Role of RND Superfamily Efflux Pumps in the Biology of Staphylococci

我们的项目评估了两个先前未被发现的编码外排泵的基因的功能，这两个基因在葡萄球菌中是保守的，属于抗性-结节分裂（RND）转运蛋白超家族。葡萄球菌，包括金黄色葡萄球菌和表皮葡萄球菌，是人类的共生体，我们假设RND外排泵FarE和FemT支持定义该属特征的生理过程；即宿主来源的抗菌脂肪酸（FarE）的外排，以及葡萄球菌肽聚糖（FemT）特有的脂联肽聚糖交叉桥结构的输出。我们进一步提出，RND外排泵内在地在蛋白质复合体中起作用，要么促进底物的偶联合成和输出，要么与输出所需的辅助蛋白质形成复合体。***我们之前鉴定出不同转录的farE和farR是金黄色葡萄球菌对抗菌脂肪酸耐药的效应和调节因子，并观察到抗菌脂肪酸诱导了farE的表达。因此，我们的调节farE的工作模型表明，FarR与farE启动子的结合是由不饱和抗菌脂肪酸代谢的特异性产物调节的，并且膜传感器蛋白可以感知并响应膜扰动或细胞质膜组成的变化，也可能参与调节。虽然farE是可诱导的，但femT外排泵与一个基因femX相邻，该基因启动葡萄球菌肽聚糖特有的五甘氨酸交叉桥结构的合成。因此，我们预计femT将在生长细胞中高度表达，但可能在细胞壁施加压力的条件下上调表达。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，并检测肽聚糖氨基酸组成的改变，以及对细胞壁施加压力的生长条件的敏感性增加；我们还将构建一个femT-farE双突变体来评估相关外排泵之间的功能冗余程度。用荧光显微镜观察表达FarE或FemT的细菌，确定这些外排泵是否位于分裂隔，那里有活性膜和肽聚糖合成。同样，使用与FemT或FarE融合的生物素连接酶，我们将识别在生长细胞中被生物素化的蛋白质，因为它们靠近FarE或FemT。***意义：我们的项目将为葡萄球菌的生物学提供新的见解，从中我们期望开发新的调控途径，外排泵功能和蛋白质相互作用网络，这些网络控制着革兰氏阳性细胞质膜和细胞包膜的维持/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. **************