Role of RND Superfamily Efflux Pumps in the Biology of Staphylococci

我们的计划评估了两个以前未描述的编码外排泵的基因的功能，这两个基因在葡萄球菌中保守，属于耐药结节分裂(RND)超家族转运蛋白。葡萄球菌，包括金黄色葡萄球菌和表皮葡萄球菌，是人类的共生菌，我们假设RND外排泵FARE和FemT支持定义该属特征的生理过程，即宿主衍生的抗菌脂肪酸(FARE)的外流，以及金黄色葡萄球菌(FemT)所特有的脂联肽聚糖(FemT)的输出。我们进一步认为，RND外排泵在蛋白质复合体中具有内在的功能，要么促进底物的偶联合成和输出，要么在与输出所需的辅助蛋白质的复合体中发挥作用。 我们以前发现在金黄色葡萄球菌中差异转录的FARE和FARR是抗菌素脂肪酸抗性的效应因子和调节因子，并观察到FARE的表达是由抗菌素脂肪酸诱导的。因此，我们的FARE调控工作模型提出，FARR与FARE启动子的结合受不饱和抗微生物脂肪酸代谢特异性产物的调节，感受膜扰动或细胞膜组成变化并对其做出反应的膜传感器蛋白也可能参与调节。虽然FARE是可诱导的，但FemT外排泵与一个基因FemX相邻，该基因启动了葡萄球菌肽聚糖所特有的五甘氨酸交叉桥结构的合成。因此，我们预计Femt将在生长中的细胞中高表达，但可能会在对细胞壁施加压力的条件下上调。为了评估FemT在肽聚糖前体运输中的预测作用，我们将构建FemT缺失突变体，并检测肽聚糖的氨基酸组成变化，以及对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FemT-Fare双重突变体，以评估相关外排泵之间的功能冗余程度。表达FARE或FemT的细菌的荧光显微镜将被用来确定这些外排泵是否定位于分区隔膜，那里有活跃的膜和肽聚糖合成。同样，使用与FemT或FemT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，因为它们与FemT或FemT接近。 意义：我们的计划将为葡萄球菌的生物学提供新的见解，我们希望从中发展出调控途径、外排泵功能和蛋白质相互作用网络的新知识，这些网络管理着革兰氏阳性细胞膜和细胞被膜的维持/内稳态。

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.