NEW MECHANISMS FOR REGULATING VIRULENCE GENE EXPRESSION

描述（改编自申请人摘要）：毒力基因 在许多病原菌中的表达受环境控制， 在宿主内外都有刺激。 对于人类病原体弧菌 霍乱，霍乱毒素（CT）的协同表达，毒素协同调节 菌毛（TCP），和其他一些毒力因子的强烈影响 受温度、pH值、渗透压和氧气等环境参数影响 张力 为了阐明这种调节的性质，转录 CT操纵子（ctx）启动子与大肠杆菌的融合 将霍乱弧菌染色体中的β-半乳糖苷酶（lacZ）基因用作 筛选以鉴定影响融合蛋白表达的基因， 对各种环境刺激的反应。 使用这个屏幕，两个V。 已经鉴定出了cya和crp这两种胆固醇基因，它们负调节 特定生长条件下的毒力基因表达。 产品 腺苷酸环化酶催化环腺苷酸的合成 （cAMP），最重要和普遍存在的细胞内调节因子之一， 分子。 与CRP产物cAMP受体蛋白（CRP）一起， 系统作为肠道细菌基因表达的全局调节器 通过控制碳和能源的利用， 环境 观察到crp突变体在经典和El Tor生物型霍乱弧菌O 1的CT表达被去抑制， TCP在各种体外生长条件下， 在幼鼠霍乱模型中的发病机制是首次明确的 证明cAMP-CRP在这些过程中发挥作用。 主 建议工作的重点是阐明分子和生理 cAMP-CRP调控V. 胆汁。 这将在具体目标#1中通过确定 调节基因受cAMP-CRP的影响，并通过识别位点 在特异性目标#2中，通过检查CRP结合的关系， 不同生长条件下细胞内cAMP水平之间的关系， CT和TCP的表达。 因为对crp突变体的分析表明 除了cAMP-CRP，其他未知因素也影响 毒力基因表达的环境控制，第三特异性 该提议的目的是再次利用ctx-lacZ融合筛选， 在两种生物型菌株中鉴定这些基因，并阐明它们的 行动机制。 这项工作的长期目标是 最终了解来自环境的刺激是如何传递到 霍乱弧菌中的基因表达水平，以促进 制定新的战略来控制和预防细菌性疾病。

DESCRIPTION (Adapted from the applicant's abstract): Virulence gene expression in many pathogenic bacteria is controlled by environmental stimuli both inside and outside of the host. For the human pathogen Vibrio cholerae, the coordinate expression of cholera toxin (CT), toxin-coregulated pilus (TCP), and a number of other virulence factors is strongly influenced by environmental parameters such as temperature, pH, osmolarity, and oxygen tension. To elucidate the nature of this regulation, a transcriptional fusion between the promoter of the CT operon (ctx) and the Escherichia coli b-galactosidase (lacZ) gene in the V. cholerae chromosome was used as a screen to identify genes that influence the expression of the fusion in response to various environmental stimuli. Using this screen, two V. cholerae genes have been identified, cya and crp, that negatively regulate virulence gene expression under particular growth conditions. The product of the cya gene, adenylate cyclase, catalyzes the synthesis of cyclic AMP (cAMP), one of the most important and ubiquitous intracellular regulatory molecules. Together with the crp product, cAMP-receptor protein (CRP), this system serves as a global regulator of gene expression in enteric bacteria by controlling the utilization of carbon and energy sources in the environment. The observation that crp mutants in both the classical and El Tor biotypes of V. cholerae O1 are derepressed for the expression of CT and TCP under a variety of growth conditions in vitro and are defective for pathogenesis in the infant mouse cholera model is the first clear demonstration that cAMP-CRP plays a role in these processes. The primary focus of the proposed work is to elucidate the molecular and physiological mechanisms by which cAMP-CRP controls the expression of CT and TCP in V. cholerae. This will be accomplished in SPECIFIC AIM #1 by determining which regulatory genes are influenced by cAMP-CRP and by identifying the site(s) to which CRP binds, and in SPECIFIC AIM #2 by examining the relationship between the intracellular levels of cAMP under various growth conditions and the expression of CT and TCP. Since analysis of the crp mutants suggests that in addition to cAMP-CRP, other as yet unknown factors also influence the environmental control of virulence gene expression, the third SPECIFIC AIM of this proposal is to again exploit the ctx-lacZ fusion screen to identify these genes in both biotype strains and to shed light on their mechanisms of action. The long-term objectives of this work are to ultimately understand how stimuli from the environment are transmitted to the level of gene expression in V. cholerae so as to facilitate the development of new strategies to control and prevent bacterial diseases.