Defining novel mechanisms of clonal emergence in Group A Streptococcus

项目摘要 密切相关的菌株或克隆的出现和传播是许多细菌引起的特征。 严重的人类疾病。主要的人类病原体A组链球菌（GAS）表现出这种行为 并且长期以来一直是研究细菌克隆出现的模式生物。GAS分为EMM类型 基于编码细胞表面抗吞噬M蛋白的emm基因的变异。本 范式是GAS克隆出现是由于基因重组事件而发生的，这要么允许 获得新的毒力因子或增加现有毒力因子的产量，特别是那些 由NGA操纵子编码。通过对来自不同时间和地理的> 1，000个emm 4菌株进行测序， 来源，我们已经确定，一个新的emm 4克隆已取代以前循环emm 4菌株在 过去十年“新出现的”菌株没有发生显著的基因重组，不含新的 毒力因子编码基因，并且相对于 被“取代”的品种。然而，在两个菌株中，新出现的emm 4 GAS比替代的emm 4菌株毒力更强。 动物模型和在人血液中生长期间。因此，这种新发现的克隆出现不符合 目前对GAS克隆出现的理解。R21提案的目标是开始建立新的 emm 4 GAS增殖的潜在机制。在具体目标1中，我们将确定 新出现的EMM 4菌株相对于替代菌株具有增加的定殖/传播能力。这 aim将使用原代人类细胞以及新建立的GAS传播动物模型。在 具体目标2，我们将利用我们现有的转录组学数据，这些数据表明新出现的菌株具有 编码参与细胞表面氧化应激的蛋白质的基因的转录水平显著更高 反应和肽聚糖周转。我们将确定新出现的GAS菌株是否增加了 抗氧化应激和人类嗜中性粒细胞的挑战，利用活性氧作为一种抗氧化剂。 主要的杀人机制此外，将探讨新出现的菌株和替代菌株之间的细胞壁差异 使用互补成像技术和通过测试对细胞包膜活性先天性 抗菌剂特定基因在观察到的表型差异中的特定作用将使用以下方法进行评估： 插入诱变方法或通过修饰基因表达， 具有本质意义设计这些研究是为了促进下游的后续设计和执行。 细菌流行病的分子基础的调查，广泛的发病机制的一个关键方面， 多种医学上重要的病原体。

PROJECT SUMMARY The emergence and spread of closely related strains or clones are characteristic of many bacteria causing serious disease in humans. The major human pathogen group A Streptococcus (GAS) displays such behavior and has long been a model organism for studying clonal emergence in bacteria. GAS is divided into emm types based on variation in the emm gene which encodes for the cell-surface, anti-phagocytic M protein. The present paradigm is that GAS clonal emergence occurs due to genetic recombination events which either allow for acquisition of a novel virulence factor or for increased production of existing virulence factors, particularly those encoded by the nga-slo operon. By sequencing >1,000 emm4 strains from diverse temporal and geographic sources, we have identified that a new emm4 clone has replaced previously circulating emm4 strains over the past decade. The “emergent” strains have not undergone significant genetic recombination, do not contain new virulence factor encoding genes, and have significantly lower transcript levels of the nga-slo operon relative to the “replaced” strains. However, emergent emm4 GAS are more virulent than replaced emm4 strains in both animal models and during growth in human blood. Thus, this newly identified clonal emergence does not fit the current understanding of GAS clonal emergence. It is the goal of this R21 proposal to begin to establish novel mechanisms underlying the proliferation of emergent emm4 GAS. In specific aim 1, we will determine whether emergent emm4 strains have increased colonization/transmission capacities relative to replaced strains. This aim will employ both primary human cells as well as a newly established animal model of GAS transmission. In specific aim 2, we will leverage our existing transcriptomic data which show that emergent strains have significantly higher transcript levels of genes encoding proteins putatively involved in cell surface oxidative stress response and peptidoglycan turnover. We will determine whether the emergent GAS strains have augmented resistance to oxidative stress and to challenge by human neutrophils, which utilize reactive oxygen species as a major killing mechanism. Moreover, cell wall differences between emergent and replaced strains will be explored using complementary imaging techniques and by testing susceptibility to cell-envelope active innate antimicrobials. The specific role of particular genes in observed phenotypic differences will be assessed using either an insertional mutagenesis approach or by modifying gene expression when the candidate genes are essential. These studies have been devised to facilitate the subsequent design and execution of downstream investigations of the molecular underpinning of bacterial epidemics, a key aspect of pathogenesis for a wide variety of medically important pathogens.