keyhani NO, Roseman S. Physiological aspects of chitin catabolism in marine bacteria. Biochim Biophys Acta - Gen Subj 1999; 1473:108–122 [View Article]
    [Google Scholar]
  22. Froelich B, Oliver J. Increases in the amounts of Vibrio spp. in oysters upon addition of exogenous bacteria. Appl Environ Microbiol 2013; 79:5208–5213 [View Article][PubMed]
    [Google Scholar]
  23. Kaneko T, Colwell RR. Adsorption of Vibrio parahaemolyticus onto chitin and copepods. Appl Microbiol 1975; 29:269–274 [View Article][PubMed]
    [Google Scholar]
  24. Meibom KL, Li XB, Nielsen AT, Wu C-Y, Roseman S et al. The Vibrio cholerae chitin utilization program. Proc Natl Acad Sci U S A 2004; 101:2524–2529 [View Article]
    [Google Scholar]
  25. Nalin DR, Daya V, Reid A, Levine MM, Cisneros L. Adsorption and growth of Vibrio cholerae on chitin. Infect Immun 1979; 25:768–770 [View Article][PubMed]
    [Google Scholar]
  26. Huq A, Small EB, West PA, Huq MI, Rahman R et al. Ecological relationships between Vibrio cholerae and planktonic crustacean copepods. Appl Environ Microbiol 1983; 45:275–283 [View Article][PubMed]
    [Google Scholar]
  27. Lo Scrudato M, Blokesch M. A transcriptional regulator linking quorum sensing and chitin induction to render Vibrio cholerae naturally transformable. Nucleic Acids Res 2013; 41:3644–3658 [View Article][PubMed]
    [Google Scholar]
  28. Antonova ES, Hammer BK. Genetics of natural competence in Vibrio cholerae and other Vibrios . Microbiol Spectr 2015; 3: [View Article][PubMed]
    [Google Scholar]
  29. Meibom KL, Blokesch M, Dolganov NA, Wu C-Y, Schoolnik GK. Chitin induces natural competence in Vibrio cholerae . Science 2005; 310:1824–1827 [View Article]
    [Google Scholar]
  30. Mondal M, Nag D, Koley H, Saha DR, Chatterjee NS. The Vibrio cholerae extracellular chitinase ChiA2 is important for survival and pathogenesis in the host intestine. PLoS ONE 2014; 9:e103119 [View Article][PubMed]
    [Google Scholar]
  31. Conner JG, Teschler JK, Jones CJ, Yildiz FH. Staying alive: Vibrio cholerae’s cycle of environmental survival, transmission, and dissemination. Microbiol Spectr 2016; 4: [View Article][PubMed]
    [Google Scholar]
  32. Keyhani NO, Roseman S. The chitin catabolic cascade in the marine bacterium Vibrio furnissii: Molecular cloning, isolation, and characterization of a periplasmic β-N-acetylglucosaminidase. J Biol Chem 1996; 271:33425–33432 [View Article][PubMed]
    [Google Scholar]
  33. Keyhani NO, Roseman S. The chitin catabolic cascade in the marine bacterium Vibrio furnissii: Molecular cloning, isolation, and characterization of a periplasmic chitodextrinase. J Biol Chem 1996; 271:33414–33424 [View Article][PubMed]
    [Google Scholar]
  34. Chitlaru E, Roseman S. Molecular cloning and characterization of a novel β-N-acetyl-d-glucosaminidase from Vibrio furnissii . J Biol Chem 1996; 271:33433–33439 [View Article][PubMed]
    [Google Scholar]
  35. Jung B-. O, Roseman S, Park JK. The central concept for chitin catabolic cascade in marine bacterium, Vibrios. Macromol Res 2008; 16:1–5 [View Article]
    [Google Scholar]
  36. Hirano T, Okubo M, Tsuda H, Yokoyama M, Hakamata W et al. Chitin heterodisaccharide, released from chitin by chitinase and chitin oligosaccharide deacetylase, enhances the chitin-metabolizing ability of Vibrio parahaemolyticus . J Bacteriol 2019; 201:e00270-19 [View Article][PubMed]
    [Google Scholar]
  37. Suginta W, Vongsuwan A, Songsiriritthigul C, Prinz H, Estibeiro P et al. An endochitinase A from Vibrio carchariae: Cloning, expression, mass and sequence analyses, and chitin hydrolysis. Arch Biochem Biophys 2004; 424:171–180 [View Article][PubMed]
    [Google Scholar]
  38. Suginta W, Chuenark D, Mizuhara M, Fukamizo T. Novel β-N-acetylglucosaminidases from Vibrio harveyi 650: Cloning, expression, enzymatic properties, and subsite identification. BMC Biochem 2010; 11:40 [View Article][PubMed]
    [Google Scholar]
  39. Wortman AT, Somerville CC, Colwell RR. Chitinase determinants of Vibrio vulnificus: Gene cloning and applications of a chitinase probe. Appl Environ Microbiol 1986; 52:142–145 [View Article][PubMed]
    [Google Scholar]
  40. Kirn TJ, Jude BA, Taylor RK. A colonization factor links Vibrio cholerae environmental survival and human infection. Nature 2005; 438:863–866 [View Article][PubMed]
    [Google Scholar]
  41. Bhowmick R, Ghosal A, Das B, Koley H, Saha DR et al. Intestinal adherence of Vibrio cholerae involves a coordinated interaction between colonization factor GbpA and mucin. Infect Immun 2008; 76:4968–4977 [View Article][PubMed]
    [Google Scholar]
  42. Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV et al. The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. PLoS Pathog 2012; 8:e1002373 [View Article][PubMed]
    [Google Scholar]
  43. Loose JSM, Forsberg Z, Fraaije MW, Eijsink VGH, Vaaje-Kolstad G. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase. FEBS Lett 2014; 588:3435–3440 [View Article][PubMed]
    [Google Scholar]
  44. Jude BA, Martinez RM, Skorupski K, Taylor RK. Levels of the secreted Vibrio cholerae attachment factor GbpA are modulated by quorum-sensing-induced proteolysis. J Bacteriol 2009; 191:6911–6917 [View Article][PubMed]
    [Google Scholar]
  45. Hayes CA, Dalia TN, Dalia AB. Systematic genetic dissection of chitin degradation and uptake in Vibrio cholerae . Environ Microbiol 2017; 19:4154–4163 [View Article][PubMed]
    [Google Scholar]
  46. Li X, Roseman S. The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase. Proc Natl Acad Sci USA 2004; 101:627–631 [View Article][PubMed]
    [Google Scholar]
  47. Dalia AB. RpoS is required for natural transformation of Vibrio cholerae through regulation of chitinases. Environ Microbiol 2016; 18:3758–3767 [View Article][PubMed]
    [Google Scholar]
  48. Connell TD, Metzger DJ, Lynch J, Folster JP. Endochitinase is transported to the extracellular milieu by the eps-encoded general secretory pathway of Vibrio cholerae . J Bacteriol 1998; 180:5591–5600 [View Article][PubMed]
    [Google Scholar]
  49. Soysa HSM, Aunkham A, Schulte A, Suginta W. Single-channel properties, sugar specificity, and role of chitoporin in adaptive survival of Vibrio cholerae type strain O1. J Biol Chem 2020; 295:9421–9432 [View Article][PubMed]
    [Google Scholar]
  50. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012; 19:455–477 [View Article][PubMed]
    [Google Scholar]
  51. Page AJ, De Silva N, Hunt M, Quail MA, Parkhill J et al. Robust high-throughput prokaryote de novo assembly and improvement pipeline for Illumina data. Microb Genom 2016; 2:e000083 [View Article][PubMed]
    [Google Scholar]
  52. Seemann T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article][PubMed]
    [Google Scholar]
  53. O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D et al. Reference sequence (RefSeq) database at NCBI: Current status, taxonomic expansion, and functional annotation. Nucleic Acids Res 2016; 44:D733–D745
    [Google Scholar]
  54. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S et al. Roary: Rapid large-scale prokaryote pan genome analysis. Bioinformatics 2015; 31:3691–3693 [View Article][PubMed]
    [Google Scholar]
  55. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009; 25:1972–1973 [View Article][PubMed]
    [Google Scholar]
  56. Page AJ, Taylor B, Delaney AJ, Soares J, Seemann T et al. SNP-sites: Rapid efficient extraction of SNPs from multi-FASTA alignments. Microb Genom 2016; 2:e000056 [View Article][PubMed]
    [Google Scholar]
  57. Nguyen L-. T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 2015; 32:268–274 [View Article][PubMed]
    [Google Scholar]
  58. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410 [View Article][PubMed]
    [Google Scholar]
  59. Jones P, Binns D, Chang H-Y, Fraser M, Li W et al. InterProScan 5: Genome-scale protein function classification. Bioinformatics 2014; 30:1236–1240 [View Article][PubMed]
    [Google Scholar]
  60. Carver TJ, Rutherford KM, Berriman M, Rajandream M-. A, Barrell BG et al. ACT: The Artemis comparison tool. Bioinformatics 2005; 21:3422–3423 [View Article][PubMed]
    [Google Scholar]
  61. Sullivan MJ, Petty NK, Beatson SA. Easyfig: A genome comparison visualizer. Bioinformatics 2011; 27:1009–1010 [View Article][PubMed]
    [Google Scholar]
  62. Harris SR, Feil EJ, Holden MTG, Quail MA, Nickerson EK et al. Evolution of MRSA during hospital transmission and intercontinental spread. Science 2010; 327:469–474 [View Article][PubMed]
    [Google Scholar]
  63. Carver T, Böhme U, Otto TD, Parkhill J, Berriman M. BamView: Viewing mapped read alignment data in the context of the reference sequence. Bioinformatics 2010; 26:676–677 [View Article][PubMed]
    [Google Scholar]
  64. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P et al. Artemis: Sequence visualization and annotation. Bioinformatics 2000; 16:944–945 [View Article][PubMed]
    [Google Scholar]
  65. Dorman MJ, Kane L, Domman D, Turnbull JD, Cormie C et al. The history, genome and biology of NCTC 30: A non-pandemic Vibrio cholerae isolate from World War One. Proc R Soc B: Biol Sci 2019; 286:20182025
    [Google Scholar]
  66. R Core Team R: a Language and Environment for Statistical Computing R Foundation for Statistical Computing, Vienna, Austria; 2018
    [Google Scholar]
  67. Wickham H. ggplot2: Elegant Graphics for Data Analysis Springer-Verlag New York; 2016
    [Google Scholar]
  68. Hadfield J, Croucher NJ, Goater RJ, Abudahab K, Aanensen DM et al. Phandango: An interactive viewer for bacterial population genomics. Bioinformatics 2018; 34:292–293 [View Article][PubMed]
    [Google Scholar]
  69. Reddi G, Pruss K, Cottingham KL, Taylor RK, Almagro-Moreno S. Catabolism of mucus components influences motility of Vibrio cholerae in the presence of environmental reservoirs. PLoS ONE 2018; 13:e0201383 [View Article][PubMed]
    [Google Scholar]
  70. Stauder M, Huq A, Pezzati E, Grim CJ, Ramoino P et al. Role of GbpA protein, an important virulence-related colonization factor, for Vibrio cholerae’s survival in the aquatic environment. Environ Microbiol Rep 2012; 4:439–445 [View Article][PubMed]
    [Google Scholar]
  71. Vaitkevicius K, Lindmark B, Ou G, Song T, Toma C et al. A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing. Proc Natl Acad Sci U S A 2006; 103:9280–9285 [View Article][PubMed]
    [Google Scholar]
  72. Sikora AE, Zielke RA, Lawrence DA, Andrews PC, Sandkvist M. Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases. J Biol Chem 2011; 286:16555–16566 [View Article][PubMed]
    [Google Scholar]
  73. Farfán M, Miñana D, Fusté MC, Lorén JG. Genetic relationships between clinical and environmental Vibrio cholerae isolates based on multilocus enzyme electrophoresis. Microbiology 2000; 146:2613–2626 [View Article]
    [Google Scholar]
  74. Farfán M, Miñana-Galbis D, Fusté MC, Lorén JG. Allelic diversity and population structure in Vibrio cholerae O139 Bengal based on nucleotide sequence analysis. J Bacteriol 2002; 184:1304–1313 [View Article][PubMed]
    [Google Scholar]
  75. Keymer DP, Miller MC, Schoolnik GK, Boehm AB. Genomic and phenotypic diversity of coastal Vibrio cholerae strains is linked to environmental factors. Appl Environ Microbiol 2007; 73:3705–3714 [View Article][PubMed]
    [Google Scholar]
  76. Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T et al. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae . Lancet 2003; 361:743–749 [View Article][PubMed]
    [Google Scholar]
  77. Debnath A, Mizuno T, Miyoshi S. Regulation of chitin-dependent growth and natural competence in Vibrio parahaemolyticus . Microorganisms 2020; 8:1303 [View Article]
    [Google Scholar]
  78. Svitil AL, Chadhain S, Moore JA, Kirchman DL. Chitin degradation proteins produced by the marine bacterium Vibrio harveyi growing on different forms of chitin. Appl Environ Microbiol 1997; 63:408–413 [View Article][PubMed]
    [Google Scholar]
  79. Wang Z, Hervey WJ, Kim S, Lin B, Vora GJ. Complete genome sequence of the bioluminescent marine bacterium Vibrio harveyi ATCC 33843 (392 [MAV]. Genome Announc 3:e01493–14 [View Article][PubMed]
    [Google Scholar]
  80. Suginta W, Sirimontree P, Sritho N, Ohnuma T, Fukamizo T. The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions. Int J Biol Macromol 2016; 93:1111–1117 [View Article][PubMed]
    [Google Scholar]
  81. Songsiriritthigul C, Pantoom S, Aguda AH, Robinson RC, Suginta W. Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: Implications for the catalytic mechanism. J Struct Biol 2008; 162:491–499 [View Article][PubMed]
    [Google Scholar]
  82. Vezzulli L, Stauder M, Grande C, Pezzati E, Verheye HM et al. gbpA as a novel qPCR target for the species-specific detection of Vibrio cholerae O1, O139, non-O1/non-O139 in environmental, stool, and historical continuous plankton recorder samples. PLoS ONE 2015; 10:e0123983 [View Article][PubMed]
    [Google Scholar]
  83. Nasreen T, Hussain NAS, Islam MT, Orata FD, Kirchberger PC et al. Simultaneous quantification of Vibrio metoecus and Vibrio cholerae with its O1 serogroup and toxigenic subpopulations in environmental reservoirs. Pathogens 2020; 9:1053 [View Article]
    [Google Scholar]
  84. Chowdhury F, Mather AE, Begum YA, Asaduzzaman M, Baby N et al. Vibrio cholerae serogroup O139: Isolation from cholera patients and asymptomatic household family members in Bangladesh between 2013 and 2014. PLoS Negl Trop Dis 2015; 9:e0004183 [View Article][PubMed]
    [Google Scholar]
  85. Gardner AD, Venkatraman KV. The antigens of the cholera group of Vibrios. J Hyg (Lond) 1935; 35:262–282 [View Article][PubMed]
    [Google Scholar]
  86. Hasan NA, Choi SY, Eppinger M, Clark PW, Chen A et al. Genomic diversity of 2010 Haitian cholera outbreak strains. Proc Natl Acad Sci USA 2012; 109:E2010–E2017
    [Google Scholar]
  87. Aydanian A, Tang L, Chen Y, Morris JG Jr, Olsen P et al. Genetic relatedness of selected clinical and environmental non-O1/O139 Vibrio cholerae . Int J Infect Dis 2015; 37:152–158 [View Article][PubMed]
    [Google Scholar]
  88. Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose Pbad promoter. J Bacteriol 1995; 177:4121–4130 [View Article][PubMed]
    [Google Scholar]
  89. Dorman MJ, Kane L, Domman D, Turnbull JD, Cormie C et al. Table s1 from the history, genome and biology of NCTC 30: A non-pandemic Vibrio cholerae isolate from World War one. Figshare 2019
    [Google Scholar]
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