Model of early internalization and establishment of intracellular niche.

<p>The model shows that binding and internalization of <i>C</i>. <i>pneumoniae</i> EBs is mediated by EGFR and requires receptor activation. Activated EGFR stimulates the PI3 kinase located at the plasma membrane, thus generating PI(3,4,5)P, which is converted to PI3P. The internalized EB remains in a PI3P-positive endosome, which acquires several Rab GTPases e.g. Rab4, Rab5, Rab7, Rab11 and Rab14. Loss of Rab4 and Rab7 subsequently mark the early inclusion as a recycling endosome. In addition the Rab11/Rab14 adaptor protein Rab11-Fip2 is recruited, which is essential for intracellular positioning of the inclusion. This is achieved by interaction of Fip2 with the actin-based motor protein myosin Vb.</p

The Chlamydia pneumoniae Invasin Protein Pmp21 Recruits the EGF Receptor for Host Cell Entry

Infection of mammalian cells by the strictly intracellular pathogens Chlamydiae requires adhesion and internalization of the infectious Elementary Bodies (EBs). The components of the latter step were unknown. Here, we identify Chlamydia pneumoniae Pmp21 as an invasin and EGFR as its receptor. Modulation of EGFR surface expression evokes correlated changes in EB adhesion, internalization and infectivity. Ectopic expression of EGFR in EGFR-negative hamster cells leads to binding of Pmp21 beads and EBs, thus boosting the infection. EB/Pmp21 binding and invasion of epithelial cells results in activation of EGFR, recruitment of adaptors Grb2 and c-Cbl and activation of ERK1/2, while inhibition of EGFR or MEK kinase activity abrogates EB entry, but not attachment. Binding of Grb2 and c-Cbl by EGFR is essential for infection. This is the first report of an invasin-receptor interaction involved in host-cell invasion by any chlamydial species

Entry of <i>C</i>. <i>pneumoniae</i> EBs is PI3K dependent.

<p><b>(A)</b> Quantification of colocalization of PI(3,4,5)P and EGFR in cells transiently transfected with Btk-PH-GFP. Cells were infected at MOI 5, fixed with PFA at the indicated time points (5–60 min), and stained for endogenous EGFR using anti-EGFR, anti-rabbit Alexa594 and DAPI. Confocal images of 30 individual cells were used to analyze colocalization (n = 3). <b>(B)</b> The arrow marks a bacterial entry site at 5 min p.i. visualized by confocal imaging. The bacterial DNA stained with DAPI colocalizes with the “ring-like” signal of PI(3,4,5)P detected with Btk-PH-GFP. Furthermore, the entry site is marked by PI3P detected with mCherry-2xFYVE and the endogenous EGFR surrounding the internalized EB. <b>(C)</b> Quantification of <i>C</i>. <i>pneumoniae</i> internalization in cells pretreated with LY29 (50 μmol) for 2 h prior to infection. At 2 hpi internalization was measured by comparison numbers of internal and external EBs in 30 imaged cells (n = 4). <b>(D)</b> Quantification of EBs (stained with DAPI) colocalizing with PI(3,4,5)P (labeled by Btk-PH-GFP) at 5 min p.i. in cells pretreated with AG1478 (2 μmol) or LY29 (50 μmol) for 2 h (n = 3). <b>(E)</b> Quantification of colocalization of PI3P and EGFR in infected cells expressing GFP-2xFYVE. EGFR was stained as before (n = 3). <b>(F)</b> Colocalization of PI3P, EGFR and internalized EBs at 15 min p.i. by expression of GFP-2xFYVE and detection of EGFR. <b>(G)</b> Quantification of internalization in cells pretreated for 2 h with the SHIP2 inhibitor AS1949490 (10 μmol). *** <i>P</i> value ≤0.001. Bar 1 μm.</p

Acquisition of Rab11 and Rab11-Fip2—A novel strategy for <i>Chlamydia pneumoniae</i> early survival

<div><p>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 <i>Chlamydia pneumoniae</i> 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 <i>C</i>. <i>pneumoniae</i> 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 <i>C</i>. <i>pneumoniae</i> establishes the intracellular niche essential for its survival.</p></div

The acquisition of myosin Vb by the Rab11-Fip2 adaptor protein is essential for the <i>C</i>. <i>pneumoniae</i> infection.

<p><b>(A)</b> Quantification of colocalization of EGFR-positive EBs with GFP-MyosinVb from 5 min to 60 min p.i. (n = 3). <b>(B)</b> Confocal images of colocalization of GFP-MyosinVb and EGFR with <i>C</i>. <i>pneumoniae</i> EBs at 15 min p.i. White arrows indicate colocalization. Bar 1 μm. <b>(C)</b> Immunoblot analysis of cells transiently transfected for 72 h with control or myosin Vb miRNA plasmids and lysed in phospho-Lysis buffer. Samples were fractionated by SDS/PAGE and probed with antibodies against myosin Vb to monitor knockdown; β-actin served as loading control. The pixel intensity of bands was analyzed with ImageJ. The arrow marks the specific myosin Vb band. <b>(D)</b> Relative internalization of EBs into cells transfected for 72 h with control or MyosinVb miRNA plasmids was measured by q-PCR at 2 hpi as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006556#ppat.1006556.g004" target="_blank">Fig 4</a> (n = 6). <b>(E)</b> Quantification of internalization of EBs into PI3P endosomes at 30 min p.i. Confocal images of 30 individual cells transiently expressing mCherry-2xFYVE and GFP or GFP-MyoVbtail were analyzed (n = 3). <b>(F)</b> Quantification of the inclusion diameter in cells expressing in GFP or GFP-MyoVbtail cells at 48 hpi. Inclusions were stained with anti-Cpn0147 and anti-rabbit Alexa594 and their diameters were measured in 30 individual cells (n = 3). *** <i>P</i> value ≤0.001, n.s. <i>P</i> value ≤0.01 <b>(G)</b> Confocal images of cells quantified in (F). Arrows mark the smaller inclusions in cells expressing GFP-MyoVbtail. Green lines mark the outline of the inclusions used for quantification. Bar 5 μm.</p

The early <i>C</i>. <i>pneumoniae</i> inclusion is a recycling endosome.

<p><b>(A)</b> Quantification of colocalization of EGFR-positive <i>C</i>. <i>pneumoniae</i> EBs with GFP-Rab11, GFP-Rab14, GFP-Rab4 at 5–60 min p.i. in transiently transfected cells over the course of (n = 3). <b>(B)</b> Confocal images of colocalization of EBs (visualized with DAPI) with GFP-Rab11 and EGFR (left), or with GFP-Rab14 and EGFR (right) at 15 min p.i. White arrows indicate colocalization. Bar 1μm. <b>(C, D)</b> HEp-2 cells stably expressing wild-type GFP-Rab11 or the dominant-negative (S25N) or constitutively active (Q70L) GFP-Rab11 variant. <b>(C)</b> Quantification of EB internalization at 2 hpi in 30 individual cells (n = 4). <b>(D)</b> Quantification of infection at 48 hpi in 40 visual fields (n = 4). <i>P</i> values: * ≤ 0.1; *** ≤0.001. n.s.–not significantly different.</p

Development of early <i>C</i>. <i>pneumoniae</i> inclusion is dependent on Akt/PIKfyve activity.

<p><b>(A)</b> Quantification of colocalization of Rab7 and EGFR with chlamydial EBs during the first hour p.i. in cells transiently transfected with GFP-Rab7 (n = 3). <b>(B)</b> Confocal images of GFP-Rab7 colocalizing with EBs in PI3P-positive endosomes (visualized by mCherry-2xFYVE and DAPI, respectively) at 15 min (top row) and 30 min p.i. (bottom row). White arrows indicate colocalization. Bar 1 μm. <b>(C–E)</b> Quantification of EB internalization in cells pretreated with the indicated inhibitor for 2 h prior to infection. Internalization was analyzed in 30 individual cells. <b>(C)</b> Entry of EBs into cells pretreated with the Akt inhibitor MK22 (3 μmol) or DMSO for 2 h prior to infection. Internalization was quantified microscopically at 2 h p.i. (n = 3). <b>(D)</b> Effects of pretreatment with the PIKfyve inhibitor YM201636 (800 nmol) for 2 h prior to infection on EB internalization (n = 3). <b>(E)</b> Quantification of GFP-Rab7 colocalization with EBs in PI3P-positive endosomes (visualized by mCherry-2xFYVE and DAPI) at 30 min p.i. in cells pretreated with MK22, the PIKfyve inhibitor or DMSO (n = 3). *** <i>P</i> value ≤0.001, n.s. <i>P</i> value <0.05.</p

The Rab11-binding domain of Fip2 is essential for <i>C</i>. <i>pneumoniae</i> infection.

<p><b>(A)</b> Schematic representation of Fip2. <b>(B, C)</b> Quantification of EB internalization <b>(B)</b> or chlamydial infection <b>(C)</b> in HEp-2 cells stably expressing the indicated GFP-Fip2 mutant variants. <b>(B)</b> Analysis of <i>C</i>. <i>pneumoniae</i> EB internalization at 2 hpi in 30 individual cells (n = 3). <b>(C)</b> Quantification of infection based on the numbers of inclusions found in 40 visual fields at 48 hpi (n = 4). <b>(D-G)</b> Colocalization analysis of EBs in PI3P-positive endosomes (visualized with mCherry-2xFYVE and DAPI) in HEp-2 cells stably expressing GFP-Fip2 mutant variants at 15 min to 60 min p.i. For each time point the intracellular distribution of inclusions was assessed in 30 cells and the localization pattern was classified as follows: P, endosome in the cell periphery; N, endosomes in the perinuclear region; vA, vesicular aggregates in cell periphery. <b>(D)</b> Wild-type GFP-Fip2. <b>(E)</b> GFP-Fip2ΔC2. <b>(F)</b> GFP-Fip2ΔRBD. <b>(F)</b> GFP-Fip2ΔMyoBD. *** <i>P</i> value ≤0.001, ** <i>P</i> value ≤0.01, * <i>P</i> value ≤0.05, n.s. <i>P</i> value ≤0.01.</p