Mechanism and function of membrane homeostasis of sortase modulated by an evolutionarily conserved protein involved in pilus assembly in Gram-positive bacteria

革兰氏阳性菌聚集了一类独特的共价连接的蛋白质聚合物，称为索尔特酶组装的菌毛或菌毛，对多菌相互作用(或共聚集)、黏附和 细菌毒性。索尔特酶介导的菌毛组装是一个两步过程：菌毛蛋白的聚合 由菌毛特异的索酸酶催化，随后是由 家政服务分类酶。这些分类酶如何协调它们的酶活性来组装菌毛和 细胞表面的最佳长度还不是很清楚。在口腔细菌中，口腔放线菌是关键 口腔生物膜发育中的定殖者，2型菌毛，由菌毛轴FIMA和顶端组成 共聚集因子CAFA是放线菌与口腔链球菌共聚集所必需的。菌毛特异的分类酶SrtC2聚合菌毛聚合物，然后通过 家政分类酶srtA。重要的是，缺乏srtA的突变细胞会产生非常长的菌毛，但不能 介导细菌共聚集。为了阐明srtA对菌毛长度的调节机制，我们试图 描述srtA下游的SafA基因，因为功能相关的基因往往聚集在 细菌基因组。值得注意的是，SafA的缺失会导致细胞形态的改变，废除 多个微生物的相互作用和产生超长的菌毛，所有的表型都与缺失的 SrtA。通过棒状杆菌SafA的异位表达挽救了这些缺陷表型 白喉和马氏棒状杆菌在金黄色葡萄球菌Safa突变体中的作用，表明在 放线杆菌。细菌双杂交分析表明SafA与看家分类酶相互作用 主席先生。有趣的是，在口腔细菌牙科双歧杆菌中，看家分类酶SrtE包含一个 在其C末端融合了同源SafA序列。总而言之，假设Safa坚持认为 跨放线杆菌通过进化保守机制实现的srtA膜动态平衡 调整毛发组件和毛发长度。为了检验这一假说，本文提出了三个具体目标。按基因 和生化方法，我们的目的是阐明srtA膜动态平衡的机制 进化保守蛋白SafA(Aim 1)，并研究SafA的保守性及其与 家政分类酶srtA(目标2)。利用线虫和豚鼠的感染模型，我们的目标是 研究SAFA在细菌毒力中的作用(目标3)。除了提供博士前培训工具外，这 提出的研究不仅将增进我们对细菌毒力因子表面组装的理解，而且 也可能为抗毒力策略提供有吸引力的靶点。

Gram-positive bacteria assemble a unique class of covalently-linked protein polymers known as sortase-assembled pili or fimbriae that are important for polymicrobial interactions (or coaggregation), adhesion, and bacterial virulence. Sortase-mediated pilus assembly is a two-step process: polymerization of pilus proteins catalyzed by pilus-specific sortase is followed by cell wall anchoring of pilus polymers catalyzed by the housekeeping sortase. How these sortases coordinate their enzymatic activities to assemble pili with an optimal length on the cell surface is not well understood. In the oral bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, type 2 fimbriae, made of the fimbrial shaft FimA and the tip coaggregation factor CafA, are essential for Actinomyces coaggregation with oral streptococci. The pilus-specific sortase SrtC2 polymerizes fimbrial polymers that are then anchored to the cell wall by the housekeeping sortase SrtA. Importantly, mutant cells lacking srtA produce exceedingly long pili but fail to mediate bacterial coaggregation. To elucidate a mechanism of SrtA modulation of pilus length, we sought to characterize safA, a gene immediately downstream of srtA, since functionally related genes tend to cluster in the bacterial genome. Remarkably, deletion of safA causes altered cell morphology, abolishment of polymicrobial interactions, and production of exceedingly long pili, all phenotypes consistent with deletion of srtA. These defective phenotypes were rescued with ectopic expression of safA from Corynebacterium diphtheriae and Corynebacterium matruchotii in the A. oris safA mutant, suggesting a common mechanism in Actinobacteria. Bacterial two-hybrid analysis demonstrated that SafA interacts with the housekeeping sortase SrtA. Intriguingly, in the oral bacterium Bifidobacterium dentium, the housekeeping sortase SrtE contains a fused homologous SafA sequence at its C-terminus. Altogether, it is hypothesized that SafA maintains membrane homeostasis of SrtA through an evolutionarily conserved mechanism across Actinobacteria to modulate pilus assembly and pilus length. Three specific aims are proposed to test this hypothesis. By genetic and biochemical approaches, we aim to elucidate the mechanism of SrtA membrane homeostasis by the evolutionary conserved protein SafA (Aim 1) and investigate the conservation of SafA and its co-evolution with the housekeeping sortase SrtA (Aim 2). Using nematode and guinea pig models of infection, we aim to examine the role of SafA in bacterial virulence (Aim 3). Besides providing a predoctoral training vehicle, this proposed study will not only advance our understanding of surface assembly of bacterial virulence factors, but also may provide attractive targets for anti-virulence strategies.