Assessing the Structure of Regulatory Protein Genes in Strains of Genovariants of Vibrio cholerae O1 Biovar El Tor, Isolated in Different Years of the Seventh Cholera Pandemic

The aim of the work was to conduct a comparative analysis of the structure of genes encoding regulatory proteins in strains of Vibrio cholerae O1 biovar El Tor genovariants isolated in cholera endemic and non-endemic territories in different years of the seventh cholera pandemic, and their phylogenetic analysis. Materials and methods. Nucleotide sequences of complete genomes of 75 toxigenic genovariants of V. cholerae O1 El Tor with ctxB1 and ctxB7 alleles listed in NCBI GenBank, ENA, VGARus were used. Bioinformatics analysis was performed using the Blast algorithm (http://blast.ncbi), MEGA X. Phylogenetic analysis was carried out using the REALPHY server. Results and discussion. The structure of 13 loci (toxT, aphA, aphB, luxO, luxT, gntR, hapR, lysR_vc2383, lysR_vc1617, hns, vieA, carR, carS) was studied. Stable preservation of the aphB and carS genes in the genome of all strains has been established, and toxT, aphA, and luxT have been found in 99 % of them. All genovariants have an altered gntR gene (G565A) encoding a protein that represses gluconate fermentation. The majority of genovariants have an intact hapR gene, unlike the reference strain N16961, which has a thymine deletion at the position 219 in this gene. Some foreign strains include additional mutations in this gene. High variability of the luxO gene has been revealed. Mutations in other regulatory genes have been piling up gradually and stably persisted. During phylogenetic analysis, all the studied strains isolated in different endemic countries and imported to the Russian Federation and neighboring states fell under four clusters in accordance with the structure of regulatory genes. Thus, highly virulent genovariants currently isolated in the endemic territory and imported to the Russian Federation have the genotype hapR (insT219), lysR_vc2383 (C110T), hns (G319A), lysR_vc1617 (A650C), vieA (C235T), carR (G265A).

1. Popova A.Yu., Noskov A.K., Ezhlova E.B., Kruglikov V.D., Monakhova E.V., Chemisova O.S., Lopatin A.A., Ivanova S.M., Podoynitsyna O.A., Vodop’yanov A.S., Levchenko D.A., Savina I.V. [Epidemiological situation on cholera in the Russian Federation in 2023 and forecast for 2024]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2024; (1):76–88. DOI: 10.21055/0370-1069-2024-1-76-88.

2. Kruglikov V.D., Gaevskaya N.E., Monakhova E.V., Moskvitina E.A., Agafonova V.V., Savina I.V., Podoynitsyna O.A., Selyanskaya N.A., Vodop’yanov A.S., Duvanova O.V., Men’shikova E.A., Ezhova M.I., Shipko E.S., Evteev A.V., Kaz’mina V.S., Bodraya P.V., Sokirkina E.N. [Analysis of peculiarities of epidemiological situation on cholera in the World and in the Russian Federation in 2024 and forecast of its development in 2025]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2025; (1):35–47. DOI: 10.21055/0370-1069-2025-1-35-47.

3. Nair G.B., Faruque S.M., Bhuiyan N.A., Kamruzzaman M., Siddique A.K., 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; 40(9):3296–9. DOI: 10.1128/JCM.40.9.3296-3299.2002.

4. Son M.S., Megli C.J., Kovacikova G., Qadri F., Taylor R.K. Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes. J. Clin. Microbiol. 2011; 49:3739–49. DOI: 10.1128/JCM.01286-11.

5. Alam M., Islam A., Bhuiyan N.A., Rahim N., Hossain A., Khan G.Y., Ahmed D., Watanabe H., Izumiya H., Faruque A.S., Akanda A.S., Islam S., Sack R.B., Huq A., Colwell R.R., Cravioto A. Clonal transmission, dual peak, and off-season cholera in Bangladesh. Infect. Ecol. Epidemiol. 2011; 1:7273. DOI: 10.3402/iee.v1i0.7273.

6. Satchell K.J., Jones C.J., Wong J., Queen J., Agarwal S., Yildiz F.H. Phenotypic analysis reveals that the 2010 Haiti cholera epidemic is linked to a hypervirulent strain. Infect. Immun. 2016; 84(9):2473–81. DOI: 10.1128/IAI.00189-16.

7. Russell R., Wang H., Benitez J.A., Silva A.J. Deletion of gene encoding the nucleoid-associated protein H-NS unmasks hidden regulatory connections in El Tor biotype Vibrio cholerae. Microbiology. 2018; 164(7):998–1003. DOI: 10.1099/mic.0.000672.

8. Matson J.S., Withey J.H., DiRita V.J. Regulatory networks controlling Vibrio cholerae virulence gene expression. Infect. Immun. 2007; 75(12):5542–9. DOI: 10.1128/IAI.01094-07.

9. Faruque S.M., Nair G.B., editors. Vibrio cholerae: Genomics and Molecular Biology. Norfolk: Caister Academic Press; 2008. 218 p.

10. Ramamurthy T., Nandy R.K., Mukhopadhyay A.K., Dutta S., Mutreja A., Okamoto K., Miyoshi S.-I., Nair G.B., Ghosh A. Virulence regulation and innate host response in the pathogenicity of Vibrio cholerae. Front. Cell. Infect. Microbiol. 2020; 10:572096. DOI: 10.3389/fcimb.2020.572096.

11. Carignan B.M., Brumfield K.D., Son M.S. Single nucleotide polymorphisms in regulator-encoding genes have an additive effect on virulence gene expression in a Vibrio cholerae clinical isolate. mSphere. 2016; 1(5):e00253-16. DOI: 10.1128/mSphere.00253-16.

12. Childers B.M., Weber G.G., Prouty M.G., Castaneda M.M., Peng F., Klose K.E. Identification of residues critical for the function of the Vibrio cholerae virulence regulator ToxT by scanning alanine mutagenesis. J. Mol. Biol. 2007; 367(5):1413–30. DOI: 10.1016/j.jmb.2007.01.061.

13. Kovacikova G., Lin W., Skorupski K. Dual regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive LysR-type regulator AlsR in Vibrio cholerae. Mol. Microbiol. 2005; 57(2):420–33. DOI: 10.1111/j.1365-2958.2005.04700.x.

14. Yang M., Frey E.M., Liu Z., Bishar R., Zhu J. The virulence transcriptional activator AphA enhances biofilm formation by Vibrio cholerae by activating the expression of the biofilm regulator VpsT. Infect. Immun. 2010; 78(2):697–703. DOI: 10.1128/IAI.00429-09.

15. Kovacikova G., Lin W., Skorupski K. The LysR-type virulence activator AphB regulates the expression of genes in Vibrio cholerae in response to low pH and anaerobiosis. J. Bacteriol. 2010; 192(16):4181–91. DOI: 10.1128/JB.00193-10.

16. Li Y., Yan J., Li J., Xue X., Wang Y., Cao B. A novel quorum sensing regulator LuxT contributes to the virulence of Vibrio cholerae. Virulence. 2023; 14(1):2274640. DOI: 10.1080/21505594.2023.2274640.

17. Kovacikova G., Skorupski K. Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter. Mol. Microbiol. 2002; 46(4):1135–47. DOI: 10.1046/j.1365-2958.2002.03229.x.

18. Joelsson A., Liu Z., Zhu J. Genetic and phenotypic diversity of quorum-sensing systems in clinical and environmental isolates of Vibrio cholerae. Infect. Immun. 2006; 74(2):1141–7. DOI: 10.1128/IAI.74.2.1141-1147.2006.

19. Wang H., Ayala J.C., Benitez J.A., Silva A.J. RNA-seq analysis identifies new genes regulated by the histone-like nucleoid structuring protein (H-NS) affecting Vibrio cholerae virulence, stress response and chemotaxis. PLoS One. 2015; 10(2):e0118295. DOI: 10.1371/journal.pone.0118295.

20. Rakibora Y., Dunham D.T., Seed K.D., Freddolino L. Nucleoid-associated proteins shape the global protein occupancy and transcriptional landscape of a clinical isolate of Vibrio cholerae. mSphere. 2024; 9(7):e0001124. DOI: 10.1128/msphere.00011-24.

21. Liu Y., Xu T., Wang Q., Huang J., Zhu Y., Liu X., Liu R., Yang B., Zhou K. Vibrio cholerae senses human enteric α-defensin 5 through a CarSR two-component system to promote bacterial pathogenicity. Commun. Biol. 2022; 5(1):559. DOI: 10.1038/s42003-02203525-3.

22. Plekhanov N.A., Fedorov A.V., Cheldyshova N.B., Kuratashvili A.Yu., Zadnova S.P. [Comparative analysis of the structure of regulatory genes of Vibrio cholerae serotype О1 biotype El Tor strains]. Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii [Journal of Microbiology, Epidemiology and Immunobiology]. 2024; 101(4):520–9. DOI: 10.36233/0372-9311-527.