Contribution of catabolite control protein A to group A streptococcal virulence

描述（由申请人提供）：革兰氏阳性菌感染每年在美国造成数万人死亡。最近发现转录调节因子分解代谢物控制蛋白A（CcpA）影响许多革兰氏阳性病原体（包括A组链球菌（GAS））在标准实验室培养基中生长期间的毒力基因表达谱。CcpA最初在芽孢杆菌属物种中被鉴定，其中它结合称为分解代谢物反应元件（cre）位点的DNA序列，连同其辅因子含组氨酸的在丝氨酸残基46处磷酸化的磷蛋白（HPr-Ser 46-P）。CcpA、HPr和调节HPr磷酸化状态的激酶/磷酸化酶（HPrK/P）的直向同源物存在于所有主要的革兰氏阳性人类病原体中，并且有积累的数据表明CcpA-（HPr-Ser 46-P）-HPrK/P轴有助于不同细菌物种的感染性。该项目的长期目标是明确定义CcpA-（HPr-Ser 46-P）-HPrK/P轴如何影响GAS毒力，并确定CcpA在感染过程中影响基因表达的机制。该提议的具体目的是为了检验由初步数据产生的假设，这些数据表明：1）在小鼠肌肉中感染期间，CcpA的失活改变了GAS基因表达; 2）在小鼠肌炎模型中，CcpA的失活降低了GAS毒力; 3）GAS cre位点的中心CpG的突变降低了CcpA的表达。（HPr-Ser 46-P）DNA结合亲和力;和4）在HPr-Ser 46-P水平低或不存在的条件下，CcpA影响基因表达。在目标1中，将在多种小鼠感染模型中进行野生型和CcpA灭活GAS菌株的基因表达分析，以检验CcpA是GAS感染期间关键毒力因子调节剂的假设。在目的2中，将在各种小鼠感染模型中将源自不同M蛋白血清型亲本菌株的CcpA同基因突变菌株的毒力与其亲本野生型菌株进行比较，以检验CcpA对多种M蛋白血清型的GAS菌株的感染性有积极贡献的假设。在目标3中，将使用体外和体内方法的组合来检验中心cre位点CpG对于CcpA对GAS基因表达的影响是关键的这一假设。在目标4中，将在不同的代谢条件下比较CcpA和HPr-Ser 46-P失活菌株的基因表达谱，以检验CcpA在HPr-Ser 46-P不存在的情况下可以影响GAS基因表达的假设。（HPr-Ser 46-P）-HPrK/P轴在主要革兰氏之间-阳性细菌病原体意味着拟议研究的成功完成可能会对疾病发病机制产生新的见解，这些疾病发病机制与广泛的病原菌有关。这些信息可以成为开发新的治疗或预防干预措施的基础。

DESCRIPTION (provided by applicant): Infections by Gram-positive bacteria cause tens of thousands of deaths in the United States each year. The transcriptional regulator catabolite control protein A (CcpA) has recently been found to influence the virulence gene expression profile during growth in standard laboratory medium of numerous Gram-positive pathogens including group A Streptococcus (GAS). CcpA was originally identified in Bacillus species where it binds DNA sequences termed catabolite response element (cre) sites in conjunction with its co-factor histidine-containing phosphoprotein phosphorylated at serine residue 46 (HPr-Ser46-P). Orthologues of CcpA, HPr, and the kinase/phosphorylase that regulates HPr phosphorlyation status (HPrK/P) are present in all major Gram- positive human pathogens, and there are accumulating data suggesting that the CcpA-(HPr-Ser46-P)-HPrK/P axis contributes to the infectivity of diverse bacterial species. The long term goals of this project are to clearly define how the CcpA-(HPr-Ser46-P)-HPrK/P axis contributes to GAS virulence and to ascertain mechanisms by which CcpA influences gene expression during infection. The specific aims of this proposal have been designed to test hypotheses arising from preliminary data demonstrating that: 1) Inactivation of CcpA altered GAS gene expression during infection in mouse muscle; 2) Inactivation of CcpA decreased GAS virulence in a mouse myositis model; 3) Mutation of the central CpG of a GAS cre site decreased CcpA-(HPr-Ser46-P) DNA binding affinity; and 4) CcpA influenced gene expression under conditions in which HPr-Ser46-P levels are low or absent. In aim 1, gene expression analysis of wild-type and CcpA-inactivated GAS strains will be performed in multiple mouse models of infection to test the hypothesis that CcpA is a key virulence factor regulator during GAS infection. In aim 2, the virulence of CcpA isogenic mutant strains derived from different M protein serotype parental strains will be compared to their parental wild-type strains in various mouse models of infection to test the hypothesis that CcpA positively contributes to the infectivity of GAS strains of multiple M protein serotypes. In aim 3, a combination of in vitro and in vivo methods will be used to test the hypothesis that the central cre site CpG is critical for the effect of CcpA on GAS gene expression. In aim 4, the gene expression profiles of CcpA and HPr-Ser46-P inactivated strains will be compared under different metabolic conditions to test the hypothesis that CcpA can affect GAS gene expression in the absence of HPr-Ser46-P. The highly conserved nature of the CcpA-(HPr-Ser46-P)-HPrK/P axis amongst major Gram-positive bacterial pathogens means that the successful completion of the proposed research may generate new insights into disease pathogenesis that are relevant to a broad array of pathogenic bacteria. Such information could form the basis for the development of novel therapeutic or preventive interventions.