1887

Abstract

is a common cause of nosocomial diarrhoea. Toxins TcdA and TcdB are considered to be the main virulence factors and are encoded by the PaLoc region, while the binary toxin encoded in the CdtLoc region also contributes to pathogenicity. Variant toxinotypes reflect the genetic diversity of a key toxin-encoding 19 kb genetic element (the PaLoc). Here, we present analysis of a comprehensive collection of all known major toxinotypes to address the evolutionary relationships of the toxin gene variants, the mechanisms underlying the origin and development of variability in toxin genes and the PaLoc, and the relationship between structure and function in TcdB variants. The structure of both toxin genes is modular, composed of interspersed blocks of sequences corresponding to functional domains and having different evolutionary histories, as shown by the distribution of mutations along the toxin genes and by incongruences of domain phylogenies compared to overall cluster organization. In TcdB protein, four mutation patterns could be differentiated, which correlated very well with the type of TcdB cytopathic effect (CPE) on cultured cells. Mapping these mutations to the three-dimensional structure of the TcdB showed that the majority of the variation occurs in surface residues and that point mutation at residue 449 in alpha helix 16 differentiated strains with different types of CPE. In contrast to the PaLoc, phylogenetic trees of the CdtLoc were more consistent with the core genome phylogenies, but there were clues that CdtLoc can also be exchanged between strains.

Funding
This study was supported by the:
  • Federation of European Microbiological Societies (Award FEMS Research fellowship)
    • Principle Award Recipient: Sandra Janezic
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2020-10-08
2024-03-29
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References

  1. Leffler DA, Lamont JT. Clostridium difficile infection. N Engl J Med 2015; 373:1539–1548 [View Article][PubMed]
    [Google Scholar]
  2. Smits WK, Lyras D, Lacy DB, Wilcox MH, Kuijper EJ. Clostridium difficile infection. Nat Rev Dis Primers 2016; 2:16020 [View Article]
    [Google Scholar]
  3. Chumbler NM, Rutherford SA, Zhang Z, Farrow MA, Lisher JP et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol 2016; 1:15002 [View Article]
    [Google Scholar]
  4. Pruitt RN, Lacy DB. Toward a structural understanding of Clostridium difficile toxins A and B. Front Cell Infect Microbiol 2012; 2:28 [View Article][PubMed]
    [Google Scholar]
  5. Gerding DN, Johnson S, Rupnik M, Aktories K. Clostridium difficile binary toxin CDT: mechanism, epidemiology, and potential clinical importance. Gut Microbes 2014; 5:15–27 [View Article][PubMed]
    [Google Scholar]
  6. Kuehne SA, Collery MM, Kelly ML, Cartman ST, Cockayne A et al. Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain. J Infect Dis 2014; 209:83–86 [View Article][PubMed]
    [Google Scholar]
  7. Braun V, Hundsberger T, Leukel P, Sauerborn M, von Eichel-Streiber C. Definition of the single integration site of the pathogenicity locus in Clostridium difficile . Gene 1996; 181:29–38 [View Article][PubMed]
    [Google Scholar]
  8. Martin-Verstraete I, Peltier J, Dupuy B. The regulatory networks that control Clostridium difficile toxin synthesis. Toxins 2016; 8:153 [View Article][PubMed]
    [Google Scholar]
  9. Carter GP, Lyras D, Allen DL, Mackin KE, Howarth PM et al. Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator. J Bacteriol 2007; 189:7290–7301 [View Article][PubMed]
    [Google Scholar]
  10. Stare BG, Delmée M, Rupnik M. Variant forms of the binary toxin CDT locus and tcdC gene in Clostridium difficile strains. J Med Microbiol 2007; 56:329–335 [View Article][PubMed]
    [Google Scholar]
  11. Rupnik M. Heterogeneity of large clostridial toxins: importance of Clostridium difficile toxinotypes. FEMS Microbiol Rev 2008; 32:541–555 [View Article][PubMed]
    [Google Scholar]
  12. Rupnik M, Avesani V, Janc M, von Eichel-Streiber C, Delmée M. A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 1998; 36:2240–2247 [View Article][PubMed]
    [Google Scholar]
  13. Rupnik M, Janezic S. An update on Clostridium difficile toxinotyping. J Clin Microbiol 2016; 54:13–18 [View Article][PubMed]
    [Google Scholar]
  14. Cairns MD, Preston MD, Hall CL, Gerding DN, Hawkey PM et al. Comparative genome analysis and global phylogeny of the toxin variant Clostridium difficile PCR ribotype 017 reveals the evolution of two independent sublineages. J Clin Microbiol 2017; 55:865–876 [View Article][PubMed]
    [Google Scholar]
  15. He M, Miyajima F, Roberts P, Ellison L, Pickard DJ et al. Emergence and global spread of epidemic healthcare-associated Clostridium difficile . Nat Genet 2013; 45:109–113 [View Article][PubMed]
    [Google Scholar]
  16. Knight DR, Squire MM, Collins DA, Riley TV. Genome analysis of Clostridium difficile PCR Ribotype 014 lineage in australian pigs and humans reveals a diverse genetic repertoire and signatures of long-range interspecies transmission. Front Microbiol 2016; 7:2138 [View Article][PubMed]
    [Google Scholar]
  17. Knight DR, Riley TV. Genomic delineation of zoonotic origins of Clostridium difficile . Front Public Health 2019; 7:164 [View Article][PubMed]
    [Google Scholar]
  18. Collins J, Robinson C, Danhof H, Knetsch CW, Leeuwen HC van et al. Dietary trehalose enhances virulence of epidemic Clostridium difficile . Nature 2018; 553:291–294 [View Article][PubMed]
    [Google Scholar]
  19. Dingle KE, Didelot X, Quan TP, Eyre DW, Stoesser N et al. A Role for tetracycline selection in recent evolution of agriculture-assocor tetracycline selection in recent evolution of agriculture-associated Clostridium difficile PCR Ribotype 078. MBio 2019; 10:e02790-18 [View Article][PubMed]
    [Google Scholar]
  20. Ramírez-Vargas G, López-Ureña D, Badilla A, Orozco-Aguilar J, Murillo T et al. Novel clade C-I Clostridium difficile strains escape diagnostic tests, differ in pathogenicity potential and carry toxins on extrachromosomal elements. Sci Rep 2018; 8:13951 [View Article][PubMed]
    [Google Scholar]
  21. Dingle KE, Elliott B, Robinson E, Griffiths D, Eyre DW et al. Evolutionary history of the Clostridium difficile pathogenicity locus. Genome Biol Evol 2014; 6:36–52 [View Article][PubMed]
    [Google Scholar]
  22. Elliott B, Dingle KE, Didelot X, Crook DW, Riley TV. The complexity and diversity of the pathogenicity locus in Clostridium difficile clade 5. Genome Biol Evol 2014; 6:evu2483170 [View Article][PubMed]
    [Google Scholar]
  23. Monot M, Eckert C, Lemire A, Hamiot A, Dubois T et al. Clostridium difficile: new insights into the evolution of the pathogenicity locus. Sci Rep 2015; 5:15023 [View Article][PubMed]
    [Google Scholar]
  24. Dingle KE, Griffiths D, Didelot X, Evans J, Vaughan A et al. Clinical Clostridium difficile: clonality and pathogenicity locus diversity. PLoS One 2011; 6:e19993 [View Article][PubMed]
    [Google Scholar]
  25. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008; 18:821–829 [View Article][PubMed]
    [Google Scholar]
  26. Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N et al. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 2006; 38:779–786 [View Article][PubMed]
    [Google Scholar]
  27. Stabler RA, He M, Dawson L, Martin M, Valiente E et al. Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol 2009; 10:R102 [View Article][PubMed]
    [Google Scholar]
  28. Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res 2018; 3:124 [View Article][PubMed]
    [Google Scholar]
  29. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal omega. Mol Syst Biol 2011; 7:539 [View Article][PubMed]
    [Google Scholar]
  30. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. mega6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013; 30:2725–2729 [View Article][PubMed]
    [Google Scholar]
  31. Bruen TC, Philippe H, Bryant D. A simple and robust statistical test for detecting the presence of recombination. Genetics 2006; 172:2665–2681 [View Article][PubMed]
    [Google Scholar]
  32. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 2014; 42:W252–W258 [View Article][PubMed]
    [Google Scholar]
  33. Chandrasekaran R, Lacy DB. The role of toxins in Clostridium difficile infection. FEMS Microbiol Rev 2017; 41:723–750 [View Article][PubMed]
    [Google Scholar]
  34. Reinert DJ, Jank T, Aktories K, Schulz GE. Structural basis for the function of Clostridium difficile toxin B. J Mol Biol 2005; 351:973–981 [View Article][PubMed]
    [Google Scholar]
  35. Janezic S, Marín M, Martín A, Rupnik M. A new type of toxin A-negative, toxin B-positive Clostridium difficile strain lacking a complete tcdA gene. J Clin Microbiol 2015; 53:JCM.02211–.02214 [View Article][PubMed]
    [Google Scholar]
  36. Wren BW. A family of clostridial and streptococcal ligand-binding proteins with conserved C-terminal repeat sequences. Mol Microbiol 1991; 5:797–803 [View Article][PubMed]
    [Google Scholar]
  37. Zeiser J, Gerhard R, Just I, Pich A. Substrate specificity of clostridial glucosylating toxins and their function on colonocytes analyzed by proteomics techniques. J Proteome Res 2013; 12:1604–1618 [View Article][PubMed]
    [Google Scholar]
  38. Chaves-Olarte E, Löw P, Freer E, Norlin T, Weidmann M et al. A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins. J Biol Chem 1999; 274:11046–11052 [View Article][PubMed]
    [Google Scholar]
  39. Mehlig M, Moos M, Braun V, Kalt B, Mahony DE et al. Variant toxin B and a functional toxin A produced by Clostridium difficile C34. FEMS Microbiol Lett 2001; 198:171–176 [View Article][PubMed]
    [Google Scholar]
  40. Soehn F, Wagenknecht-Wiesner A, Leukel P, Kohl M, Weidmann M et al. Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864--implications for transcription, expression and enzymatic activity of toxins A and B. Mol Gen Genet 1998; 258:222–232 [View Article][PubMed]
    [Google Scholar]
  41. Brouwer MSM, Roberts AP, Hussain H, Williams RJ, Allan E et al. Horizontal gene transfer converts non-toxigenic Clostridium difficile strains into toxin producers. Nat Commun 2013; 4:2601 [View Article][PubMed]
    [Google Scholar]
  42. Janezic S, Indra A, Rattei T, Weinmaier T, Rupnik M. Recombination drives evolution of the Clostridium difficile 16S-23S rRNA intergenic spacer region. PLoS One 2014; 9:e106545 [View Article][PubMed]
    [Google Scholar]
  43. Stubbs S, Rupnik M, Gibert M, Brazier J, Duerden B et al. Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile . FEMS Microbiol Lett 2000; 186:307–312 [View Article][PubMed]
    [Google Scholar]
  44. Metcalf DS, Weese JS. Binary toxin locus analysis in Clostridium difficile . J Med Microbiol 2011; 60:1137–1145 [View Article][PubMed]
    [Google Scholar]
  45. Riedel T, Wittmann J, Bunk B, Schober I, Spröer C et al. A Clostridioides difficile bacteriophage genome encodes functional binary toxin-associated genes. J Biotechnol 2017; 250:23–28 [View Article][PubMed]
    [Google Scholar]
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