Molecular Biology and Virulence of CTX Phage

描述（由申请人提供）：CTXphi是一种编码霍乱毒素的丝状噬菌体。这是导致严重腹泻疾病霍乱的革兰氏阴性菌霍乱弧菌的主要毒力因子。CTXphi是第一个显示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，在溶原状态下，由于CTXphi抑制因子RstR的活性，大多数CTXphi基因不表达。一般来说，CTXphi的整合形式是作为穿插相关遗传元件RS1的前噬菌体DNA串联阵列的一部分。RS1编码一种蛋白RstC，该蛋白可以对抗RstR抑制，并导致CTX前噬菌体基因（包括编码霍乱毒素的基因ctxAB）的表达显著增强。

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.