Phosphorylation of Mycobacterial PcaA Inhibits Mycolic Acid Cyclopropanation CONSEQUENCES FOR INTRACELLULAR SURVIVAL AND FOR PHAGOSOME MATURATION BLOCK
Phosphorylation of Mycobacterial PcaA Inhibits Mycolic Acid Cyclopropanation CONSEQUENCES FOR INTRACELLULAR SURVIVAL AND FOR PHAGOSOME MATURATION BLOCK
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DOI：
10.1074/jbc.m112.373209
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发表时间：
2012-07-27
影响因子：
4.8
通讯作者：
Kremer, Laurent
中科院分区：
文献类型：
--
作者：
Corrales, Rosa Milagros;Molle, Virginie;Kremer, Laurent
致病性分枝杆菌在巨噬细胞内存活的方式是寄生在吞噬体中，阻止吞噬体成熟并与溶酶体融合。虽然几种细菌成分被认为可以调节吞噬体加工，但参与这一过程的分子调节机制仍然难以捉摸。我们研究了吞噬体成熟阻滞（PMB）是否可以通过丝氨酸/苏氨酸磷酸化信号来调节。在这里，我们证明了分枝杆菌丝氨酸/苏氨酸激酶在体外和分枝杆菌中磷酸化了mycolic酸环丙烷合成酶PcaA，而不是MmaA2，在Thr-168和Thr-183位点。PcaA的磷酸化与甲基转移酶活性的显著降低有关，这与这两种磷酸化受体的战略性结构定位一致。利用BCG δ pcaA突变体，我们发现pcaA是人类单核细胞源性巨噬细胞细胞内存活和防止吞噬体成熟所必需的。通过产生PcaA磷酸化（T168A/T183A）或拟磷（T168D/T183D）突变体，进一步评估PcaA磷酸化的生理相关性。与野生型和磷酸化型pcaA等位基因相比，在Delta pcaA突变体中引入拟磷型pcaA等位基因未能恢复亲本的霉菌酸谱和相应的形态型。重要的是，PcaA拟磷菌株作为δ PcaA突变体，在人巨噬细胞中表现出存活率降低，并且不能阻止吞噬体成熟。我们的研究结果为霉菌酸环丙烷环在PMB中的重要性提供了新的见解，并首次提供了丝氨酸/苏氨酸激酶依赖性调节霉菌酸组成和PMB的机制的证据。
Pathogenic mycobacteria survive within macrophages by residing in phagosomes, which they prevent from maturing and fusing with lysosomes. Although several bacterial components were seen to modulate phagosome processing, the molecular regulatory mechanisms taking part in this process remain elusive. We investigated whether the phagosome maturation block (PMB) could be modulated by signaling through Ser/Thr phosphorylation. Here, we demonstrated that mycolic acid cyclopropane synthase PcaA, but not MmaA2, was phosphorylated by mycobacterial Ser/Thr kinases at Thr-168 and Thr-183 both in vitro and in mycobacteria. Phosphorylation of PcaA was associated with a significant decrease in the methyltransferase activity, in agreement with the strategic structural localization of these two phosphoacceptors. Using a BCG Delta pcaA mutant, we showed that PcaA was required for intracellular survival and prevention of phagosome maturation in human monocyte-derived macrophages. The physiological relevance of PcaA phosphorylation was further assessed by generating PcaA phosphoablative (T168A/T183A) or phosphomimetic (T168D/T183D) mutants. In contrast to the wild-type and phosphoablative pcaA alleles, introduction of the phosphomimetic pcaA allele in the Delta pcaA mutant failed to restore the parental mycolic acid profile and cording morphotype. Importantly, the PcaA phosphomimetic strain, as the Delta pcaA mutant, exhibited reduced survival in human macrophages and was unable to prevent phagosome maturation. Our results add new insight into the importance of mycolic acid cyclopropane rings in the PMB and provide the first evidence of a Ser/Thr kinase-dependent mechanism for modulating mycolic acid composition and PMB.