Defining novel mechanisms of clonal emergence in Group A Streptococcus

项目概要 密切相关的菌株或克隆的出现和传播是许多细菌的特征，导致 人类的严重疾病。主要人类病原体 A 组链球菌 (GAS) 表现出这种行为 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。现在的 范式是 GAS 克隆的出现是由于基因重组事件而发生的，这些事件要么允许 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 个 emm4 菌株进行测序 根据消息来源，我们发现新的 emm4 克隆已经取代了之前流行的 emm4 菌株。 过去十年。 “新兴”菌株没有经历显着的基因重组，不含有新的菌株 毒力因子编码基因，并且 nga-slo 操纵子的转录水平相对于 “被取代”的菌株。然而，在两种情况下，新出现的 emm4 GAS 都比被取代的 emm4 菌株毒性更强。 动物模型和人类血液中的生长过程。因此，这种新发现的克隆的出现并不符合 目前对 GAS 克隆出现的理解。 R21 提案的目标是开始建立新颖的 紧急 emm4 GAS 增殖的机制。在具体目标 1 中，我们将确定是否 相对于被取代的菌株，新出现的 emm4 菌株具有增强的定植/传播能力。这个 目标将采用原代人类细胞以及新建立的气体传播动物模型。在 具体目标 2，我们将利用现有的转录组数据，这些数据表明新出现的菌株已 编码与细胞表面氧化应激有关的蛋白质的基因转录水平显着升高 反应和肽聚糖周转。我们将确定新出现的 GAS 菌株是否已增强 抵抗氧化应激和人类中性粒细胞的挑战，其中性粒细胞利用活性氧作为 主要杀伤机制。此外，将探讨新出现的菌株和替代菌株之间的细胞壁差异 使用互补成像技术并通过测试对细胞包膜活性先天的敏感性 抗菌剂。将使用以下方法评估特定基因在观察到的表型差异中的具体作用 插入诱变方法或当候选基因存在时修改基因表达 必不可少的。这些研究旨在促进下游下游的后续设计和执行 对细菌流行病的分子基础的研究，这是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.