Acquisition of Rab11 and Rab11-Fip2—A novel strategy for Chlamydia pneumoniae early survival

The initial steps in chlamydial infection involve adhesion and internalization into host cells and, most importantly, modification of the nascent inclusion to establish the intracellular niche. Here, we show that Chlamydia pneumoniae enters host cells via EGFR-dependent endocytosis into an early endosome with a phosphatidylinositol 3-phosphate (PI3P) membrane identity. Immediately after entry, the early chlamydial inclusion acquires early endosomal Rab GTPases including Rab4, Rab5, Rab7, as well as the two recycling-specific Rabs Rab11 and Rab14. While Rab5, Rab11 and Rab14 are retained in the vesicular membrane, Rab4 and Rab7 soon disappear. Loss of Rab7 enables the C. pneumoniae inclusion to escape delivery to, and degradation in lysosomes. Loss of Rab4 and retention of Rab11/ Rab14 designates the inclusion as a slowly recycling endosome—that is protected from degradation. Furthermore, we show that the Rab11/ Rab14 adaptor protein Rab11-Fip2 (Fip2) is recruited to the nascent inclusion upon internalization and retained in the membrane throughout infection. siRNA knockdown of Fip2 demonstrated that the protein is essential for internalization and infection, and expression of various deletion variants revealed that Fip2 regulates the intracellular positioning of the inclusion. Additionally, we show that binding to Rab11 and Fip2 recruits the unconventional actin motor protein myosin Vb to the early inclusion and that together they regulate the relocation of the nascent inclusion from the cell periphery to the perinuclear region, its final destination. Here, we characterize for the first time inclusion identity and inclusion-associated proteins to delineate how C. pneumoniae establishes the intracellular niche essential for its survival.衣原体感染的初始步骤包括黏附与内化进入宿主细胞，而最为关键的环节是对新生包涵体（nascent inclusion）进行修饰，以建立可供其寄生的细胞内微生态位（intracellular niche）。本研究证实，肺炎衣原体（Chlamydia pneumoniae）通过依赖表皮生长因子受体（EGFR）的内吞作用进入宿主细胞，并进入具有磷脂酰肌醇3-磷酸（PI3P）膜特征的早期内体中。入侵后即刻，新生衣原体包涵体即可获得早期内体相关的Rab GTP酶（Rab GTPases），包括Rab4、Rab5、Rab7，以及两种特异性参与膜蛋白回收过程的Rab家族蛋白Rab11与Rab14。尽管Rab5、Rab11和Rab14会持续保留在囊泡膜上，但Rab4与Rab7会迅速消失。Rab7的缺失使得肺炎衣原体包涵体能够逃逸溶酶体递送途径并避免被降解；而Rab4的缺失与Rab11/Rab14的保留，则将该包涵体定义为一种可免于降解的慢速回收内体。此外，本研究发现，Rab11/Rab14的适配蛋白Rab11-Fip2（Fip2）会在肺炎衣原体内化后被招募至新生包涵体，并在整个感染周期中持续定位于囊泡膜上。通过小干扰RNA（siRNA）敲低Fip2的实验证实，该蛋白对于肺炎衣原体的内化与感染过程不可或缺；而对各类缺失变体的表达分析则表明，Fip2可调控包涵体的细胞内定位。研究还显示，结合Rab11与Fip2可将非常规肌动蛋白马达蛋白肌球蛋白Vb（myosin Vb）招募至早期包涵体，二者协同调控新生包涵体从细胞外周向其最终归宿——核周区域的转运过程。本研究首次系统表征了衣原体包涵体的膜特性及其相关蛋白，以阐明肺炎衣原体如何建立其生存所必需的细胞内微生态位。