Contribution of the Putative ScfCDE Importer to the Pathophysiology of Group A Streptococcal Disease

细菌病原体必须适应体内生态位的变化，才能在宿主内定居并持续存在， 经常克服营养免疫来获得必要的营养。体能所需基因的识别 可以为新的治疗方法提供靶点。A组链球菌(GAS)是一种严格的 人类病原体通常与无症状携带者和自限性感染有关，但有时 侵入无菌部位并导致危及生命的疾病(如中毒性休克综合征、坏死性筋膜炎)。那里 是明确的证据表明，生理学与人类GAS的发病机制有关，并允许病原体 在一系列主机环境中激增。M1T1株5448株体内气体适合性的TN-SEQ筛选 一种软组织感染的小鼠模型确定了编码可能的ABC的皮下适合性基因scfCDE 进口蛋白在链球菌和大多数Firmicum中保守，其中SCFC是底物结合蛋白，ScfD是 膜透性酶和单链构象蛋白ATPase。尽管它的作用尚未确定，但同源性表明它可能 运输色氨酸等芳香氨基酸(L-色氨酸)。ScfCDE基因座还包含两个未表征的 可能起作用的小开放阅读框(SmORF)。初步数据显示，scfDE是 在小鼠软组织中的气体竞争适合性，两者都是气体在人类血液中生存所必需的，一种Δscfd 渗透酶突变体在小鼠软组织模型中的高度减弱以及在软组织中scfDE的适用性缺陷 组织可以使用营养限制介质(CDM，mRPMI)进行体外复制。我们已出版的转录本 结果表明，在天冬酰胺的存在下，scfCDE被抑制，在血红素胁迫时被诱导，并被抑制。 由全球毒力监管机构MGA。然而，这一基因座还有待于在病理生理学的背景下进行研究。 在革兰氏阳性病原菌中，这种保守的ABC导入蛋白的功能仍然是个谜。我们 假设scfCDE为一个重要的ABC导入子编码，该导入子对于 GAS以获取宿主组织中的关键营养物质。本探索性R21的目标是调查基本角色 SCFCDE在GAS的病理生理学中的作用，期望这项工作将促进我们对 这个基因是如何影响气体和其他病原体的毒力的。在目标1中，我们将研究泛函 ScfCDE通过识别ScfCDE可能运输的潜在底物在GAS中的作用 色氨酸等芳香氨基酸(L-色氨酸)。我们还将探索互动伙伴和新陈代谢 在低营养条件下，ΔscfD突变体通过Tn-seq参与scfCDE的途径 模仿软组织表型。在目标2中，我们将通过表征scfCDE基因座的特征来探索scfCDE基因座的表达 不同介质条件下scfCDE的转录结构和表达模式。这个 两个小ORF(SmORF)在该基因座中的贡献将被检测在ScfCDE中的潜在作用 功能。最后，在目标3中，我们将表征ScfCDE对宿主反应和粘膜定植的影响 采用已建立的体内和体外GAS感染模型。

Bacterial pathogens must adapt to changing in vivo niches in order to colonize and persist within their host, often overcoming nutritional immunity to obtain essential nutrients. Identification of genes required for fitness in the host can provide targets for new therapeutic approaches. The Group A Streptococcus (GAS) is a strict human pathogen typically associated with asymptomatic carriage and self-limiting infection but can sometimes invade sterile sites and causing life-threatening diseases (e.g., toxic shock syndrome, necrotizing fasciitis). There is clear evidence that physiology is linked to pathogenesis for GAS in the human and allows the pathogen to proliferate in a range of host environments. An in vivo Tn-seq screen of GAS fitness in the M1T1 strain 5448 in a murine model of soft tissue infection identified the subcutaneous fitness locus scfCDE encoding a putative ABC importer conserved in Streptococcus and most Firmicutes, where ScfC is the substrate-binding protein, ScfD the membrane permease, and ScfE the ATPase. Although its role has not established, homology suggests it may transport aromatic amino acids such as tryptophan (L-trp). The scfCDE locus also contains two uncharacterized small open reading frames (smORFs) that might play a role. Preliminary data shows that scfDE are required for GAS competitive fitness in murine soft tissue, both scfDE are required for GAS survival in human blood, a ΔscfD permease mutant is highly attenuated in the murine soft tissue model and the fitness defect for scfDE in soft tissue can be replicated in vitro using nutrient-limiting media (CDM, mRPMI). Our published transcriptomic results reveal that scfCDE is repressed in the presence of asparagine, induced during heme stress, and repressed by the global virulence regulator Mga. Still, this locus has yet to be studied in the context of the pathophysiology of GAS and the function of this conserved ABC importer remains enigmatic in Gram-positive pathogens. We hypothesize that scfCDE encodes for an essential ABC importer that is important the ability of GAS to obtain key nutrients in host tissues. The goal in this exploratory R21 is to investigate the basic role of scfCDE in the pathophysiology of GAS, with the expectation that this work will advance our understanding of how this locus contributes to virulence in GAS and other pathogens. In Aim 1, we will investigate the functional role of ScfCDE in GAS by identifying potential substrates that ScfCDE may transport with an emphasis on aromatic amino acids such as tryptophan (L-trp). We will also explore interacting partners and metabolic pathways that scfCDE could be involved in through Tn-seq of the ΔscfD mutant in a low-nutrient medium that mimics the soft tissue phenotype. In Aim 2, we will explore expression of the scfCDE locus by characterizing the transcriptional architecture and expression patterns of scfCDE's under different media conditions. The contribution of the two small ORFs (smORFs) in the locus will be examined for potential roles in ScfCDE function. Finally, in Aim 3, we will characterize the impact of ScfCDE on host response and mucosal colonization using established in vivo and ex vivo models of GAS infection.