Structural and Functional Changes in the Genome of Avirulent El Tor Biovar Vibrio cholerae ctxA + tcpA + Strains

Molecular Genetics, Microbiology and Virology volume 35, pages134–144(2020)Cite this article

Abstract

High genomic variability of cholera pathogen, which is capable of persisting in the human organism and aquatic environments, underlies the diversity of virulent properties of its strains. However, the mechanism of emergence of the El Tor biovar Vibrio cholerae O1 strains with altered virulence remains poorly understood. A total of 21 El Tor biovar V. cholerae O1 strains were studied. Protein electrophoresis, enzyme-linked immunosorbent assay (GM1-ELISA), whole-genome sequencing, genomic analysis, and SNP genotyping were employed to study their properties. The virulence of the strains was assessed via intestinal infection of model animals. This paper reports that the genome of avirulent V. cholerae O1 strains of the El Tor biovar isolated from the aquatic environment contains the CTXφ prophage and VPI-1 pathogenicity island, which contain the ctxAB and tcpA-F genes, respectively, encoding the key pathogenicity factors, namely, the cholera toxin and toxin-coregulated pili. Comparative analysis of the nucleotide sequences of these mobile elements' genomes from two avirulent strains (89 and 147) of the ctxA+tcpA+ genotype and five clinical virulent isolates of the same genotype has not revealed any differences between them. At the same time, a change in the nucleotide sequence of the toxR global regulator gene located in the core region of pathogen’s genome has for the first time been detected in avirulent strains. This was a single nucleotide deletion (T in position 357), which resulted in the formation of a TGA stop codon. The consequence of such mutation is the production of defective transmembrane DNA-binding protein ToxR that positively regulates the toxT main virulence regulator gene expression due to the loss of 176 amino acids. It has been demonstrated that the loss of toxR protein function resulted in a significant decrease of the mRNA levels of the key regulatory (toxR and toxT) and structural (ctxA, ctxB, and tcpA) virulence genes in the studied strains. As a result, the expression of the cholera toxin genes in the mutant strains 89 and 147 was more than 20 times lower compared to the virulent strains. Whole-genome SNP analysis of the strains 89 and 147, as well as of 19 virulent strains including different genetic variants of the pathogen was carried out to infer their phylogenetic relationships. A new mechanism for the virulence change in cholera vibrio strains has been identified. A change in the nucleotide sequence of the toxR global regulator gene has been demonstrated in the avirulent strains of the ctxA+tcpA+ genotype. This mutation resulted in the formation of a defective transmembrane DNA-binding protein ToxR, which caused a dramatic decrease in the production of the toxin required for the development of the cholera disease.

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REFERENCES

  1. 1

    Moskvitina, E.A., Tyuleneva, E.G., Samorodova, A.V., Kruglikov, V.D., Titova, S.V., Ivanova, S.M., et al., Epidemiological situation on Cholera across the globe and in the Russian Federation in 2007-2016. Forecast for 2017, Probl. Osobo Opasnykh Infekts., 2017, no. 1, pp. 13–20. https://doi.org/10.21055/0370-1069-2017-1-13-20

  2. 2

    Mironova, L.V., Current conceptions concerning the objective laws of a Cholera epidemic process: ecological and molecular biological aspects, Epidemiol. Infect. Dis., 2018, vol. 23, no. 5, pp. 242–250. https://doi.org/10.18821/1560-9529-2018-23-5-242-250

    Article  Google Scholar 

  3. 3

    Dziejman, M., Balon, E., Boyd, D., Fraser, C.M., Heidelberg, J.F., and Mekalanos, J.J., Comparative genomic analysis of Vibrio cholerae: genes that correlate with cholera endemic and pandemic disease, Proc. Natl. Acad. Sci. U. S. A., 2002, vol. 99, no. 3, pp. 1556–1561. https://doi.org/10.3410/f.1004686.53905

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4

    Smirnova, N.I., Zadnova, S.P., Agafonov, D.A., Shashkova, A.V., Cheldyshova, N.B., and Cherkasov, A.V., Comparative molecular-genetic analysis of mobile elements in natural strains of Cholera agent, Russ. J. Genet., 2013, vol. 49, no. 9, pp. 898–908. https://doi.org/10.7868/S0016675813090087

    CAS  Article  Google Scholar 

  5. 5

    Karaolis, D.K., Johnson, J.A., Bailey, C.C., Boedeker, E.C., Kaper, J.B., and Reeves, P.R., A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains, Proc. Natl. Acad. Sci. U. S. A., 1998, vol. 95, no. 6, pp. 3134–3139. https://doi.org/10.1073/pnas.95.6.3134

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Waldor, M.K. and Mekalanos, J.J., Lysogenic conversion by a filamentous phage encoding cholera toxin, Science, 1996, vol. 272, no. 5270, pp. 1910–1914. https://doi.org/10.1126/science.272.5270.1910

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Karaolis, D.K., Somara, S., Maneval, D.R., Jr., Johnson, J.A., and Kaper, J.B., A bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria, Nature, 1999, vol. 399, no. 6734, pp. 375–379. https://doi.org/10.1038/20715

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Hu, D., Liu, B., Feng, L., Ding, P., Guo, X., Wang, M., et al., Origins of the current seventh cholera pandemic, Proc. Natl. Acad. Sci. U. S. A., 2016, vol. 113, no. 48, pp. 7730–7739. https://doi.org/10.1073/pnas.1608732113

    CAS  Article  Google Scholar 

  9. 9

    Mutreja, A., Kim, D.W., Thomson, N., Connor, T.R., Lee, J.H., Kariuki, S., et al., Evidence for multiple waves of global transmission within the seventh cholera pandemic, Nature, 2011, vol. 477, no. 7365, pp. 462–465. https://doi.org/10.1038/nature10392

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10

    Smirnova, N.I., Goryaev, A.A., and Kutyrev, V.V., The evolution of the Vibrio cholerae genome during the modern period, Mol. Genet., Microbiol. Virol., 2010, vol. 25, no. 4, pp. 148–157. https://doi.org/10.3103/S0891416810040026

    Article  Google Scholar 

  11. 11

    Nair, G.B., Faruque, S.M., Bhuiyan, N.A., Kamruzzaman, M., Siddique, A.K., and Sack, D.A., New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh, J. Clin. Microbiol., 2002, vol. 40, no. 9, pp. 3296–3299. https://doi.org/10.1128/jcm.40.9.3296-3299.2002

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Satchell, K.J., Jones, C.J., Wong, J., Queen, J., Agarwal, S., and Yildiz, F.H., Phenotypic analysis reveals that the 2010 Haiti cholera epidemic is linked to a hypervirulent strain, Infect. Immun., 2016, vol. 84, no. 9, pp. 2473–2481. https://doi.org/10.1128/iai.00189-16

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Hasan, N.A., Choi, S.Y., Eppinger, M., Clark, P.W., Chen, A., Alam, M., et al., Genomic diversity of 2010 Haitian cholera outbreak strains, Proc. Natl. Acad. Sci. U. S. A., 2012, vol. 109, no. 29, pp. 2010–2017. https://doi.org/10.1073/pnas.1207359109

    Article  Google Scholar 

  14. 14

    Matson, J.S., Withey, J.H. and DiRita, V.J., Regulatory networks controlling Vibrio cholerae virulence gene expression, Infect. Immun., 2007, vol. 75, no. 12, pp. 5542–5549. https://doi.org/10.3389/fmicb.2018.02686

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Cotter, P.A. and DiRita, V.J., Bacterial virulence gene regulation: an evolutionary perspective, Annu. Rev. Microbiol., 2000, vol. 54, pp. 519–565.

    CAS  Article  Google Scholar 

  16. 16

    Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, vol. 227, no. 5259, pp. 680–685. https://doi.org/10.1038/227680a0

    CAS  Article  Google Scholar 

  17. 17

    Holmgren, J. and Svennerholm, A.M., Cholera and the immune response, Prog. Allergy, 1983, vol. 33, pp. 106–119. https://doi.org/10.1007/978-3-540-88452-1_12

    CAS  Article  PubMed  Google Scholar 

  18. 18

    Dutta, N.K. and Habbu, M.K., Experimental cholera in infant rabbits: a method for chemotherapeutic investigation, Br. J. Pharmacol. Chemother., 1955, vol. 10, no. 2, pp. 153–159. https://doi.org/10.1111/j.1476-5381.1955.tb00074.x

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Livak, K.J. and Schmittgen, T.D., Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods, 2001, vol. 25, no. 4, pp. 402–408. https://doi.org/10.1006/meth.2001.1262

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Fykse, E.M., Skogan, G., Davies, W., Olsen, J.S., and Blatny, J.M., Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification, Appl. Environ. Microbiol., 2007, vol. 73, no. 5, pp. 1457–1466. https://doi.org/10.1128/AEM.01635-06

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Smirnova, N.I., Agafonov, D.A., Kul’shan’, T.A., Shchelkanova, E.Yu., Krasnov, Ya.M., Lozovsky, Yu.V., et al., Effect of CTXφ prophage deletion in cholera agent on expression of regulatory genes controlling virulence and biofilm formation, Russ. J. Genet., 2017, vol. 53, no. 3, pp. 302–313. https://doi.org/10.1134/S1022795417020119

    CAS  Article  Google Scholar 

  22. 22

    Cholera Toxin Transcriptional Activator. https://www. uniprot.org/uniprot/P15795.

  23. 23

    Crawford, J.A., Kaper, J.B., and DiRita, V.J., Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae, Mol. Microbiol., 1998, vol. 29, no. 1, pp. 235–246. https://doi.org/10.1046/j.1365-2958.1998.00925.x

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Li, C.C., Crawford, J.A., DiRita, V.J., and Kaper, J.B., Molecular cloning and transcriptional regulation of ompT, a ToxR-repressed gene in Vibrio cholerae, Mol. Microbiol., 2000, vol. 35, no. 1, pp. 189–203. https://doi.org/10.1046/j.1365-2958.2000.01699.x

    Article  PubMed  Google Scholar 

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ACKNOWLEDGMENTS

The authors are grateful to L.F. Livanova, PhD, of the Microbe Russian Research Anti-Plague Institute for her assistance with animal experiments.

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  1. Microbe Russian Research Anti-Plague Institute, 410005, Saratov, Russia

    N. I. Smirnova, D. A. Agafonov, E. Yu. Shchelkanova, D. A. Rybal’chenko, A. A. Kritsky, Zh. V. Al’khova, Ya. M. Krasnov, E. Yu. Agafonova & V. V. Kutyrev

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  1. N. I. Smirnova
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  2. D. A. Agafonov
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  3. E. Yu. Shchelkanova
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  6. Zh. V. Al’khova
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STATEMENT ON THE WELFARE OF ANIMALS

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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The authors declare that they have no conflict of interest.

Additional information

Translated by E. Martynova

Smirnova N.I., https://orcid.org/0000-0002-7115-6286; e-mail: rusrapi@microbe.ru

Agafonov D.A., https://orcid.org/0000-0001-9273-6063; e-mail: rusrapi@microbe.ru

Shchelkanova E.Yu., https://orcid.org/0000-0003-0672-8820; e-mail: rusrapi@microbe.ru

Rybal’chenko D.A., https://orcid.org/0000-0002-3117-8229; e-mail: rusrapi@microbe.ru

Kritsky A.A., https://orcid.org/0000-0002-5506-4285; e-mail: rusrapi@microbe.ru

Al’khova Zh.V., https://orcid.org/0000-0001-7681-5773; e-mail: rusrapi@microbe.ru

Krasnov Ya.M., https://orcid.org/0000-0002-4909-2394; e-mail: rusrapi@microbe.ru

Agafonova E.Yu., https://orcid.org/0000-0001-9988-6312; e-mail: rusrapi@microbe.ru

Kutyrev V.V., https://orcid.org/0000-0003-3788-3452; e-mail: rusrapi@microbe.ru

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Smirnova, N.I., Agafonov, D.A., Shchelkanova, E.Y. et al. Structural and Functional Changes in the Genome of Avirulent El Tor Biovar Vibrio cholerae ctxA + tcpA + Strains. Mol. Genet. Microbiol. Virol. 35, 134–144 (2020). https://doi.org/10.3103/S0891416820030106

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Keywords:

  • cholera pathogen
  • virulence
  • genome variability
  • regulatory and structural genes
  • mutations