Defining novel mechanisms of clonal emergence in Group A Streptococcus

项目总结 密切相关的菌株或克隆的出现和传播是许多引起 人类患上严重的疾病。人类主要病原体A组链球菌(GAS)表现出这样的行为 长期以来一直是研究细菌克隆出现的模式生物。天然气分为EMM类型 基于编码细胞表面M蛋白的emm基因的变异，抗吞噬M蛋白。现在 范式是，气体克隆的出现是由于基因重组事件，这要么允许 获得新的毒力因子或增加现有毒力因子的产生，特别是那些 由NGA-SLO操纵子编码。通过对来自不同时间和地理的1,000株emm4菌株进行测序 消息来源，我们已经确定一个新的emm4克隆已经取代了以前流行的emm4毒株。 过去十年。新出现的菌株没有发生重大的基因重组，不包含新的 毒力因子编码基因，并且ngA-slo操纵子的转录水平显著低于 “被取代的”菌株。然而，新出现的emm4毒株在这两种情况下的毒力都比替换的emm4毒株更强。 在动物模型和人类血液的生长过程中。因此，这种新发现的克隆出现并不符合 目前对GAS克隆出现的理解。这份R21提案的目标是开始建立小说 浮现emm4气体扩散的潜在机制。在具体目标1中，我们将确定是否 与替换菌株相比，新出现的emm4菌株具有更强的定植/传播能力。这 AIM将使用原代人类细胞以及新建立的气体传输动物模型。在……里面 具体目标2，我们将利用我们现有的转录数据，这些数据表明新出现的菌株具有 编码可能与细胞表面氧化应激有关的蛋白的基因转录水平显著升高 反应和肽聚糖周转。我们将确定紧急气体压力是否有所增加 对氧化应激和人类中性粒细胞挑战的抵抗力，中性粒细胞利用活性氧物种作为 主要的杀戮机制。此外，还将探索新出现的菌株和替换的菌株之间的细胞壁差异。 使用互补成像技术并通过测试对细胞膜天然活性的敏感性 抗菌剂。特定基因在观察到的表型差异中的具体作用将通过 要么是插入突变方法，要么是当候选基因 必不可少的。这些研究的目的是为了促进后续的下游设计和实施 细菌流行的分子基础研究是广泛致病机制的关键方面 各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.