A novel regulatory system promotes group A Streptococcus survival in human blood

项目总结 A组链球菌(GAS；化脓性链球菌)在人类中造成巨大的发病率(7亿感染 每年)和死亡率(每年550,000人死亡)，感染的范围从轻度和自体 限制(如咽炎)至严重侵袭性(如坏死性筋膜炎)。我们已经确认了FasX，即 SRNA是一种新的四组分调控系统(FasBCAX)的组成部分，它在转录后调节 产生关键毒力因子的气体。FasBCAX系统提供的调节影响气体 毒力，增强气体对人体血液杀菌特性的抵抗力(见初步数据)和 小鼠侵袭性感染模型中的致死性。然而，我们的知识仍然存在很大差距。 关于这一模式监管系统的运作，包括FasX如何减少人体内的气体死亡 血液增加约30倍，以及FasBCA蛋白如何发挥功能以增强FasX的表达~100倍。我们 将通过追求以下目标来填补我们知识中的监管、机械和毒性空白： 目的1：确定FasX增强人体血液气体抵抗力的机制。在这个目标中， 我们将验证我们的初步数据，支持FasX-Regon是目前的两倍大小 认识到，确定哪些FasX调节的毒力因子/S负责耐药表型和 它们被FasX的调节是否改变了宿主分子与气细胞表面的结合，并且，鉴于我们的 初步数据表明，耐药表型是通过抑制吞噬细胞而发生的，测试是否 中性粒细胞激活、吞噬和/或氧化爆发受到抑制。 目的2：确定FasBCA蛋白增加FasX sRNA丰度的机制。这个 Fas基因由共转录的FasBCA和单独转录的FasX组成。FASB和FASC是 与感受器激酶同源，而FASA与反应调节同源。初步数据为 与FASB和FASC形成异二聚体一致，而不是经典的传感器中出现的同源二聚体 激活剂。在这个目标中，我们将揭示FasBCA蛋白是如何相互作用来增强FasX和FasX的转录的 其他调控目标，并将开始描述能够调节Fas系统活动的宿主因素。 这项研究的完成将极大地扩展关于sRNA在GAS中的功能的已知内容， 以及其他乳杆菌病原体，其中sRNA机制数据有限。我们将生成基本的 通过描绘一个从未描述过的调控系统的科学洞察力，在这个系统中，两个传感器激酶 异二聚体以激活活性。我们还将通过描绘分子基础来产生临床洞察力 在FasX抑制血液中气体杀死的能力背后，一种对这一能力至关重要的表型 流行的人类病原体导致严重的侵袭性疾病。

PROJECT SUMMARY The group A Streptococcus (GAS; S. pyogenes) causes significant human morbidity (>700 million infections annually) and mortality (>550,000 deaths annually), with a spectrum of infections that range from mild and self- limiting (e.g. pharyngitis) to severely invasive (e.g. necrotizing fasciitis). We have identified that FasX, the sRNA component of a novel four-component regulatory system (FasBCAX), post-transcriptionally regulates the production of key GAS virulence factors. The regulation afforded by the FasBCAX system influences GAS virulence, enhancing GAS resistance to the bactericidal properties of human blood (see preliminary data) and lethality in a mouse invasive infection model. However, there remains significant gaps in our knowledge regarding the functioning of this model regulatory system, including how FasX reduces GAS killing in human blood by ~30-fold, and how the FasBCA proteins function to enhance fasX expression ~100-fold. We will fill the regulatory, mechanistic, and virulence gaps in our knowledge by pursuing the following aims: Aim 1: Determine the mechanism by which FasX enhances GAS resistance to human blood. In this aim, we will verify our preliminary data that supports the FasX-regulon being twice the size of that currently appreciated, identify which FasX-regulated virulence factor/s are responsible for the resistance phenotype and whether their regulation by FasX modifies the binding of host molecules to the GAS cell surface, and, given our preliminary data that the resistance phenotype occurs via the inhibition of phagocytic cells, test whether neutrophil activation, phagocytosis, and/or oxidative burst is inhibited. Aim 2: Determine the mechanism by which the FasBCA proteins enhance FasX sRNA abundance. The Fas locus consists of the co-transcribed fasBCA and the separately transcribed fasX. FasB and FasC are homologous to sensor kinases while FasA is homologous to response regulators. Preliminary data are consistent with FasB and FasC forming heterodimers, rather than homodimers as classically occurs for sensor kinases. In this aim, we will unravel how the FasBCA proteins interact to enhance the transcription of fasX and other regulatory targets, and will initiate delineation of host factors capable of modulating Fas system activity. Completion of this research will greatly expand what is known about sRNA function in GAS, an organism, along with fellow Lactobacillales pathogens, in which sRNA mechanistic data is limited. We will generate basic science insights by delineating a never-before-described regulatory system in which two sensor kinases heterodimerize to activate activity. We will also generate clinical insights by delineating the molecular basis behind the ability of FasX to inhibit GAS killing in blood, a phenotype that is critical to the ability of this prevalent human pathogen to cause severe invasive disease.