Genomic Variability of Pathogenicity Islands in Nontoxigenic Strains of Vibrio cholerae O1 Biotype El Tor

The results of comparative sequence analysis of pathogenicity islands (PIs), VPI-1 and VPI-2, in 43 nontoxigenic Vibrio cholerae strains are presented. It was demonstrated that these strains, differing from each other in the PI genome composition and structure, formed three genetically distinct groups (ctxA−tcpA+VPI-2+, ctxA−tcpA+VPI-2del+, and ctxA−tcpA−VPI-2del+) that differed in adaptive potential. It was found that the level of PI genomic variability caused by point mutations and deletions of different lengths was different. Nucleotide sequences of the VPI-1 structural and regulatory pathogenicity genes present in the chromosome of nontoxigenic ctxA−tcpA+ vibrios were mostly identical to that of the reference toxigenic strain. Only a small number of polymorphic sites (5) with single nucleotide substitutions in the acfB, асfС, tcpА, tcpF, and tcpH genes were identified. At the same time, high VPI-2 genomic instability, manifested in the formation of deletions of different lengths (31 131–49 986 kb) and the occurrence of multiple single nucleotide substitutions in all conserved genes, was observed. It was first demonstrated that the loss of VPI-2 DNA correlated with the appearance of multiple substitutions in its genes of utilization of amino sugars and sialic acids, located in the nan-nag region (57–274 substitutions), including the nanH gene. Twelve previously undescribed nanH allelic variants were identified. It seems likely that high genetic diversity of these genes that are important for the vibrio survival in different ecological niches is one of the reasons for high adaptive potential of V. cholerae O1 biotype El Tor сtxA−tcpA−VPI-2del+, ubiquitous in an aquatic environment. On the basis of sequence analysis of the nanH gene included in VPI-2, as well as six housekeeping genes, cluster analysis that determined the phylogenetic relationships of different groups of nontoxigenic strains with each other and with toxigenic strains was performed. The genetic closeness of nontoxigenic ctxA−tcpA+VPI-2+ and ctxA−tcpA+VPI-2del+ vibrios to the causative agent of cholera was discovered. The novel data obtained in the present study on the PI structure in nontoxigenic vibrios not only shed light on the molecular mechanisms of genetically diverse strains with different adaptive potentials. Nontoxigenic strains with the determined genome structure that do not require the removal of key virulence genes upon genome editing can be useful in creating a new generation of live cholera vaccines.