NEW MECHANISMS FOR REGULATING VIRULENCE GENE EXPRESSION

描述(改编自申请人的摘要)：毒力基因 许多致病菌的表达受环境的控制 宿主内部和外部的刺激。对于人类病原体弧菌 霍乱毒素(CT)的协同表达、毒素协同调控 Pilus(Tcp)和其他一些毒力因子受到强烈影响。 通过环境参数，如温度、pH、渗透压和氧气 紧张。为了阐明这一规定的性质，一份转录 CT操纵子启动子(CTX)与大肠杆菌的融合 以霍乱弧菌染色体上的B-半乳糖苷酶(LacZ)基因为研究对象。 筛选影响融合基因表达的基因 对各种环境刺激的反应。使用此屏幕，两个V。 霍乱弧菌的cyA和crp基因已被鉴定为负性调节基因。 毒力基因在特定生长条件下的表达。该产品 在CyA基因中，腺苷环化酶催化合成环状AMP (CAMP)，最重要和最普遍的细胞内调节因子之一 分子。与CRP产物cAMP受体蛋白(CRP)一起，这 系统是肠道细菌基因表达的全球调节器 通过控制碳和能源的利用， 环境。C反应蛋白在经典细胞和EL细胞中的突变观察 霍乱弧菌O1株Tor生物型的CT和 TcP在各种体外生长条件下均有缺陷 幼年小鼠霍乱发病机制首次明确 证明cAMP-CRP在这些过程中发挥作用。初级阶段 建议的工作重点是阐明分子和生理上的 CAMP-CRP调控沙门氏菌CT和TcP表达的机制 霍乱弧菌。这将在特定的AIM#1中通过确定 调节基因受cAMP-C反应蛋白和识别位点的影响(S) 在特定的AIM#2中，通过检查该关系 不同生长条件下细胞内cAMP水平与 CT和Tcp的表达。因为对CRP突变体的分析表明 除了cAMP-CRP外，其他未知因素也会影响 毒力基因表达的环境调控，第三个特异性 这项提议的目的是再次利用CTX-LacZ融合屏幕来 在两个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 霍乱弧菌的基因表达水平，以促进霍乱弧菌 制定控制和预防细菌性疾病的新战略。

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.