Role of ScfAB in the Pathophysiology of the Group A Streptococcus

细菌病原体必须适应体内不断变化的环境，才能在体内感染期间持续存在 他们的主人。识别病原体生存和适应所需的“功能”基因 宿主有助于更好地理解发病机制，告知基因组注释，并指导新的 治疗方法。TN-SEQ是一种强大的遗传方法，使研究人员能够集体识别 使用相关的感染模型研究对活体健康很重要的基因。化脓性链球菌(群) 链球菌，GAS)是一种严格的人类病原体，被列为导致急性生命的十大原因之一 威胁世界各地的细菌感染，导致从自我限制的表面性疾病 皮肤和喉部感染导致严重的软组织和无菌部位的侵袭性疾病。我们的团队已经 建立了在临床上使用GAS进行全基因组基因筛查的工具和经验 相关的M1T1株5448，我们现在已经完成了第一次在小鼠体内感染GAS的TN-SEQ 局部GAS软组织感染模型用于确定溃烂病变中健康所必需的基因 队形。在我们的数据集中，我们识别了两个未注释的基因(这里称为皮下健身基因 SCFA和SCFB)，这对术后24小时和48小时的病变中的气体适合性是极其重要的 感染。这两个基因在GAS中都没有特征，但被预测编码与膜相关的蛋白 在菲米库特人中高度保守。文献中对scfAB同源物的唯一研究发现它们 在变形链球菌的酸胁迫反应和生物膜形成中起关键作用；提示ScfAB具有如下功能 一种膜渗透酶复合体。我们的初步研究发现，在SCFA中定义了GAS M1T1 5448突变体 和scfb在体内被野生型击败，并在单一菌株后存活减弱。 软组织模型中的感染。我们假设scfAB基因在增强 GAS在小鼠软组织感染期间的适应，以及可能在其他宿主环境中的适应，通过 到目前为止，可能涉及膜转运的机制尚不清楚。此R21提案的主要目标是 探讨SCFA和ScfB在GAS生理和发病机制中的作用，以期对这项工作有所裨益 将促进我们对它们可能如何促成其他重要的G+病原体的理解。因此，我们寻求 通过两个目的来确定ScfAB在GAS病理生理学中的贡献：1)建立定位， ScfAB在气细胞生理学中的调节和作用；2)研究scfAB对GAS的影响 发病机制和定植。

Bacterial pathogens must adapt to changing environments in vivo in order to persist during infection within their host. Identifying genes that are "functionally required" for survival and fitness of the pathogen within the host helps to better understand pathogenesis, inform genome annotations, and guide the development of new therapeutic approaches. Tn-seq is a powerful genetic approach that allows researchers to identify en masse genes that are important for fitness in vivo using relevant models of infection. Streptococcus pyogenes (Group A Streptococcus, GAS) is a strict human pathogen and is listed among the top 10 causes of acutely life threatening bacterial infections worldwide, causing a wide array of diseases from self-limiting superficial infections of the skin & throat to severe invasive diseases of soft tissues & sterile sites. Our group has established the tools and experience to perform genome-wide genetic screens in GAS using the clinically relevant M1T1 strain 5448, and we have now completed the first in vivo Tn-seq of GAS infection in a murine model of localized GAS soft tissue infection to identify genes necessary for fitness during ulcerative lesion formation. Amongst our dataset, we identified two unannotated genes (called here subcutaneous fitness genes scfA and scfB) that were extremely important for GAS fitness in the lesion at both 24 and 48 hours post infection. Both genes are uncharacterized in GAS, but are predicted to encode membrane-associated proteins and are highly conserved among Firmicutes. The only study on scfAB homologs in the literature found them to be critical for the acid stress response and biofilm formation in S. mutans; suggesting that ScfAB functioned as a membrane permease complex. Our preliminary studies found that defined GAS M1T1 5448 mutants in scfA and scfB were outcompeted by wild type in vivo and were attenuated for survival following single strain infection in the soft tissue model. We hypothesize that the scfAB genes play an integral role in enhancing adaptation of GAS during murine soft tissue infection, and potentially in other host environments, through an as yet unknown mechanism that might involve membrane transport. The primary goal in this R21 proposal is to explore the role of ScfA and ScfB in GAS physiology and pathogenesis, with the expectation that this work will advance our understanding of how they might contribute to other important G+ pathogens. Thus, we seek to establish the contribution of scfAB to the pathophysiology of GAS via two aims: 1) Establish the localization, regulation and function of scfAB in GAS cell physiology, and 2) Investigate the impact of scfAB on GAS pathogenesis and colonization.