Capmany Anahí, Gambarte Tudela Julián, Alonso Bivou Mariano, Damiani María T
Laboratorio de Bioquímica e Inmunidad, Área de Química Biológica, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas, Mendoza, Argentina.
Front Microbiol. 2019 Apr 3;10:666. doi: 10.3389/fmicb.2019.00666. eCollection 2019.
, an obligate intracellular bacterium, intercepts different trafficking pathways of the host cell to acquire essential lipids for its survival and replication, particularly from the Golgi apparatus via a Rab14-mediated transport. Molecular mechanisms underlying how these bacteria manipulate intracellular transport are a matter of intense study. Here, we show that utilizes Akt/AS160 signaling pathway to promote sphingolipids delivery to the chlamydial inclusion through Rab14-controlled vesicular transport. provokes Akt phosphorylation along its entire developmental life cycle and recruits phosphorylated Akt (pAkt) to the inclusion membrane. As a consequence, Akt Substrate of 160 kDa (AS160), also known as TBC1D4, a GTPase Activating Protein (GAP) for Rab14, is phosphorylated and therefore inactivated. Phosphorylated AS160 (pAS160) loses its ability to promote GTP hydrolysis, favoring Rab14 binding to GTP. Akt inhibition by an allosteric isoform-specific Akt inhibitor (iAkt) prevents AS160 phosphorylation and reduces Rab14 recruitment to chlamydial inclusions. iAkt further impairs sphingolipids acquisition by inclusion and provokes lipid retention at the Golgi apparatus. Consequently, treatment with iAkt decreases chlamydial inclusion size, bacterial multiplication, and infectivity in a dose-dependent manner. Similar results were found in AS160-depleted cells. By electron microscopy, we observed that iAkt generates abnormal bacterial forms as those reported after sphingolipids deprivation or Rab14 silencing. Taken together, our findings indicate that targeting the Akt/AS160/Rab14 axis could constitute a novel strategy to limit chlamydial infections, mainly for those caused by antibiotic-resistant bacteria.
衣原体是一种专性细胞内细菌，它拦截宿主细胞的不同运输途径以获取生存和复制所需的必需脂质，特别是通过Rab14介导的运输从高尔基体获取。这些细菌如何操纵细胞内运输的分子机制是深入研究的课题。在这里，我们表明衣原体利用Akt/AS160信号通路通过Rab14控制的囊泡运输促进鞘脂向衣原体包涵体的递送。衣原体在其整个发育生命周期中都会引发Akt磷酸化，并将磷酸化的Akt（pAkt）募集到包涵体膜上。结果，160 kDa的Akt底物（AS160），也称为TBC1D4，一种Rab14的GTP酶激活蛋白（GAP），被磷酸化并因此失活。磷酸化的AS160（pAS160）失去促进GTP水解的能力，有利于Rab14与GTP结合。变构异构体特异性Akt抑制剂（iAkt）对Akt的抑制可防止AS160磷酸化，并减少Rab14向衣原体包涵体的募集。iAkt进一步损害包涵体对鞘脂的获取，并导致脂质在高尔基体中滞留。因此，用iAkt处理以剂量依赖性方式减小衣原体包涵体大小、细菌增殖和感染性。在AS160缺失的细胞中也发现了类似的结果。通过电子显微镜，我们观察到iAkt产生异常细菌形态，类似于鞘脂剥夺或Rab14沉默后报道的形态。综上所述，我们的研究结果表明，靶向Akt/AS160/Rab14轴可能构成一种限制衣原体感染的新策略，主要针对由抗生素耐药细菌引起的感染。
