Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier II, CNRS UMR 5235, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
J Biol Chem. 2012 Dec 21;287(52):43607-19. doi: 10.1074/jbc.M112.418913. Epub 2012 Nov 6.
The Staphylococcus aureus serine/threonine protein kinase Stk1 (also known as PknB) affects different key pathways such as cell wall metabolism, antibiotic susceptibility, and regulation of virulence. Here we report that the catabolite control protein A (CcpA), a highly conserved regulator of carbon catabolite repression and virulence in a number of gram-positive pathogens, was efficiently phosphorylated in vitro and in vivo by Stk1 in S. aureus, whereas the CcpA homologues of Bacillus subtilis and Bacillus anthracis were not affected by the Stk1 orthologue PrkC. Mass spectrometry and mutational analyses identified Thr-18 and Thr-33 as the phosphoacceptors; both are located in the DNA binding domain of this protein. Electrophoretic mobility shift assays demonstrated that the CcpA DNA binding activity was completely abrogated for the phosphorylated CcpA. The physiological relevance of CcpA phosphorylation was assessed by generating CcpA phosphoablative (T18A/T33A) or phosphomimetic (T18D/T33D) mutants. In contrast to the wild-type and phosphoablative ccpA alleles, introduction of the phosphomimetic ccpA allele in a ΔccpA mutant failed to restore the parental biofilm formation profile and the transcription of citZ and hla to levels seen with the wild type. The strong up regulation of ccpA transcripts and CcpA level in the ccpA mutant trans-complemented with the phosphomimetic CcpA variant suggest furthermore that CcpA acts as a negative regulator of its own expression. Together, these findings demonstrate that Stk1-driven phosphorylation of CcpA inhibits its DNA binding activity toward its regulon in S. aureus, representing a novel regulatory mechanism of CcpA activity in addition to the well known regulation via HprKP/Hpr in this clinically important pathogen.
金黄色葡萄球菌丝氨酸/苏氨酸蛋白激酶 Stk1（也称为 PknB）影响不同的关键途径，如细胞壁代谢、抗生素敏感性和毒力调节。在这里，我们报告说，代谢物控制蛋白 A (CcpA) 是许多革兰氏阳性病原体中碳分解代谢物阻遏和毒力的高度保守调节剂，可被金黄色葡萄球菌中的 Stk1 在体外和体内有效磷酸化，而枯草芽孢杆菌和炭疽芽孢杆菌的 CcpA 同源物不受 Stk1 同源物 PrkC 的影响。质谱和突变分析鉴定出 Thr-18 和 Thr-33 为磷酸受体；这两个位点都位于该蛋白的 DNA 结合域。电泳迁移率变动分析表明，磷酸化 CcpA 的 CcpA DNA 结合活性完全丧失。通过生成 CcpA 磷酸缺失（T18A/T33A）或磷酸模拟（T18D/T33D）突变体来评估 CcpA 磷酸化的生理相关性。与野生型和磷酸缺失 ccpA 等位基因相比，在 ΔccpA 突变体中引入磷酸模拟 ccpA 等位基因未能将亲本生物膜形成谱和 citZ 和 hla 的转录恢复到与野生型相同的水平。在 ccpA 突变体中引入磷酸模拟 ccpA 等位基因后，ccpA 转录物和 CcpA 水平的强烈上调进一步表明 CcpA 作为其自身表达的负调节剂发挥作用。总之，这些发现表明，Stk1 驱动的 CcpA 磷酸化抑制了其在金黄色葡萄球菌中对其调控基因的 DNA 结合活性，代表了 CcpA 活性的一种新的调节机制，除了在这种临床上重要的病原体中通过 HprKP/Hpr 进行的众所周知的调节之外。
