PRD-containing Virulence Regulators of Pathogenic Streptococci

细菌病原体在感染过程中必须迅速适应有限的营养。的磷酸烯醇式丙酮 磷酸转移酶系统（PTS）是一种保守的磷酸化中继，其将糖转运与糖转运偶联。 蛋白质磷酸化，并作为细菌细胞中碳水化合物流动的监测器。A组链球菌 (GAS)是一种重要的人类限制性病原体，在不同宿主中引起广泛的急性疾病 组织，每年导致全球超过50万人死亡。与此相关的更新，GAS和其他 致病性链球菌依靠碳水化合物摄取系统在宿主中存活。Mga 调节编码免疫逃避因子、粘附素和糖利用操纵子的毒力基因，我们 在上一个资助期内，已经确定Mga是由PTS直接磷酸化的， PTS调节结构域（PRD）内的组氨酸改变了Mga调节的基因表达和毒力， 气体旁系同源物存在于GAS（RofA样蛋白，RALP）中以及其他G+病原体如S. 肺炎克雷伯氏菌（MgaSpn）和B.炭疽菌（AtxA）。Mga与AtxA形成PRD家族的范例- 在G+病原体中含有毒力调节因子（PCVRs）， 通过全球毒力调节途径感知并影响疾病进程。根据我们的大量 根据已发表的数据，我们认为，随着GAS在宿主中遇到糖源的变化或限制，PTS 磷酸化PRD-1中的Mga以阻断二聚化并抑制其活性，从而有效地关闭Mga。我们 GAS中PTS的分子解剖表明，葡萄糖水平显著改变PTS介导的Mga 磷酸化和Mga调节子。然而，许多问题仍然存在。这次更新的目标将是 继续我们对Mga作为PCVR原型的开创性研究，同时将我们的范围扩大到其他假定的 PCVRs。在目标1中，我们将通过定义PTS和非PTS来描述GAS中的葡萄糖摄取如何影响Mga。 使用最新技术水平研究影响Mga磷酸化和活性的葡萄糖摄取PTS组分 监测磷酸组氨酸的LC-MS/MS方法。在目标2中，我们将确定非葡萄糖PTS 糖通过GAS中的Mga信号传导，使用具有特定PTS EIIC突变体的PTS糖中的生长补充 用Tn-seq来绘制不同PTS糖中的Mga特异性遗传相互作用。目标3将重点关注是否 通过探索PTS是否直接磷酸化，Mga的旁系同源物和同源物是功能性PCVRs 并影响GAS中的旁系同源物（RofA，RivR）、S. dysgalactiae（DmgB）和S. 肺炎克雷伯氏菌（MgaSpn）。最后，目标4将研究PTS调节对Mga和RALP旁系同源物的作用 在代表已知影响Mga的葡萄糖水平的两种宿主环境中;血流感染（高 葡萄糖），和使用鼠IL 17-/-IL 17-的粘膜定植（低葡萄糖）。 NALT模型。推进我们对PCVRs的理解可能会导致治疗严重感染的新策略 由这些重要的病原体引起。

Bacterial pathogens must rapidly adapt to limiting nutrients during infection. The phosphoenolpyruvate phosphotransferase system (PTS) is a conserved phosphorelay that couples sugar transport with phosphorylation and serves as a monitor of carbohydrate flow in the bacterial cell. The Group A Streptococcus (GAS) is a significant human-restricted pathogen causing a wide array of acute diseases in different host tissues, resulting in over half a million deaths worldwide each year. Relevant to this renewal, GAS and other pathogenic streptococci depend upon carbohydrate uptake systems for their ability to survive in the host. Mga regulates virulence genes encoding immune evasion factors, adhesins, and sugar utilization operons, and we have established over the previous grant period that Mga is directly phosphorylated by the PTS at conserved histidines within PTS regulatory domains (PRD) that alter Mga-regulated gene expression and virulence in GAS. Paralogs exist in GAS (RofA-like proteins, RALPs) as well as orthologs in other G+ pathogens such as S. pneumoniae (MgaSpn) and B. anthracis (AtxA). Mga, with AtxA, form the paradigms for a family of PRD- Containing Virulence Regulators (PCVRs) in G+ pathogens that that allow sugar availability to be sensed by global virulence regulatory pathways and influence the disease process. Based on our substantial published data, we posit that as GAS encounter changing or limiting sugar sources in the host, the PTS phosphorylates Mga in PRD-1 to block dimerization and inhibit its activity, effectively shutting Mga off. Our molecular dissection of the PTS in GAS has shown that glucose levels significantly alter PTS-mediated Mga phosphorylation and the Mga regulon. However, many questions still remain. The goal of this renewal will be to continue our pioneering studies on Mga as an archetype PCVR, while expanding our scope to other putative PCVRs. In Aim 1, we will delineate how glucose uptake in GAS impacts Mga by defining the PTS and non- PTS components for glucose uptake that influence Mga phosphorylation and activity using state-of-the-art LC-MS/MS approaches to monitor phosphohistidines. In Aim 2, we will determine how non-glucose PTS sugars signal through Mga in GAS using growth in PTS sugars with specific PTS EIIC mutants complemented with Tn-seq to map Mga-specific genetic interactions in different PTS sugars. Aim 3 will focus on whether paralogs and homologs of Mga are functional PCVRs by exploring whether the PTS directly phosphorylates and influences the activity of paralogs in GAS (RofA, RivR), homologs in S. dysgalactiae (DmgB) and S. pneumoniae (MgaSpn). Finally, Aim 4 will investigate the role of PTS regulation on Mga and RALP paralogs in two host environments representing glucose levels known to affect Mga; bloodstream infection (high glucose) using ex vivo and in vivo models, and mucosal colonization (low glucose) using a murine IL17-/- NALT model. Advancing our understanding of PCVRs could lead to novel strategies to treat severe infections caused by these important pathogens.