Abstract
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.
Highlights
Chlamydia pneumoniae is the causative agent of a variety of acute and chronic diseases of the upper and lower respiratory tract including pneumonia, asthma, bronchitis and sinusitis, and is associated with ~50% of cases of chronic obstructive pulmonary disease [1]
We show that Rab5, Rab11 and Rab14 are retained in the vesicular membrane, while Rab4 and Rab7 subsequently disappear
When we monitored the C. pneumoniae infection process from 5 to 60 min p.i. in cells transfected with the PI(3,4,5)P-specific biomarker Btk-PH-GFP, we found that 91% of elementary bodies (EBs) that colocalized with epidermal growth factor receptor (EGFR) at 5 min p.i. showed a ring of PI(3,4,5)P surrounding the bacterial DNA
Summary
Chlamydia pneumoniae is the causative agent of a variety of acute and chronic diseases of the upper and lower respiratory tract including pneumonia, asthma, bronchitis and sinusitis, and is associated with ~50% of cases of chronic obstructive pulmonary disease [1]. Binding to EGFR results in receptor phosphorylation, which activates downstream signaling cascades and recruits the endocytosis adaptor proteins c-Cbl and Grb to the bacterial entry sites [3]. In a previous study we made the surprising observation that the internalized bacteria remain associated with activated EGFR even after reaching their final destination in the perinuclear region [3]. Ligand-mediated activation of EGFR either leads to degradation of the receptor via the lysosomal pathway or its recycling to the plasma membrane [4]. In order to establish the early inclusion, C. pneumoniae must somehow intervene in EGFR-mediated events so as to avoid EGFR-triggered degradation or rerouting back to the plasma membrane
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