Role of RND Superfamily Efflux Pumps in the Biology of Staphylococci

我们的程序评估了两个以前未知的基因编码外排泵的功能，这些外排泵在葡萄球菌中是保守的，属于耐药-结瘤-分裂（RND）转运蛋白超家族。葡萄球菌，包括S.金黄色葡萄球菌和表皮葡萄球菌是人类的寄生虫，我们假设RND外排泵FarE和FemT支持定义该属特征的生理过程;即宿主来源的抗微生物脂肪酸（FarE）的外排和葡萄球菌肽聚糖（FemT）特有的脂质连接的肽聚糖跨桥结构的输出。我们进一步提出，RND外排泵固有的功能内的蛋白质复合物，以促进耦合合成和出口的底物，或在复杂的出口所需的辅助蛋白质。 我们以前确定了分歧转录farE和farR作为一个效应器和调节器的耐药性抗菌脂肪酸在S。金黄色葡萄球菌，并观察到抗微生物脂肪酸诱导farE的表达。因此，我们的工作模式，为farE的调节提出，结合的farR的farE启动子是由特定的不饱和的抗微生物脂肪酸的代谢产物调制，和膜传感器蛋白的传感器和响应膜扰动，或细胞质膜的组成的变化也可能参与调节。而farE是可诱导的，femT外排泵是相邻的基因femX，启动合成的五甘氨酸跨桥结构，这是唯一的葡萄球菌肽聚糖。因此，我们预期femT将在生长的细胞中高度表达，但可能在对细胞壁施加压力的条件下上调。为了评估FemT在肽聚糖前体转运中的预测作用，我们将构建一个femT缺失突变体，并测定肽聚糖的氨基酸组成改变，以及对细胞壁施加应力的生长条件的敏感性增加;我们还将构建一个femT-farE双突变体，以评估相关外排泵之间的功能冗余程度。表达与荧光报告基因融合的FarE或FemT的细菌的荧光显微镜检查将用于确定这些外排泵是否定位于分裂隔膜，其中存在活性膜和肽聚糖合成。类似地，使用与FemT或FarE融合的生物素连接酶，我们将鉴定生长细胞中生物素化的蛋白质，因为它们接近FarE或FemT。 重要性：我们的计划将提供新的洞察葡萄球菌的生物学，从中我们希望开发新的知识的监管途径，外排泵功能，和蛋白质相互作用网络，管理维护/革兰氏阳性细胞质膜和细胞包膜的稳态。

Our program evaluates the function of two previously uncharacterized genes encoding efflux pumps which are conserved among Staphylococci, and belong to the resistance -nodulation-division (RND) superfamily of transporters. Staphylococci, including S. aureus and S. epidermidis, are commensals of humans, and we hypothesize that the RND efflux pumps FarE and FemT, support physiological processes that are defining traits of this genus; namely efflux of host derived antimicrobial fatty acids (FarE), and export of a lipid-linked peptidoglycan cross-bridge structure that is unique to Staphylococcal peptidoglycan (FemT). We further propose that RND efflux pumps inherently function within a protein complex, either to promote coupled synthesis and export of a substrate, or in complex with accessory proteins required for export. We previously identified the divergently transcribed farE and farR as an effector and regulator of resistance to antimicrobial fatty acids in S. aureus, and observed that expression of farE was induced by antimicrobial fatty acids. Thus, our working model for regulation of farE proposes that binding of FarR to the farE promoter is modulated by products specific to metabolism of unsaturated antimicrobial fatty acids, and that membrane sensor proteins which sense and respond either to membrane perturbation, or changes in composition of the cytoplasmic membrane may also be involved in regulation. Whereas farE is inducible, the femT efflux pump is adjacent to a gene femX, which initiates synthesis of a pentaglycine cross-bridge structure that is unique to staphylococcal peptidoglycan. Thus, we expect that femT will be highly expressed in growing cells, but may be up-regulated in response to conditions that impose stress on the cell wall. To evaluate a predicted role for FemT in transport of a peptidoglycan precursor, we will construct a femT deletion mutant, and assay for altered amino acid composition of peptidoglycan, and increased sensitivity to growth conditions that impose stress on the cell wall; we will also construct a femT-farE double mutant to evaluate the extent of functional redundancy between related efflux pumps. Fluorescence microscopy of bacteria expressing FarE or FemT fused to fluorescent reporters, will be used to determine whether these efflux pumps localize to the division septum, where there is active membrane and peptidoglycan synthesis. Similarly, using a biotin ligase fused to either FemT or FarE, we will identify proteins that are biotinylated in growing cells, due to their proximity to FarE or FemT. Significance: Our program will provide new insight into the biology of the Staphylococci, from which we expect to develop new knowledge of regulatory pathways, efflux pump function, and protein interaction networks that govern maintenance/homeostasis of the Gram-positive cytoplasmic membrane and cell envelope.