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）引起广泛的疾病，包括咽炎，脓疱疮和坏死性筋膜炎。GAS引起这种疾病多样性的能力部分是由于特定毒力因子亚群的协调表达。小调控rna （sRNAs）在GAS和相关病原体的调控中是一个鲜为人知的领域。该研究的目的是表征毒力调节的GAS sRNA FASX的作用机制，作为鉴定新型抗菌药物操纵的新靶点的一种手段。由于以下观察结果，这项研究引起了传染病界的兴趣。首先，初步数据表明FASX调控GAS毒力。其次，FASX的生长阶段依赖性转录与GAS在感染阶段之间的过渡中的作用是一致的。第三，FASX或任何GAS sRNA调节表达的机制尚不清楚。第四，尽管在编码rna结合蛋白Hfq同源物的病原体中，rna介导的调控已经得到了很好的研究，但在那些缺乏Hfq同源物的病原体中（例如链球菌、肠球菌和分枝杆菌属病原体），我们知之甚少。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剂。为了确定在GAS中fasX介导的调节的呼吸，我们将使用二维液相色谱质谱分析来比较临床GAS分离物与其等基因fasX突变衍生物的蛋白质组。蛋白质组将在体外和离体进行比较，离体条件是在人血浆（侵袭性感染模型）和人唾液（咽感染模型）中生长。（ii） FASX结合mrna和/或蛋白质来调节毒力因子的产生。我们将链霉素结合RNA适配体与FASX融合，使杂交RNA能够保留在链霉素亲和矩阵中。我们将通过下拉试验分离与FASX杂交相互作用的GAS mrna和/或蛋白质。fasx结合mrna和/或蛋白质的身份将分别通过使用定制微阵列或质谱分析来确定，并使用体外结合试验来确认。（iii） FASX内的特定核苷酸必须具有活性。为了便于研究FASX的调控靶点和作用机制，我们将对FASX进行定点诱变。根据突变体FASX等位基因恢复突变株2221FASX的链激酶活性的能力，FASX核苷酸将被评分为无作用、中等作用或主要作用。候选FASX:mRNA相互作用将进行生物信息学分析，以突出互补碱基配对区域。假定的碱基配对将在体内通过将预测与FASX杂交的mRNA区域融合到lacZ报告基因上，并在FASX存在和不存在时测量2-gal活性来验证。

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.