Delineation of the molecular mechanisms underlying group A Streptococcus virulenc

描述(申请人提供)：人类致病菌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导致这种疾病多样性的能力在一定程度上是由于毒力因子的特定子集的协调表达。小调节RNA(SRNAs)代表了GAS和相关病原体调节的一个鲜为人知的领域。这项拟议研究的目的是表征毒力调节气体SRNA FASX的作用机制，作为一种手段来确定新的抗菌剂操纵的新靶点。由于以下观察结果，这项研究引起了传染病学界的兴趣。首先，初步数据表明，FASX调节毒力。其次，FASX依赖生长阶段的转录与GAS在感染阶段之间的转换中的作用是一致的。第三，FASX，甚至任何GAS SRNA调控表达的机制尚不清楚。第四，虽然在编码RNA结合蛋白Hfq同源物的病原体中已经进行了很好的研究，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的病原体)，人们对此知之甚少。我们将通过检验以下假设来实现我们的目标：(I)FASX是毒力因子的主要调节因子。为了鉴定FASX在GAS中介导的呼吸调节，我们将使用二维液相色谱质谱分析来比较临床分离的GAS及其等基因FasX突变衍生物的蛋白质组。蛋白质组将在体外和体外进行比较，体外条件是在人血浆(侵入性感染模型)和人唾液(咽部感染模型)中生长。(Ii)FASX结合mRNAs和/或蛋白质以调节毒力因子的产生。我们已经将链霉素结合的RNA适配子融合到FASX上，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我们的FASX杂交种相互作用的GAS mRNAs和/或蛋白质。FASX结合的mRNAs和/或蛋白质的特性将分别通过使用定制微阵列或通过质谱仪分析来确定，并使用体外结合分析进行确认。(3)FASX内的特定核苷酸是活性所必需的。为了便于研究Fas X的调控靶点和作用机制/S，我们将对Fas X进行定点突变。根据突变的FasX等位基因恢复FasX突变株2221FASX的链激酶活性的能力，FASX核苷酸将被评为在活性中没有作用、中等作用或主要作用。候选FASX：mRNA的相互作用将被生物信息学分析，以突出互补碱基配对的区域。推测的碱基配对将通过将预测与FASX杂交的mRNA区域融合到LacZ报告基因，并在存在和不存在FASX的情况下测量2-Gal的活性来在体内进行测试。 与公共卫生相关：在美国，每年约有3000万例毒气咽炎病例。这项拟议的研究将提供对气体和相关病原体毒力调控这一未被充分研究的领域的分子洞察力。通过将FASX调控途径的知识转化为基于新型抗菌剂对这些途径的抑制的新的治疗和/或预防制度的长期目标，可以增强公共健康。

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.