The irvA-dependent pathway: a link between stress adaptation and virulence

描述(申请人提供)：这项拟议的研究旨在研究变形链球菌通过IRVA依赖途径进行基因调控的遗传和生化机制。本项目的目标将通过三个目标来实现：1)分析IRVA诱导的机制；2)鉴定IRVA调节子；3)表征组成IRVA依赖途径的遗传调控因子。1)为了分析IRVA的诱导机制，我们将对SMu1275的关键残基进行详细的生化研究，以确定其内源性自切割能力如何调节其调控IRVA表达的能力。2)我们将利用染色质免疫沉淀和微阵列(芯片)来鉴定IRVA调节子。使用专门设计的Affymetrix Genechips(R)，我们将能够在体内检测大多数或所有IRVA结合位点。对这些基因的转录分析将确定Irva作为与胁迫和毒力相关表型相关基因的转录调节因子的作用。3)为了表征IRVA依赖途径的基因调控，我们首先要确定能够诱导IRVA的基因。然后，遗传分析将检查IRVA作为压力和毒力相关表型的中介因子在这些菌株中的作用。最后，将确定与IRVA依赖途径共同调节的基因。本项目的目标是以IRVA依赖的途径为模型，研究压力适应与变形链球菌重要毒力因子调控之间的联系。由于变形链球菌的长期存在需要胁迫反应机制和毒力因子基因表达的协调，这些数据可能会对变形链球菌的致龋性产生新的见解。

DESCRIPTION (provided by applicant): The proposed research aims to investigate the genetic and biochemical mechanisms of gene regulation in Streptococcus mutans through the irvA-dependent pathway. The goals of this project will be achieved in three aims by: 1) Analyzing the mechanism of irvA induction 2) Identifying the IrvA regulon 3) Characterizing the genetic regulators that comprise the irvA-dependent pathway. 1) To analyze the mechanism of irvA induction, we will perform detailed biochemical studies of key residues of SMu1275 to determine how its endogenous autocleavage capacity can modulate its ability to control irvA expression. 2) To identify the IrvA regulon, we will employ chromatin immunoprecipitation and microarray (ChIP-chip). Using specially designed Affymetrix Genechips(R), we will be able to detect most or all of the IrvA binding sites in vivo. Transcription analysis of these genes will determine the role of IrvA as a transcription regulator of genes associated with stress and virulence related phenotypes. 3) To characterize the genetic regulators of the irvA-dependent pathway, we will first identify the genes that are capable of inducing irvA. Genetic analyses will then examine the role of irvA as a mediator of stress and virulence related phenotypes in these strains. Finally, genes coregulated with the irvA-dependent pathway will be identified. The goal of this project is to use the irvA-dependent pathway as a model to examine the connection between stress adaptation and the regulation of important virulence factors of Streptococcus mutans. Since the long-term persistence of S. mutans requires the coordination of stress response mechanisms and virulence factor gene expression, these data may yield new insights into S. mutans cariogenicity.