Molecular Biology and Virulence of CTX Phage

描述（由申请人提供）：CTXphi是编码霍乱毒素的丝状噬菌体。这是霍乱弧菌的主要毒力因子，霍乱弧菌是导致严重的霍乱弧菌病的革兰氏阴性细菌。CTXphi是第一个显示出介导毒力基因水平转移的丝状噬菌体。CTXphi整合到霍乱弧菌染色体中，并且在溶原状态下，由于CTXphi阻遏物RstR的活性，大多数CTXphi基因不表达。一般来说，CTXphi的整合形式被发现是散布有相关遗传元件RS 1的原噬菌体DNA串联阵列的一部分。RS 1编码一种蛋白质RstC，该蛋白质可以对抗RstR抑制并导致CTX原噬菌体基因（包括ctxAB，编码霍乱毒素的基因）的表达显著增强。 这项工作的长期目标是了解CTXphi生命周期中的分子事件以及这种噬菌体在霍乱发病机制中的作用。拟议的研究将探索噬菌体生命周期的3个核心过程：i）噬菌体DNA位点特异性整合到细菌染色体中; ii）整合后大多数噬菌体基因表达的抑制; iii）环境和遗传刺激激活噬菌体基因表达和病毒体产生。目的1中鉴定介导CTXphi DNA整合到霍乱弧菌染色体中的机制和因子的实验将揭示染色体编码的重组酶XerC和XerD如何与噬菌体和染色体序列相互作用以实现CTXphi整合。这些研究将阐明噬菌体整合的新机制，并可能阐明ctxAB扩增的机制。目标2中的实验：表征RstR的调节和作用模式将阐明CTXphi如何维持在静止状态。rstR自动调节和RstR水平的环境因素的调制将进行探讨。RstR的绑定到其不寻常的运营商也将进行研究。目的3中确定RstC作用模式的实验将探索RstC如何抑制RstR介导的阻遏。将研究RstC结合RstR和/或RstR结合位点的能力，并测量感染期间rstC的表达。所有这些研究将产生对噬菌体生物学基本方面的见解。此外，它们可能揭示噬菌体基因表达或拷贝数的变化可能有助于霍乱弧菌致病性的方式。

DESCRIPTION (provided by applicant): CTXphi is a filamentous bacteriophage that encodes cholera toxin. This is the principal virulence factor of Vibrio cholerae, the Gram-negative bacterium that causes the severe diarrheal disease cholera. CTXphi is the first filamentous bacteriophage shown to mediate the horizontal transfer of a virulence gene. CTXphi integrates into the Vibrio cholerae chromosome and, in the lysogenic state, most CTXphi genes are not expressed due to the activity of the CTXphi repressor, RstR. Generally, the integrated form of CTXphi is found as part of tandem arrays of prophage DNA interspersed with the related genetic element RS1. RS1 encodes a protein, RstC, that can counter RstR repression and lead to markedly enhanced expression of CTX prophage genes including ctxAB, the genes encoding cholera toxin. The long-term goal of this work is to understand the molecular events in the life cycle of CTXphi and the role that this phage plays in the pathogenesis of cholera. The proposed studies will explore 3 processes central to the phage life cycle: i) the site-specific integration of phage DNA into the bacterial chromosome; ii) the repression of most phage gene expression following integration; and iii) the activation of phage gene expression and virion production by environmental and genetic stimuli. Experiments in Aim 1 to identify the mechanism and factors that mediate the integration of CTXphi DNA into the V. cholerae chromosome will reveal how the chromosome encoded recombinases XerC and XerD interact with phage and chromosome sequences to accomplish CTXphi integration. These studies will elucidate a novel mechanism of phage integration and may shed light on the mechanism of ctxAB amplification as well. Experiments in Aim 2: to characterize the regulation and mode of action of RstR will clarify how CTXphi can be maintained in a quiescent state. rstR autoregulation and modulation of RstR levels by environmental factors will be explored. RstR's binding to its unusual operators will also be studied. Experiments in Aim 3 to determine the mode of action of RstC-will explore how RstC can inactivate RstR-mediated repression. RstC's ability to bind to either RstR and/or RstR's binding sites will be investigated and the expression of rstC during infection will be measured. All of these studies will yield insights into fundamental aspects of phage biology. In addition, they may reveal ways in which changes in phage gene expression or copy number can contribute to the pathogenicity of V. cholerae.