Abstract

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.

中文翻译：

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霍乱弧菌O1生物型El Tor非毒原性菌株致病岛的基因组变异性

摘要

给出了43株非产毒霍乱弧菌菌株的致病岛（PIs），VPI-1和VPI-2的比较序列分析结果。结果表明，这些菌株在PI基因组组成和结构上互不相同，形成了三个遗传上不同的组（ctxA ^- tcpA ⁺ VPI-2 ⁺，ctxA ^- tcpA ⁺ VPI-2del ⁺和ctxA ^- tcpA ^- VPI- 2del ⁺）的适应电位不同。发现由点突变和不同长度的缺失引起的PI基因组变异性的水平是不同的。存在于非毒素ctxA ^- tcpA ⁺弧菌染色体中的VPI-1结构和调节致病性基因的核苷酸序列与参考产毒菌株的核苷酸序列基本相同。acfB，асfС，tcpА，tcpF和tcpH中只有少数具有单核苷酸取代的多态性位点（5）基因被鉴定。同时，观察到高VPI-2基因组不稳定性，表现为形成不同长度的缺失（31 131–49 986 kb），并且在所有保守基因中均发生了多个单核苷酸取代。首次证明，VPI-2 DNA的丢失与其氨基酸（唾液酸和唾液酸）利用基因位于nan-nag区域（57-274个取代）（包括nanH基因）的出现有关。鉴定了十二个先前未描述的nanH等位基因变体。这些基因的高遗传多样性对不同生态位的弧菌生存至关重要，这似乎是高适应潜力的原因之一。霍乱弧菌O1生物型El TorсtxA ^- tcpA ^- VPI-2del ⁺在水生环境中无处不在。在对VPI-2中包含的nanH基因以及六个看家基因进行序列分析的基础上，进行了聚类分析，确定了不同组的非毒素源菌株之间以及与毒素源菌株之间的系统发生关系。无毒ctxA ^- tcpA ⁺ VPI-2 ⁺和ctxA ^- tcpA ⁺ VPI-2del ⁺的遗传接近性发现霍乱的致病菌弧菌。在本研究中获得的关于非毒素弧菌中PI结构的新数据不仅阐明了具有不同适应潜能的遗传多样菌株的分子机制。具有确定的基因组结构且在基因组编辑时不需要去除关键毒力基因的非产毒菌株可用于创建新一代的活霍乱疫苗。