The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus.
The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus.
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金黄色葡萄球菌膜通透酶VraG的胞外环与GraS相互作用以感测阳离子抗菌肽。
DOI：
10.1371/journal.ppat.1009338
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发表时间：
2021-03
期刊：
影响因子：
6.7
通讯作者：
Cheung AL
中科院分区：
文献类型：
--
作者：
Cho J;Costa SK;Wierzbicki RM;Rigby WFC;Cheung AL
宿主防御蛋白（HDPs），又名防御素，是先天免疫系统的关键部分，它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制定了生存策略来感知和响应hdp。金黄色葡萄球菌的一个关键策略是一个叫做GraRS的双组分系统（TCS），它与一个由膜渗透酶VraG和一个atp酶VraF组成的外排泵相结合，类似于枯草芽孢杆菌的bcer - bceab系统，但有明显的区别。虽然膜传感器GraS的9个带负电荷的氨基酸胞外环已被证明参与传感，但主要的问题是这样一个小环如何能够传感不同的hdp。本研究的突变分析显示，vraG突变体在下游效应物mprF的激活降低、表面正电荷减少和2小时增加方面表现出了graS突变体的表型。与亲本MRSA菌株JE2相比，LL-37的杀伤效果更好。在硅分析显示，VraG包含一个单独的200个残基的胞外环（EL），位于第7和第8跨膜段之间（10个）。值得注意的是，与亲本JE2相比，VraG中EL的缺失增强了LL-37的mprF表达，增加了表面正电荷，提高了存活。由于VraG的EL中富含赖氨酸残基（16%），而GraS的EL中则以带负电的天冬氨酸残基为主（9个残基中有3个），因此我们揭示了电荷相互作用的作用，表明VraG EL中的K380是可能与GraS相互作用以干扰GraS介导的信号传导的重要残基。细菌双杂交分析也支持VraG的EL与GraS的EL相互作用。总的来说，我们展示了外排泵的一个有趣的方面，即膜渗透酶通过抑制涉及VraG EL中带电残基的GraS传感来破坏HDP信号。细菌利用TCS来感知和响应环境刺激。在金黄色葡萄球菌中，细菌对宿主HDPs的防御是从GraRS上安装的，其中GraS是膜传感器，包括两个跨膜片段，构成一个小的细胞外环和一个细胞质尾部。我们之前已经证明，金黄色葡萄球菌中HDP的关键传感部分是传感器GraS的9个残基细胞外环，其中包含3个对HDP传感至关重要的带负电荷的天冬氨酸残基。然而，预计9-残基环过于灵活，无法为金黄色葡萄球菌对不同HDPs的特异性反应提供适当的构象。编码GraRS的基因位于vraFG编码的外排系统附近，其中VraG是膜渗透酶，VraF是atp酶。我们发现VraG有助于感知由GraS介导的HDPs。更具体地说，VraG富含200个赖氨酸残基的胞外环通过K380介导的电荷相互作用干扰HDP的GraS感应。这一发现代表了外排泵的另一个有趣的方面，即膜透酶通过抑制GraS信号干扰HDP感知。
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. TCS have been used by bacteria to sense and respond to environmental stimuli. In S. aureus, the bacterial defense against host HDPs is mounted from GraRS where GraS is the membrane sensor comprising two transmembrane segments framing a small extracellular loop, and a cytoplasmic tail. We have shown previously that the critical sensing moiety for HDPs in S. aureus is the 9-residue extracellular loop of the sensor GraS that contains 3 negatively charged aspartic acid residues critical to HDP sensing. However, the 9-residue loop is predicted to be too flexible to confer proper conformation for the specificity response to divergent HDPs in S. aureus. The genes encoding GraRS lies adjacent to an efflux system encoded by vraFG where VraG is the membrane permease and VraF is the ATPase. We showed that VraG contributes to sensing of HDPs mediated by GraS. More specifically, the 200-residue lysine rich extracellular loop of VraG interferes with GraS sensing of HDP via charge interaction mediated in part by K380. This finding represents another interesting facet of efflux pump whereby the membrane permease interferes with HDP sensing by inhibiting GraS signaling.
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影响因子：
5.2
作者：
Bayer, Arnold S.;Mishra, Nagendra N.;Yang, Soo-Jin
通讯作者：
Yang, Soo-Jin
DOI：
10.1006/bbrc.2000.2277
发表时间：
2000-03-16
影响因子：
3.1
作者：
Kuroda, M;Kuwahara-Arai, K;Hiramatsu, K
通讯作者：
Hiramatsu, K
影响因子：
3.6
作者：
Li, Min;Cha, David J.;Otto, Michael
通讯作者：
Otto, Michael
不同的细菌基因表达模式和减弱的宿主免疫反应与持续耐甲氧西林抗甲氧西葡萄球菌金黄色葡萄球菌菌血症期间低水平万古霉素耐药性的演变有关。
影响因子：
4.2
作者：
Howden, Benjamin P.;Smith, Danielle J.;Mansell, Ashley;Johnson, Paul D. R.;Ward, Peter B.;Stinear, Timothy P.;Davies, John K.
通讯作者：
Davies, John K.
DOI：
10.1073/pnas.0702159104
发表时间：
2007-05-29
影响因子：
11.1
作者：
Li, Min;Lai, Yuping;Otto, Michael
通讯作者：
Otto, Michael