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Deletion of toxin–antitoxin systems in the evolution of Shigella sonnei as a host-adapted pathogen

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Pathogenic Shigella spp. are the leading cause of bacterial dysentery, with Shigella flexneri and Shigella sonnei accounting for around 90% of cases worldwide. Although S. flexneri causes most disease in low-income countries (following ingestion of contaminated food and/or water), S. sonnei predominates in wealthy countries and is mainly spread from person to person. Although both species contain a large virulence plasmid, pINV, that is essential for the organism to cause disease, little is known about its maintenance. Here, using a counterselectable marker within the virulence-encoding region of pINV, we show that the S. sonnei plasmid is less stable than that of S. flexneri, especially at environmental temperatures. GmvAT, a toxin–antitoxin system, is responsible for the difference in stability and is present in pINV from S. flexneri but absent in S. sonnei pINV. GmvT is an acetyltransferase toxin that inhibits protein translation. Loss of GmvAT and a second toxin–antitoxin system, CcdAB, from pINV reduces S. sonnei plasmid stability outside the host, reflecting the host-adapted lifestyle and person-to-person transmission of this species and differences in its epidemiology.

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Figure 1: Loss of virulence in S. sonnei is temperature-dependent and higher than in S. flexneri.
Figure 2: S. flexneri pINV has three functional TA systems.
Figure 3: S. flexneri pINV TA systems have temperature-dependent effects on plasmid maintenance.
Figure 4: GmvT blocks translation in an acetyl-CoA-dependent manner.
Figure 5: Absence of GmvAT and CcdAB in S. sonnei leads to increased pINV loss.
Figure 6: Insertion of CcdAB and GmvAT into wild-type S. sonnei stabilizes virulence at environmental temperatures.

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  • 14 July 2017

    In the PDF version of this article previously published, the year of publication provided in the footer of each page and in the 'How to cite' section was erroneously given as 2017, it should have been 2016. This error has now been corrected. The HTML version of the article was not affected.


  1. 1

    Johnson, T. J. & Nolan, L. K. Pathogenomics of the virulence plasmids of Escherichia coli. Microbiol. Mol. Biol. Rev. 73, 750–774 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Rychlik, I., Gregorova, D. & Hradecka, H. Distribution and function of plasmids in Salmonella enterica. Vet. Microbiol. 112, 1–10 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Sansonetti, P. J. Genetic and molecular basis of epithelial cell invasion by Shigella species. Rev. Infect. Dis. 13(Suppl 4), S285–S292 (1991).

    CAS  Article  Google Scholar 

  4. 4

    Cornelis, G. R. et al. The virulence plasmid of Yersinia, an antihost genome. Microbiol. Mol. Biol. Rev. 62, 1315–1352 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Pupo, G. M., Lan, R. & Reeves, P. R. Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics. Proc. Natl Acad. Sci. USA 97, 10567–10572 (2000).

    CAS  Article  Google Scholar 

  6. 6

    Buchrieser, C. et al. The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri. Mol. Microbiol. 38, 760–771 (2000).

    CAS  Article  Google Scholar 

  7. 7

    Venkatesan, M. M. et al. Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri. Infect. Immun. 69, 3271–3285 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Nhieu, G. T. & Sansonetti, P. J. Mechanism of Shigella entry into epithelial cells. Curr. Opin. Microbiol. 2, 51–55 (1999).

    CAS  Article  Google Scholar 

  9. 9

    Parsot, C. Shigella spp. and enteroinvasive Escherichia coli pathogenicity factors. FEMS Microbiol. Lett. 252, 11–18 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Sasakawa, C. et al. Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. Infect. Immun. 51, 470–475 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Kotloff, K. L. et al. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull. World Health Organ. 77, 651–666 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Thompson, C. N., Duy, P. T. & Baker, S. The rising dominance of Shigella sonnei: an intercontinental shift in the etiology of bacillary dysentery. PLoS Negl. Trop. Dis. 9, e0003708 (2015).

    Article  Google Scholar 

  13. 13

    Livio, S. et al. Shigella isolates from the global enteric multicenter study inform vaccine development. Clin. Infect. Dis 59, 933–941 (2014).

    CAS  Article  Google Scholar 

  14. 14

    DuPont, H. L., Levine, M. M., Hornick, R. B. & Formal, S. B. Inoculum size in shigellosis and implications for expected mode of transmission. J. Infect. Dis. 159, 1126–1128 (1989).

    CAS  Article  Google Scholar 

  15. 15

    Baker, K. S. et al. Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: a cross-sectional study. Lancet Infect. Dis. 15, 913–921 (2015).

    Article  Google Scholar 

  16. 16

    Genobile, D. et al. An outbreak of shigellosis in a child care centre. Commun. Dis. Intell. 28, 225–229 (2004).

    Google Scholar 

  17. 17

    Simms, I. et al. Intensified shigellosis epidemic associated with sexual transmission in men who have sex with men—Shigella flexneri and S. sonnei in England, 2004 to end of February 2015. Euro Surveill. 20, 21097 (2015).

  18. 18

    Mahoney, F. J., Farley, T. A., Burbank, D. F., Leslie, N. H. & McFarland, L. M. Evaluation of an intervention program for the control of an outbreak of shigellosis among institutionalized persons. J. Infect. Dis. 168, 1177–1180 (1993).

    CAS  Article  Google Scholar 

  19. 19

    Cohen, D. et al. Recent trends in the epidemiology of shigellosis in Israel. Epidemiol. Infect. 142, 2583–2594 (2014).

    CAS  Article  Google Scholar 

  20. 20

    Kotloff, K. L. et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, GEMS): a prospective, case–control study. Lancet 382, 209–222 (2013).

    Article  Google Scholar 

  21. 21

    Vinh, H. et al. A changing picture of shigellosis in southern Vietnam: shifting species dominance, antimicrobial susceptibility and clinical presentation. BMC Infect. Dis. 9, 204 (2009).

    Article  Google Scholar 

  22. 22

    Lima, I. F., Havt, A. & Lima, A. A. Update on molecular epidemiology of Shigella infection. Curr. Opin. Gastroenterol. 31, 30–37 (2015).

    Article  Google Scholar 

  23. 23

    Sayeed, S., Reaves, L., Radnedge, L. & Austin, S. The stability region of the large virulence plasmid of Shigella flexneri encodes an efficient postsegregational killing system. J. Bacteriol. 182, 2416–2421 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Winther, K. S. & Gerdes, K. Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA. Proc. Natl Acad Sci. USA 108, 7403–7407 (2011).

    CAS  Article  Google Scholar 

  25. 25

    Winther, K. S. & Gerdes, K. Regulation of enteric vapBC transcription: induction by VapC toxin dimer-breaking. Nucleic Acids Res. 40, 4347–4357 (2012).

    CAS  Article  Google Scholar 

  26. 26

    Van Melderen, L. et al. ATP-dependent degradation of CcdA by Lon protease. Effects of secondary structure and heterologous subunit interactions. J. Biol. Chem. 271, 27730–27738 (1996).

    CAS  Article  Google Scholar 

  27. 27

    Sayeed, S., Brendler, T., Davis, M., Reaves, L. & Austin, S. Surprising dependence on postsegregational killing of host cells for maintenance of the large virulence plasmid of Shigella flexneri. J. Bacteriol. 187, 2768–2773 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Payne, S. M. & Finkelstein, R. A. Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Infect. Immun. 18, 94–98 (1977).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Kopecko, D. J., Washington, O. & Formal, S. B. Genetic and physical evidence for plasmid control of shigella sonnei form I cell surface antigen. Infect. Immun. 29, 207–214 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Shepherd, J. G., Wang, L. & Reeves, P. R. Comparison of O-antigen gene clusters of Escherichia coli (Shigella) Sonnei and Plesiomonas shigelloides O17: Sonnei gained its current plasmid-borne O-antigen genes from P. shigelloides in a recent event. Infect. Immun. 68, 6056–6061 (2000).

    CAS  Article  Google Scholar 

  31. 31

    Sansonetti, P. J., Kopecko, D. J. & Formal, S. B. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect. Immun. 35, 852–860 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Schuch, R. & Maurelli, A. T. Virulence plasmid instability in Shigella flexneri 2a is induced by virulence gene expression. Infect. Immun. 65, 3686–3692 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Holt, K. E. et al. Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe. Nat. Genet. 44, 1056–1059 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Angelini, M., Stehling, E. G., Moretti, M. L. & da Silveira, W. D. Molecular epidemiology of Shigella spp strains isolated in two different metropolitam areas of southeast Brazil. Braz. J. Microbiol. 40, 685–692 (2009).

    Article  Google Scholar 

  35. 35

    Blocker, A. et al. Structure and composition of the shigella flexneri ‘needle complex’, a part of its type III secreton. Mol. Microbiol. 39, 652–663 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Bernard, P. & Couturier, M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA–topoisomerase II complexes. J. Mol. Biol. 226, 735–745 (1992).

    CAS  Article  Google Scholar 

  37. 37

    Chan, W. T., Espinosa, M. & Yeo, C. C. Keeping the wolves at Bay: antitoxins of prokaryotic type II toxin–antitoxin systems. Front. Mol. Biosci. 3, 9 (2016).

    Article  Google Scholar 

  38. 38

    Zychlinsky, A., Prevost, M. C. & Sansonetti, P. J. Shigella flexneri induces apoptosis in infected macrophages. Nature 358, 167–169 (1992).

    CAS  Article  Google Scholar 

  39. 39

    Bahassi, E. M., Salmon, M. A., Van Melderen, L., Bernard, P. & Couturier, M. F plasmid CcdB killer protein: ccdB gene mutants coding for non-cytotoxic proteins which retain their regulatory functions. Mol. Microbiol. 15, 1031–1037 (1995).

    CAS  Article  Google Scholar 

  40. 40

    Lu, L., Berkey, K. A. & Casero, R. A. Jr . RGFGIGS is an amino acid sequence required for acetyl coenzyme A binding and activity of human spermidine/spermine N1acetyltransferase. J. Biol. Chem. 271, 18920–18924 (1996).

    CAS  Article  Google Scholar 

  41. 41

    Vetting, M. W. et al. Structure and functions of the GNAT superfamily of acetyltransferases. Arch. Biochem. Biophys. 433, 212–226 (2005).

    CAS  Article  Google Scholar 

  42. 42

    Christensen, S. K. et al. Overproduction of the lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM–yoeB toxin–antitoxin system. Mol. Microbiol. 51, 1705–1717 (2004).

    CAS  Article  Google Scholar 

  43. 43

    Christensen, S. K., Mikkelsen, M., Pedersen, K. & Gerdes, K. Rele, a global inhibitor of translation, is activated during nutritional stress. Proc. Natl Acad. Sci. USA 98, 14328–14333 (2001).

    CAS  Article  Google Scholar 

  44. 44

    Oaks, E. V., Wingfield, M. E. & Formal, S. B. Plaque formation by virulent Shigella flexneri. Infect. Immun. 48, 124–129 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Connor, T. R. et al. Species-wide whole genome sequencing reveals historical global spread and recent local persistence in Shigella flexneri. eLife 4, e07335 (2015).

    Article  Google Scholar 

  46. 46

    Lan, R. & Reeves, P. R. Escherichia coli in disguise: molecular origins of Shigella. Microbes Infect. Instit. Pasteur 4, 1125–1132 (2002).

    CAS  Article  Google Scholar 

  47. 47

    Caboni, M. et al. An O antigen capsule modulates bacterial pathogenesis in Shigella sonnei. PLoS Pathogens 11, e1004749 (2015).

    Article  Google Scholar 

  48. 48

    Cheverton, A. M. et al. A Salmonella toxin promotes persister formation through acetylation of tRNA. Mol. Cell 63, 86–96 (2016).

    CAS  Article  Google Scholar 

  49. 49

    Falconi, M., Colonna, B., Prosseda, G., Micheli, G. & Gualerzi, C. O. Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. EMBO J. 17, 7033–7043 (1998).

    CAS  Article  Google Scholar 

  50. 50

    Marteyn, B. et al. Modulation of Shigella virulence in response to available oxygen in vivo. Nature 465, 355–358 (2010).

    CAS  Article  Google Scholar 

  51. 51

    Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    CAS  Article  Google Scholar 

  52. 52

    Cherepanov, P. P. & Wackernagel, W. Gene disruption in Escherichia coli: tcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14 (1995).

    CAS  Article  Google Scholar 

  53. 53

    Blomfield, I. C., Vaughn, V., Rest, R. F. & Eisenstein, B. I. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol. Microbiol. 5, 1447–1457 (1991).

    CAS  Article  Google Scholar 

  54. 54

    Ramadoss, N. S., Zhou, X. & Keiler, K. C. tmRNA is essential in Shigella flexneri. PLoS ONE 8, e57537 (2013).

    CAS  Article  Google Scholar 

  55. 55

    Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol 177, 4121–4130 (1995).

    CAS  Article  Google Scholar 

  56. 56

    Alikhan, N. F., Petty, N. K., Ben Zakour, N. L. & Beatson, S. A. BLAST ring image generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12, 402 (2011).

    CAS  Article  Google Scholar 

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The authors thank I. Blomfield for the gift of P1vir bacteriophage and pIB279 (sacB-neoR), W. Dias da Silveira for S. sonnei strain CS20 and K. Baker and N. Thomson for assistance with genomic analyses. The authors acknowledge the Kirkhouse Trust (Scottish charity no. 030508) for donation of laboratory equipment. This work was funded by Stopenterics EU grant no. 261472 and a Wellcome Trust Senior Investigator award to C.M.T.

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G.M. performed experiments and analysed data. G.M. and C.M.T. designed experiments, interpreted data and wrote the manuscript. C.M.T. secured funding.

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Correspondence to Christoph M. Tang.

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The authors declare no competing financial interests.

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McVicker, G., Tang, C. Deletion of toxin–antitoxin systems in the evolution of Shigella sonnei as a host-adapted pathogen. Nat Microbiol 2, 16204 (2017).

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