Impact of regulatory cross-talk on the pathophysiology of emergent acapsular group A streptococcus

项目摘要 主要的人类病原体A组链球菌（GAS）的透明质酸胶囊已被经典地 被认为对传染性至关重要。GAS根据emm基因的变异分为emm类型， 编码细胞表面的抗吞噬M蛋白。最近已经认识到，来自 缺乏胶囊的emm类型越来越多地引起严重的人类感染， 侵袭性GAS分离株是无包膜的。囊外GAS如何引起感染目前尚不清楚， 假定无囊GAS菌株产生高水平的关键细胞毒素链球菌溶血素O（Slo）。 与这一推测相反，我们发现多种无囊emm类型不会产生高水平的Slo， 因此，我们研究的长期目标是阐明无囊GAS的机制基础 病理生理学通过比较基因组学的方法，我们已经确定， 类型具有不同的MGA和EMM启动子组成，其编码关键的转录 调节多基因激活因子（Mga）和M蛋白。此外，我们还发现， CovRS双组分基因调控系统特异性影响mga和emm转录水平 相对于包裹的GAS而言是无囊的。这些和其他调查结果为这一提议奠定了基础， 目的是检验mga和emm启动子组成对无囊泡的感染性至关重要的假设。 GAS通过允许CovRS进行调节，这对于GAS感测和响应主机信号至关重要。在 具体目标1，我们将确定无囊GAS与无囊GAS相比， 包裹的GAS改变了无囊emm 4和emm 89菌株细胞表面的组成， 人抗微生物肽LL-37。我们还将评估mga和emm启动子组成的影响 对GAS与人上皮细胞和人细胞外基质成分的无囊粘附， 人血、抗吞噬作用和小鼠模型中的毒力。在具体目标2中，我们将确定 如果在无囊GAS的mga和emm启动子中观察到的变化允许CovR直接调节 并且是CovS失活影响mga和emm转录水平的机制。我们将调查 CovR与mga和emm启动子的体内相互作用，并试图鉴定关键的启动子区域 涉及使用基于荧光素酶的报告系统的基于CovR的调控。我们还将评估CovR是否直接 在无囊GAS中不依赖于Mga抑制emm。完成本文概述的研究将 显著增加了对无囊泡GAS出现的分子机制的理解 其是GAS感染的主要原因，但相对于包封菌株仍知之甚少。

PROJECT SUMMARY The hyaluronic acid capsule of the major human pathogen group A Streptococcus (GAS) has classically been considered essential to infectivity. GAS is divided into emm types based on variation in the emm gene that encodes for the cell-surface, anti-phagocytic M protein. It has recently been appreciated that GAS strains from emm types lacking capsule are increasingly causing serious human infections to the point where some 30% of invasive GAS isolates are acapsular. How acapsular GAS causes infection is not currently known although it has been postulated that acapsular GAS strains elaborate high levels of a key cytotoxin, streptolysin O (Slo). Contrary to this conjecture, we have found that multiple acapsular emm types do not produce high levels of Slo, and thus the long term goal of our research is to elucidate the mechanistic basis of acapsular GAS pathophysiology. Through a comparative genomics approach, we have identified that acapsular GAS emm types have distinct compositions of the promoters of mga and emm which encode the key transcriptional regulator multi-gene activator (Mga) and M protein, respectively. Moreover, we have discovered that the control of virulence (CovRS) two-component gene regulatory system specifically impacts mga and emm transcript levels in acapsular relative to encapsulated GAS. These, and other, findings set the stage for this proposal which seeks to test the hypothesis that mga and emm promoter composition are critical to the infectivity of acapsular GAS by allowing for regulation by CovRS, which is critical for GAS to sense and respond to host signals. In specific aim 1, we will determine how variation in mga and emm promoters of acapsular GAS compared to encapsulated GAS alters the composition of the cell surface of acapsular emm4 and emm89 strains in response to the human antimicrobial peptide LL-37. We will also assess the impact of mga and emm promoter composition on acapsular GAS adherence to human epithelial cells and human extracellular matrix components, survival in human blood, resistance to phagocytosis, and virulence in a mouse model. In specific aim 2, we will determine if the changes observed in the mga and emm promoters of acapsular GAS allow for direct regulation by CovR and is the mechanism by which CovS inactivation influences mga and emm transcript levels. We will investigate in vivo interaction of CovR with mga and emm promoters and attempt to identify promoter regions that are key to CovR-based regulation using a luciferase based reporter system. We also will assess whether CovR directly represses emm in acapsular GAS independent of Mga. Completion of the research outlined herein will significantly augment understanding of the molecular mechanisms underlying the emergence of acapsular GAS which are a major cause of GAS infection yet remain poorly understood relative to encapsulated strains.