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

革兰氏阳性菌组装一类独特的共价连接的蛋白质聚合物，称为分选酶组装的皮利或菌毛，其对于多微生物相互作用（或共聚集）、粘附和微生物降解是重要的。 细菌毒力分选酶介导的菌毛组装是一个两步过程： 在菌毛特异性分选酶催化的细胞壁锚定之后， 内务分选酶。这些分选酶如何协调它们的酶活性以组装皮利与 细胞表面上的最佳长度还没有被很好地理解。在口腔细菌口腔放线菌中， 口腔生物膜发育中的定植者，2型菌毛，由菌毛轴FimA和尖端组成 共聚集因子CafA是放线菌与口腔链球菌共聚集所必需的。菌毛特异性分选酶SrtC 2使菌毛聚合物聚合，然后将菌毛聚合物锚定到细胞壁上。 内务分选酶SrtA。重要的是，缺乏srtA的突变细胞产生极长的皮利，但不能 介导细菌共聚集。为了阐明SrtA调节菌毛长度的机制，我们试图 safA是srtA下游的一个基因，因为功能相关的基因倾向于聚集在srtA下游。 细菌基因组。值得注意的是，safA的缺失导致细胞形态改变， 多微生物相互作用，以及超长皮利的产生，所有表型与缺失的 srtA.这些有缺陷的表型被棒状杆菌safA的异位表达所拯救 白喉杆菌和马鲁霍棒状杆菌; oris safA突变体，提示了一种共同的机制， 放线菌。细菌双杂交分析表明，SafA与管家分选酶相互作用 SrtA.有趣的是，在口腔细菌牙双歧杆菌中，管家分选酶SrtE含有一种 在其C-末端融合同源SafA序列。总而言之，假设SafA维持 SrtA的膜稳态通过放线菌的进化保守机制， 调节菌毛组装和菌毛长度。提出了三个具体目标来检验这一假设。通过遗传 和生物化学方法，我们的目的是阐明SrtA膜稳态的机制， 进化保守蛋白SafA（Aim 1），研究SafA的保守性及其与 内务分选酶SrtA（目标2）。使用线虫和豚鼠感染模型，我们的目标是 研究SafA在细菌毒力中的作用（目的3）。除了提供一个博士前培训工具，这 拟议的研究不仅将促进我们对细菌毒力因子表面组装的理解， 也可以为抗毒性策略提供有吸引力的靶标。

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.