Phase variation of virulence factors in Clostridium difficile

项目总结 艰难梭菌是一种主要的公共卫生威胁，可引起从轻度腹泻到 伪膜性结肠炎。艰难梭菌的症状在很大程度上是由分泌的细胞毒素介导的， TcdA和Tcdb。这种细菌致病性的许多方面仍然知之甚少，包括如何 艰难梭菌调节毒素和其他毒力因子的产生。毒素基因的表达与 鞭毛基因通过鞭毛sigma因子sigma-D表达，该因子编码在flgB操纵子中。因此， 调节flgB操纵子表达的因素除了鞭毛外，还会影响毒素的产生 能动性。我们的长期目标是确定艰难梭菌如何协调调节鞭毛和毒素基因 表达，因为这些机制可能是发病的基础。我们最近展示了鞭毛 基因表达受控于通过可逆性DNA位点特异性DNA重组而发生的阶段变化 FlgB操纵子上游的序列。值得注意的是，DNA序列的倒置，我们称之为“鞭毛” 开关“，也影响毒力所必需的毒素的产生。我们确认了Recv就是DNA 介导鞭毛开关倒置的重组酶，我们已经开始通过以下方式定义其机制 鞭毛开关序列的方向控制着基因的表达。我们的中心假设是 鞭毛和毒素产生的阶段变化是艰难梭菌致病的关键，因此 干扰艰难梭菌的相变能力将减弱毒力的一个或多个方面。而当 鞭毛和/或运动性对感染的某些方面可能很重要，鞭毛的下调可能是至关重要的。 艰难梭菌逃避宿主免疫识别。毒素的产生同样可能有益于或有害于 艰难梭菌在感染的不同阶段。这项提议的目标是定义分子 阶段变化的潜在机制，并评估阶段变化对寄主定植和 致命性。这项提议是创新的，因为它代表了一个全新的调查领域 了解艰难梭菌疾病：鞭毛和毒素的时相变化表达。完成拟议的 有关艰难梭菌如何控制毒力因子的研究将提供急需的机械信息。 在感染过程中产生，并可能暴露出抑制肠道定植和毒素的潜在靶点 生产以促进抗击这一问题日益严重的病原体的努力。

PROJECT SUMMARY Clostridium difficile is a major public health threat, causing disease ranging from mild diarrhea to pseudomembranous colitis. C. difficile disease symptoms are largely mediated by the secreted cytotoxins, TcdA and TcdB. Many aspects of the pathogenicity of this bacterium remains poorly understood, including how C. difficile regulates the production of the toxins and other virulence factors. Toxin gene expression is linked to expression of flagellar genes via the flagellar sigma factor sigma-D, which is encoded in the flgB operon. Thus, factors that regulate the expression of the flgB operon will impact toxin production in addition to flagellar motility. Our long-term goal is to determine how C. difficile coordinately regulates flagellum and toxin gene expression, as these mechanisms are likely fundamental to pathogenesis. We recently showed that flagellar gene expression is subject to phase variation through site-specific DNA recombination of an invertible sequence upstream of the flgB operon. Notably, inversion of the DNA sequence, which we term the “flagellar switch”, also impacts the production of toxins essential for virulence. We identified RecV as the DNA recombinase that mediates inversion of the flagellar switch, and we have begun to define the mechanism by which the orientation of the flagellar switch sequence controls gene expression. Our central hypothesis is that phase variation of flagellum and toxin production is critical to C. difficile pathogenesis, and consequently interfering with the ability of C. difficile to phase vary will attenuate one or more aspects of virulence. While flagella and/or motility may be important for some aspects of infection, downregulation of flagella is likely vital for C. difficile to evade host immune recognition. Toxin production may similarly be beneficial or detrimental to C. difficile during different stages of infection. The objective of this proposal is to define the molecular mechanisms underlying phase variation and to evaluate the impact of phase variation on host colonization and virulence. This proposal is innovative because it represents an entirely new area of investigation toward understanding C. difficile disease: phase variable expression of flagella and toxins. Completion of the proposed studies will provide much needed mechanistic information on how C. difficile controls virulence factor production during infection, and may expose potential targets for inhibition of intestinal colonization and toxin production to facilitate efforts to combat this increasingly problematic pathogen.