OBJECTIVE

Genome variability of the cholera agent that is able to persist in human organism and aquatic environment is at the bottom of alteration of its virulent properties. However the mechanism of emergence of V. cholerae O1 strains, biovar EL TOR with altered virulence remains under-researched.

MATERIAL AND METHODS

A total of 21 V. cholerae O1 biovar EL TOR strains were examined. In order to study their properties, protein electrophoresis, immunoenzime analysis (GM₁-ELISA), whole-genome sequencing, genomic analysis, and SNP typing have been used. The virulence of the strains has been rated by means the intestinal infection of the model animals.

RESULTS

We have found out that the genome of isolated from aquatic environment avirulent v. Cholerae o1 biovar EL TOR strains contains CTXφ prophage and pathogenicity island VPI-1, that include ctxAB and tcpA-F genes, respectively, encoding key factors of pathogenicity — cholera toxin and toxin-coregulated pilus. Comparative analysis of the nucleotide sequences of these mobile element genomes in two avirulent strains (89 and 147) with genotype ctxA⁺tcpA⁺ and five clinical virulent isolates with the same genotype has not revealed any differences between them. However, changes in the nucleotide sequence of global regulator gene toxR, situated in the core region of the genome, have been first-time detected in avirulent strains. It is a deletion of a single nucleotide (T in the position 357), which led to formation of terminating codon TGA. Consequence of such a mutation — genesis of defective trans-membrane DNA — binding protein ToxR, performing positive control of the main virulence regulator gene toxT expression, owing to the loss of 176 amino acids. It is demonstrated that the loss of toxR protein function has resulted in significant decrease of mRNA level of the key regulatory (ToxR, toxT) and structural (ctxA, ctxB, tcpA) virulence genes in studied strains. Consequently, expression of cholera toxin genes in mutant strains 89 and 147 was more than 20 times reduced as compared to virulent strains. We have also conducted SNP-analysis of whole-genome sequences of the strains 89 and 147, as well as 19 virulent strains, including various genovariants of the agent and showed their phylogenetic relations.

CONCLUSION

Thus new mechanism of virulence alteration in cholera vibrio strains has been identified. In avirulent strains with genotype ctxA⁺tcpA⁺, changes in the nucleotide sequence of global regulator gene toxR have been detected. This mutation has resulted in the formation of a defective trans-membrane DNA — binding protein toxR, which has caused a dramatic decline in the production of the toxin necessary to cause infection with cholera.