Roles of Staphylococcus aureus Mnh1 and Mnh2 Antiporters in Salt Tolerance, Alkali Tolerance, and Pathogenesis.
Roles of Staphylococcus aureus Mnh1 and Mnh2 Antiporters in Salt Tolerance, Alkali Tolerance, and Pathogenesis.
复制标题
DOI：
10.1128/jb.00611-17
复制
发表时间：
2018-03-01
影响因子：
3.2
通讯作者：
Krulwich TA
中科院分区：
文献类型：
--
作者：
Vaish M;Price-Whelan A;Reyes-Robles T;Liu J;Jereen A;Christie S;Alonzo F 3rd;Benson MA;Torres VJ;Krulwich TA
金黄色葡萄球菌有三种阳离子/质子逆向转运蛋白。3型家族包括两个多亚基Na+/H+(MNH)逆向转运蛋白，即Mnh1和Mnh2。这些抗转运蛋白是由7个疏水的膜结合蛋白亚基组成的簇。MNH逆向转运蛋白在维持原核生物细胞质pH，使其在极端环境胁迫下存活方面起着重要作用。在本研究中，我们研究了Mnh1和Mnh2在金黄色葡萄球菌中的生理作用和催化性质。将Mnh1和Mnh2分别克隆到反转运蛋白缺陷型KNabc大肠杆菌中的pGEM3Z+载体中。反转运体的催化性能是在外翻(内翻)囊泡中测量的。MnH1反向转运蛋白在pH为7.5时表现出明显的Na+/H+离子交换。MnH_2对Na~+/H~+和K~+/H~+离子都有明显的交换作用，尤其是在pH值为8.5时。在高盐条件下，mnhA1基因的缺失导致金黄色葡萄球菌在pH 7.5~9的范围内生长速度显著降低。mnhA2的缺失也有类似的影响，但主要在pH 8.5~9.5的范围内。MnhA1和mnhA2的双缺失导致金黄色葡萄球菌的生长速度严重下降，主要是在pH值高于8.5时。在小鼠体内感染模型中，通过它们对毒力的支持，也评估了金黄色葡萄球菌中这两个反转运蛋白功能丧失的影响。MnhA1基因的缺失导致金黄色葡萄球菌对小鼠的毒力丧失，而mnh2基因的缺失不会导致毒力的变化。本研究重点研究了Mnh1和Mnh2阳离子/质子逆向转运蛋白在金黄色葡萄球菌中的催化性质和生理作用，以及它们在不同胁迫条件下的作用。Mnh1逆向转运蛋白对Na+/H+逆向转运蛋白具有催化活性，并且在维持耐盐(pH 7.5)时起重要作用，而Mnh2逆向转运蛋白对Na+/H+和K+/H+具有催化逆向转运蛋白活性，这两种逆向转运蛋白对金黄色葡萄球菌的耐渗和耐盐都有作用。在小鼠感染模型中评估了单一缺失mnhA1或mnhA2的金黄色葡萄球菌的研究。结果表明，mnhA1在金黄色葡萄球菌毒力中起主要作用，而不是mnhA2。
Staphylococcus aureus has three types of cation/proton antiporters. The type 3 family includes two multisubunit Na+/H+ (Mnh) antiporters, Mnh1 and Mnh2. These antiporters are clusters of seven hydrophobic membrane-bound protein subunits. Mnh antiporters play important roles in maintaining cytoplasmic pH in prokaryotes, enabling their survival under extreme environmental stress. In this study, we investigated the physiological roles and catalytic properties of Mnh1 and Mnh2 in S. aureus. Both Mnh1 and Mnh2 were cloned separately into a pGEM3Z+ vector in the antiporter-deficient KNabc Escherichia coli strain. The catalytic properties of the antiporters were measured in everted (inside out) vesicles. The Mnh1 antiporter exhibited a significant exchange of Na+/H+ cations at pH 7.5. Mnh2 showed a significant exchange of both Na+/H+ and K+/H+ cations, especially at pH 8.5. Under elevated salt conditions, deletion of the mnhA1 gene resulted in a significant reduction in the growth rate of S. aureus in the range of pH 7.5 to 9. Deletion of mnhA2 had similar effects but mainly in the range of pH 8.5 to 9.5. Double deletion of mnhA1 and mnhA2 led to a severe reduction in the S. aureus growth rate mainly at pH values above 8.5. The effects of functional losses of both antiporters in S. aureus were also assessed via their support of virulence in a mouse in vivo infection model. Deletion of the mnhA1 gene led to a major loss of S. aureus virulence in mice, while deletion of mnh2 led to no change in virulence. IMPORTANCE This study focuses on the catalytic properties and physiological roles of Mnh1 and Mnh2 cation/proton antiporters in S. aureus and their contributions under different stress conditions. The Mnh1 antiporter was found to have catalytic activity for Na+/H+ antiport, and it plays a significant role in maintaining halotolerance at pH 7.5 while the Mnh2 antiporter has catalytic antiporter activities for Na+/H+ and K+/H+ that have roles in both osmotolerance and halotolerance in S. aureus. Study of S. aureus with a single deletion of either mnhA1 or mnhA2 was assessed in an infection model of mice. The result shows that mnhA1, but not mnhA2, plays a major role in S. aureus virulence.