New Mechanisms for Regulating Virulence Gene Expression

描述(申请人提供)：霍乱弧菌导致致命的流行性腹泻疾病霍乱。其主要毒力因子毒素共调节的菌毛和霍乱毒素的表达通过涉及几个激活蛋白的转录级联发生，并作为调节细菌毒力的范例。ToxT是一种AraC型调节因子，它直接激活主要毒力因子的启动子。ToxT的表达需要两对同源的跨膜激活剂ToxRS和TcpPH的协同作用。TcpPH表达的激活通过两个调节蛋白APHA和AphB之间的独特相互作用启动了级联反应。APHA是一个新的翼状螺旋转录调控家族的成员，而AphB是一种LysR型激活剂。这些不同启动子的转录激活只发生在对某些环境刺激的反应中。这项建议的长期目标是了解这一调控的分子基础，以便促进制定更好的策略来控制霍乱弧菌的传染性。要实现这些目标，需要了解调控蛋白在其同源启动子上控制基因表达的分子机制，以及最终它们如何受到环境刺激的影响。其中一个这样的刺激，细胞密度，通过群体感应调节因子HapR影响级联反应，后者抑制APHA启动子的表达。目的1旨在阐明HapR抑制APHA表达的机制。证据表明，这一过程涉及拮抗两种不同的激活剂的功能，LRP是亮氨酸反应调节蛋白，VpsR是生物膜调节因子。目的2最近发现AphB在霍乱弧菌的耐酸反应中发挥作用，并调控CADC和其他一些可能参与该反应的基因，从而揭示了pH影响级联蛋白表达的分子机制。阐明AphB与耐酸性调节的关系将为环境刺激如何影响蛋白质的活性提供新的见解。目的研究APHA和AphB在霍乱弧菌中启动毒力基因表达的独特协同机制。这项拟议的工作将促进未来的努力，以确定干扰这些调节器功能的新分子，并可能作为新的抗病毒药物。

DESCRIPTION (provided by applicant): Vibrio cholerae causes the fatal epidemic diarrheal disease cholera. The expression of its primary virulence factors, toxin-coregulated pilus and cholera toxin, occurs via a transcriptional cascade involving several activator proteins and serves as a paradigm for the regulation of bacterial virulence. ToxT, an AraC-type regulator, directly activates the promoters of the primary virulence factors. The expression of toxT requires cooperation between two homologous pairs of transmembrane activators, ToxRS and TcpPH. Activation of tcpPH expression initiates the cascade by a unique interaction between two regulatory proteins AphA and AphB. AphA is a member of a new winged-helix transcriptional regulator family and AphB is a LysR-type activator. Transcriptional activation at these various promoter occurs only in response to certain environmental stimuli. The long term goals of this proposal are to understand the molecular basis of this regulation so as to facilitate the development of better strategies to control the infectivity of V. cholerae. Achieving these goals requires an understanding of the molecular mechanisms by which the regulatory proteins function at their cognate promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. One such stimulus, cell density, influences the cascade through the quorum sensing regulator HapR which represses the expression of the aphA promoter. Aim 1 focuses on elucidating the mechanism by which HapR represses aphA expression. Evidence indicates this process involves antagonizing the function of two distinct activators, Lrp, the leucine responsive regulatory protein, and VpsR, the biofilm regulator. Aim 2 sheds new light on the molecular mechanisms by which pH influences the expression of the cascade by the recent discovery that AphB plays a role in the acid tolerance response in V. cholerae and regulates cadC and a number of other genes potentially involved in this response. Elucidating the relationship between AphB and the regulation of acid tolerance will provide new insights into how the activity of the protein is influenced by environmental stimuli. Aim 3 focuses on investigating the unique cooperative mechanism between AphA and AphB that initiates virulence gene expression in V. cholerae. This proposed work will facilitate future efforts to identify new molecules that interfere with the functions of these regulators and which may serve as novel antivirulence drugs.