Delineation of the molecular mechanisms underlying group A Streptococcus virulenc

描述（由申请人提供）：人类细菌病原体A组链球菌（GAS）可引起多种疾病，包括咽炎、脓疱病和坏死性筋膜炎。GAS引起这种疾病多样性的能力部分是由于毒力因子的特定子集的协调表达。小调控RNA（sRNA）代表了GAS和相关病原体中的一个知之甚少的调控领域。拟议研究的目标是表征毒力调节GAS sRNA FASX的作用机制，作为识别新型抗菌剂操纵的新靶点的手段。这项研究是感兴趣的传染病社区作为以下观察结果的结果。首先，初步数据表明FASX调节GAS毒力。其次，FASX的生长期依赖性转录与GAS在感染期之间的转换中的作用一致。第三，FASX或任何GAS sRNA调节表达的机制尚不清楚。第四，虽然在编码RNA结合蛋白Hfq的同源物的病原体中已经充分研究了sRNA介导的调节，但在缺乏Hfq同源物的病原体（例如链球菌属、肠球菌属和分枝杆菌属的病原体）中知之甚少。我们将通过测试以下假设来实现我们的目标：（i）FASX是GAS毒力因子的主要调节因子。为了确定呼吸的FASX介导的调节在气体中，我们将使用二维液相色谱质谱分析比较蛋白质组的临床气体分离与其同基因fasX突变衍生物。蛋白质组将在体外和离体进行比较，离体条件是在人血浆（侵入性感染模型）和人唾液（咽部感染模型）中生长。(ii)FASX结合mRNA和/或蛋白质以调节毒力因子的产生。我们已经融合了链霉素结合RNA适体FASX，使杂交RNA被保留在链霉素亲和矩阵。我们将通过下拉分析分离与我们的FASX杂交体相互作用的GAS mRNA和/或蛋白质。FASX结合mRNA和/或蛋白的身份将分别通过使用定制微阵列或通过质谱分析来确定，并使用体外结合测定来确认。(iii)FASX内的特定核苷酸是活性所必需的。为了便于研究FASX调控靶点和作用机制，我们将对fasX进行定点突变。基于突变体fasX等位基因恢复链激酶对fasX突变株2221 FASX的活性的能力，FASX核苷酸将被评分为在活性中没有作用、具有中等作用或具有主要作用。将对候选FASX：mRNA相互作用进行生物信息学分析，以突出互补碱基配对区域。通过将预测与FASX杂交的那些mRNA区域与lacZ报告基因融合，并在存在和不存在FASX的情况下测量2-gal活性，在体内测试推定的碱基配对。 公共卫生相关性：美国每年约有3000万例GAS咽炎病例。拟议的研究将为GAS和相关病原体的毒力调控研究领域提供分子见解。通过将FASX调控途径的知识转化为基于新型抗菌剂抑制这些途径的新治疗和/或预防方案的长期目标，可以增强公共卫生。

DESCRIPTION (provided by applicant): The human bacterial pathogen group A Streptococcus (GAS) causes a broad spectrum of diseases, including pharyngitis, impetigo, and necrotizing fasciitis. The ability of GAS to cause such disease diversity is in part due to the coordinated expression of specific subsets of virulence factors. Small regulatory RNAs (sRNAs) represent a poorly understood area of regulation in GAS and related pathogens. The goal of the proposed research is to characterize the mechanism of action of the virulence-regulating GAS sRNA FASX as a means to identify new targets for manipulation by novel antimicrobial agents. This research is of interest to the infectious diseases community as a consequence of the following observations. First, preliminary data indicates that FASX regulates GAS virulence. Second, the growth-phase-dependent transcription of FASX is consistent with a role in the transition of GAS between phases of infection. Third, the mechanism by which FASX, or indeed any GAS sRNA, regulates expression is unknown. Fourth, while sRNA-mediated regulation has been well-studied in pathogens that encode a homologue of the RNA-binding protein Hfq, little is known in those that lack an Hfq homologue (e.g. pathogens of the genera Streptococcus, Enterococcus, and Mycobacterium). We will achieve our goal by testing the following hypotheses: (i) FASX is a major regulator of GAS virulence factors. To identify the breath of FASX-mediated regulation in GAS we will use two-dimensional liquid chromatography mass spectrometry analysis to compare the proteomes of a clinical GAS isolate with its isogenic fasX mutant derivative. Proteomes will be compared in vitro and ex vivo, with ex vivo conditions being growth in human plasma (an invasive infection model) and human saliva (a pharyngeal infection model). (ii) FASX binds mRNAs and/or proteins to regulate virulence factor production. We have fused a streptomycin-binding RNA aptamer to FASX that enables the hybrid RNA to be retained within a streptomycin affinity matrix. We will isolate GAS mRNAs and/or proteins that interact with our FASX hybrid by performing pull-down assays. The identity of FASX-binding mRNAs and/or proteins will be determined through use of a custom microarray or by mass spec analysis, respectively, and confirmed using in vitro binding assays. (iii) Specific nucleotides within FASX are required for activity. To facilitate investigation of FASX regulatory targets and mechanism/s of action we will perform site-directed mutagenesis on fasX. FASX nucleotides will be scored as having no role, a moderate role, or a major role in activity based upon the ability of mutant fasX alleles to restore streptokinase activity to fasX mutant strain 2221FASX. Candidate FASX:mRNA interactions will be analyzed bioinformatically to highlight regions of complementary base-pairing. Putative base-pairing will be tested in vivo by fusing those mRNA regions predicted to hybridize with FASX to a lacZ reporter gene, and measuring 2-gal activity in the presence and absence of FASX. PUBLIC HEALTH RELEVANCE: Each year in the U.S. there are ~30 million cases of GAS pharyngitis. The proposed research would provide molecular insight into an understudied field of virulence regulation in GAS and related pathogens. Public health may be enhanced through the long-term goal of translating knowledge of FASX regulatory pathways into new treatment and/or preventative regimes based upon the inhibition of these pathways by novel antimicrobial agents.