New Mechanisms for Regulating Virulence Gene Expression

说明(由申请人提供)：阐明 病原菌调节毒力基因表达以响应 环境刺激相当重要，因为这一知识是核心 为了理解导致建立 细菌感染。霍乱弧菌是疫情的病原体 霍乱，并具有疾病所需的两个截然不同的致病岛： 弧菌致病岛(VPI)，它编码一种基本的定植 因子、毒素共调节的菌毛(TCP)和携带 霍乱毒素基因(CT)。我们最近发现了两个新的激活剂， APHA和AphB，其作用似乎是最初的监管步骤 在毒力转录级联反应中。APHA和AphB函数 协同激活膜结合蛋白的表达 转录激活物，TcPP和TcpH，编码在VPI上。APHA 似乎是一种新的激活剂，没有已知的同系物，AphB是 LysR家族。有趣的是，这两种蛋白质都没有编码 VPI或CTX岛，但位于染色体非 以前与发病机制有关。由于激活了tcpPH表达 APHA和AphB仅在特定的环境条件下发生，这些 蛋白质可能在正常的细胞生理和偶联功能中发挥作用 对毒力基因表达的生理反应。APHA的重要性 而AphB在发病机制中的作用反映在婴儿的显著衰减上 这两种基因中任何一种都有缺陷的突变小鼠霍乱模型。 此外，APHA和AphB对tcpPH的激活是导致 引起这两种疾病的毒力基因的差异调控 生物型，古典的和埃尔托尔的。拟议的研究将使我们能够获得 更好地了解APHA和AphB在发病机制中的作用。我们建议： (1)探讨APHA和AphB差异激活的机制 经典启动子和El Tor tcpPH启动子的表达；(2)测定 CAMP-CRP拮抗APHA和AphB负性损伤的机制 调控tcpPH的表达；(3)阐明TcpPH的分子机制。 环境刺激影响tcpPH启动子的表达；4) 确定AphB介导的tcpPH差异激活的重要性 在体内的致病机制中表达。这些研究将加强我们的 了解病原菌利用的复杂机制 调节毒力基因的表达以响应环境刺激 可以制定更好的策略来控制和预防细菌感染。

DESCRIPTION (provided by the applicant): Elucidating the mechanisms by which pathogenic bacteria regulate virulence gene expression in response to environmental stimuli is considerably important since this knowledge is central to understanding the molecular events that lead to the establishment of bacterial infections. Vibrio cholerae is the causative agent of epidemic cholera and possesses two distinct pathogenicity islands required for disease: the Vibrio pathogenicity island (VPI), which encodes an essential colonization factor, toxin-coregulated pilus (TCP), and the CTX element that carries the genes for cholera toxin (CT). We have recently identified two new activators, AphA and AphB, which function at what appears to be the initial regulatory step in the virulence transcriptional cascade. AphA and AphB function synergistically to activate the expression of the membrane bound transcriptional activators, TcpP and TcpH, which are encoded on the VPI. AphA appears to be a novel activator with no known homologs and AphB is a member of the LysR family. Interestingly, these two proteins are not encoded on either the VPI or CTX islands but are located in regions of the chromosome not previously associated with pathogenesis. Since activation of tcpPH expression by AphA and AphB occurs only under certain environmental conditions, these proteins may play a role in normal cellular physiology and function to couple physiological responses to virulence gene expression. The importance of AphA and AphB in pathogenesis is reflected by the dramatic attenuation in the infant mouse cholera model of mutants defective in either one of these genes. Furthermore, activation of tcpPH by AphA and AphB is responsible for the differential regulation of virulence genes between the two disease causing biotypes, classical and El Tor. The proposed research will allow us to gain a better understanding of the roles of AphA and AphB in pathogenesis. We propose: (1) to investigate the mechanism by which AphA and AphB differentially activate the expression of the classical and El Tor tcpPH promoters; (2) to determine the mechanism by which cAMP-CRP antagonizes AphA and AphB to negatively regulate tcpPH expression; (3) to elucidate the molecular mechanisms by which environmental stimuli influence the expression of the tcpPH promoter; and 4) to determine the importance of AphB-mediated differential activation of tcpPH expression for pathogenesis in vivo. These studies will enhance our understanding of the complex mechanisms utilized by pathogenic bacteria to regulate virulence gene expression in response to environmental stimuli so that better strategies can be developed to control and prevent bacterial infections.