Department of Microbiology and Immunology, Geisel School of Medicine, Dartmouth College, Hanover, New Hampshire, United States of America.
Department of Medicine, Geisel School of Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, United States of America.
PLoS Pathog. 2021 Mar 1;17(3):e1009338. doi: 10.1371/journal.ppat.1009338. eCollection 2021 Mar.
Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.
宿主防御蛋白 (HDPs)，又名防御素，是先天免疫系统的关键组成部分，通过插入细菌膜形成孔来杀死入侵和定植的微生物。为了确保生存，金黄色葡萄球菌等微生物已经发展出了感知和响应 HDPs 的生存策略。金黄色葡萄球菌的一个关键策略是一种称为 GraRS 的双组分系统 (TCS)，与由膜渗透酶 VraG 和 ATP 酶 VraF 组成的外排泵偶联，类似于枯草芽孢杆菌的 BceRS-BceAB 系统，但存在明显差异。虽然已经表明膜传感器 GraS 的 9 个带负电荷的氨基酸细胞外环参与了感应，但主要问题是如此小的环如何感应多种 HDPs。本研究中的突变分析表明，与亲本耐甲氧西林金黄色葡萄球菌 (MRSA) 菌株 JE2 相比，vraG 突变体在下游效应物 mprF 的激活减少、表面正电荷减少以及用 LL-37 增强 2 小时杀伤方面表现出与 graS 突变体相似的表型。计算机分析表明，VraG 含有一个位于第 7 个和第 8 个跨膜段 (共 10 个) 之间的单个 200 个残基的细胞外环 (EL)。值得注意的是，VraG 的 EL 缺失增强了 mprF 的表达，增加了表面正电荷，并提高了与亲本 JE2 相比在 LL-37 中的存活率。由于 VraG 的 EL 富含赖氨酸残基 (16%)，而 GraS 的 EL 中富含带负电荷的天冬氨酸残基 (9 个中有 3 个)，我们通过表明 VraG 的 EL 中的 K380 是一个重要的残基，可能与 GraS 相互作用来干扰 GraS 介导的信号转导，揭示了电荷相互作用的作用。细菌双杂交分析也支持了 VraG 的 EL 与 GraS 的 EL 之间的相互作用。总的来说，我们展示了外排泵的一个有趣方面，即膜渗透酶通过抑制涉及 VraG 的 EL 中带电残基的 GraS 感应来破坏 HDP 信号。
