Mycobacterium tuberculosis ClpX interacts with FtsZ and interferes with FtsZ assembly.
Mycobacterium tuberculosis ClpX interacts with FtsZ and interferes with FtsZ assembly.
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DOI：
10.1371/journal.pone.0011058
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发表时间：
2010-07-06
期刊：
影响因子：
3.7
通讯作者：
Rajagopalan M
中科院分区：
文献类型：
--
作者：
Dziedzic R;Kiran M;Plocinski P;Ziolkiewicz M;Brzostek A;Moomey M;Vadrevu IS;Dziadek J;Madiraju M;Rajagopalan M
FtsZ 在细胞中部分裂位点以 Z 环形式组装对于真细菌细胞分裂过程的启动至关重要。目前尚不清楚人类病原体结核分枝杆菌如何调节这一过程。在这里，我们发现 clpX 的表达在巨噬细胞感染和暴露于头孢氨苄抗生素时上调，在这种情况下 FtsZ 环组装被延迟。独立地，我们使用下拉、固相结合、细菌二杂交和分枝杆菌蛋白片段互补测定法证明，结核分枝杆菌 FtsZ 与 ClpX（ClpXP 蛋白酶的底物识别结构域）相互作用。 FtsZ 与 ClpX 一起孵育增加了 FtsZ 依赖 GTP 聚合的临界浓度。免疫印迹显示野生型结核分枝杆菌细胞内ClpX与FtsZ的比例约为1∶2。 ClpX 的过量产生增加了细胞长度并调节 FtsZ 在细胞中部位点的定位；然而，细胞内 FtsZ 水平不受影响。 ClpX-CFP 融合蛋白定位于细胞极和细胞中位点，并与 FtsZ-YFP 蛋白共定位。 ClpX 还与 FtsZ 突变蛋白相互作用，该突变蛋白具有结合和水解 GTP 的缺陷，并且可能与其他蛋白相互作用。综上所述，我们的结果表明结核分枝杆菌 ClpX 与 FtsZ 原聚体以化学计量相互作用，与其核苷酸结合状态无关，并负向调节 FtsZ 活性，从而调节细胞分裂。
FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.
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