Stinemetz Emily K, Gao Peng, Pinkston Kenneth L, Montealegre Maria Camila, Murray Barbara E, Harvey Barrett R
Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center, Houston, Texas, United States of America.
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas, United States of America.
PLoS One. 2017 Oct 19;12(10):e0186706. doi: 10.1371/journal.pone.0186706. eCollection 2017.
AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.
AtlA是粪肠球菌中参与细胞分裂和细胞自溶的主要肽聚糖水解酶。分泌型锌金属蛋白酶明胶酶（GelE）已被确定为通过对蛋白质底物进行翻译后修饰来调节细胞功能的重要调节因子。AtlA是GelE的已知靶点，有人提出它们之间的相互作用可调节AtlA的功能。为了研究蛋白酶介导的AtlA翻译后修饰，开发了单克隆抗体作为研究工具。流式细胞术和蛋白质印迹分析表明，在存在GelE的情况下，表面结合的AtlA主要以N端截短形式存在，而在不存在GelE的情况下，AtlA的N端结构域得以保留。我们通过N端测序确定了主要的GelE切割位点，该位点位于富含T/E的结构域I和催化区域结构域II之间的过渡区域附近。AtlA的截短对其肽聚糖水解活性没有影响。然而，我们观察到有效的AtlA介导的细胞分裂需要N端切割，而未加工的AtlA无法将正在分裂的细胞分解为单个单元。此外，我们观察到加工后的AtlA倾向于定位于野生型细胞的细胞隔膜，而ΔgelE菌株中未加工的AtlA则分散在细胞表面。综合这些结果表明，AtlA隔膜定位和随后的细胞分离可以通过单一的GelE介导的N端切割事件来调节，这为AtlA的翻译后修饰以及控制链形成和细胞分离的机制提供了新的见解。
