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.

中文翻译：

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无毒El Tor Biovar霍乱弧菌ctxA + tcpA +菌株基因组的结构和功能变化

摘要

霍乱病原体的高基因组变异性能够在人类有机体和水生环境中持续存在，这是其菌株毒性特性多样性的基础。然而，对毒力改变的埃尔托生物变种霍乱弧菌O1菌株的出现机理仍知之甚少。共研究了21株El Tor biovar霍乱弧菌O1菌株。使用蛋白质电泳，酶联免疫吸附测定（GM ₁ -ELISA），全基因组测序，基因组分析和SNP基因分型来研究其特性。通过模型动物的肠道感染评估菌株的毒力。本文报道了无毒霍乱弧菌的基因组从水生环境中分离出的El Tor生物变种的O1菌株包含CTXφ噬菌体和VPI-1致病岛，它们分别包含ctxAB和tcpA-F基因，它们编码关键的致病因子，即霍乱毒素和以毒素为核心的毒素霹雳 对来自ctxA ⁺ tcpA ⁺基因型的两个无毒株（89和147）和相同基因型的五个临床有毒分离株的这些活动元件基因组的核苷酸序列进行比较分析，没有发现它们之间的任何差异。同时，toxR核苷酸序列的变化首次在无毒菌株中检测到位于病原体基因组核心区域的全球调节基因。这是单核苷酸缺失（位置357处的T），其导致TGA终止密码子的形成。这种突变的结果是产生有缺陷的跨膜DNA结合蛋白ToxR，由于缺少176个氨基酸，该蛋白正调控toxT主要毒力调节剂基因的表达。已经证明，toxR蛋白功能的丧失会导致关键调节因子（toxR和toxT）和结构（ctxA，ctxB和tcpA）的mRNA水平显着下降。）菌株中的毒力基因。结果，突变株89和147中霍乱毒素基因的表达比有毒株低20倍以上。对菌株89和147，以及包括病原体不同遗传变异的19种强毒菌株进行了全基因组SNP分析，以推断它们的系统发生关系。已经确定了霍乱弧菌菌株毒力变化的新机制。已经在无毒的ctxA ⁺ tcpA ⁺菌株中证实了toxR全球调节基因的核苷酸序列发生了变化。基因型。这种突变导致有缺陷的跨膜DNA结合蛋白ToxR的形成，这导致霍乱病发展所需的毒素产生急剧减少。