Multisite Phosphorylation Regulates GpsB Function in Cephalosporin Resistance of Enterococcus faecalis.
Multisite Phosphorylation Regulates GpsB Function in Cephalosporin Resistance of Enterococcus faecalis.
复制标题
多位点磷酸化调节粪肠球菌头孢菌素耐药中的 GpsB 功能。
DOI：
10.1016/j.jmb.2023.168216
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发表时间：
2023
影响因子：
5.6
通讯作者：
Kristich,ChristopherJ
中科院分区：
文献类型：
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作者：
VanZeeland,NicoleE;Schultz,KathrynM;Klug,CandiceS;Kristich,ChristopherJ
肠球菌是人类的正常细菌，也是医院获得性感染的主要原因。肠球菌感染可能难以治疗，因为肠球菌具有内在和获得性抗生素耐药性，例如对头孢菌素的耐药性。在粪肠球菌中，跨膜激酶IreK是细菌PASTA激酶家族的一员，是头孢菌素耐药所必需的。IreK的活性由细胞质蛋白GpsB增强，GpsB促进IreK自磷酸化和信号传导以驱动头孢菌素抗性。先前的磷酸化蛋白质组学研究确定了GpsB上8个推定的IreK依赖性磷酸化位点，但GpsB磷酸化的功能重要性尚不清楚。在这里，我们使用遗传和生化方法来定义GpsB上的三个磷酸化位点，这些位点在功能上影响IreK活性和头孢菌素耐药性。两个位点（S80和T84）的磷酸化用于削弱GpsB在体内激活IreK的能力，表明这些位点的磷酸化充当IreK的负反馈的手段。第三个磷酸化位点（T133）发生在肠球菌GpsB同源物所特有的称为C-末端延伸的GpsB片段中。C-末端延伸在溶液中是高度移动的，表明它在很大程度上是非结构化的，并且T133的磷酸化似乎能够在S80 / T84处有效磷酸化。总的来说，我们的研究结果与GpsB的多位点磷酸化损害其激活IreK的能力，从而减少通过IreK依赖性途径的信号转导和调节表型头孢菌素耐药性的模型一致。
Enterococci are normal human commensals and major causes of hospital-acquired infections. Enterococcal infections can be difficult to treat because enterococci harbor intrinsic and acquired antibiotic resistance, such as resistance to cephalosporins. InEnterococcus faecalis, the transmembrane kinase IreK, a member of the bacterial PASTA kinase family, is essential for cephalosporin resistance. The activity of IreK is boosted by the cytoplasmic protein GpsB, which promotes IreK autophosphorylation and signaling to drive cephalosporin resistance. A previous phosphoproteomics study identified eight putative IreK-dependent phosphorylation sites on GpsB, but the functional importance of GpsB phosphorylation was unknown. Here we used genetic and biochemical approaches to define three sites of phosphorylation on GpsB that functionally impact IreK activity and cephalosporin resistance. Phosphorylation at two sites (S80 and T84) serves to impair the ability of GpsB to activate IreKin vivo, suggesting phosphorylation of these sites acts as a means of negative feedback for IreK. The third site of phosphorylation (T133) occurs in a segment of GpsB termed the C-terminal extension that is unique to enterococcal GpsB homologs. The C-terminal extension is highly mobile in solution, suggesting it is largely unstructured, and phosphorylation of T133 appears to enable efficient phosphorylation at S80 / T84. Overall our results are consistent with a model in which multisite phosphorylation of GpsB impairs its ability to activate IreK, thereby diminishing signal transduction through the IreK-dependent pathway and modulating phenotypic cephalosporin resistance.