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

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.