The lipid transfer protein CERT interacts with the Chlamydia inclusion protein IncD and participates to ER-Chlamydia inclusion membrane contact sites

Abstract

Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development.

Figure 1. CERT and VAPB localize to C. trachomatis inclusion.

(A–C) HeLa cells were infected with C. trachomatis at a high MOI for 8 h to generate a cluster of incoming bacteria in the perinuclear area of the cell (A) or at a low MOI for 24 h to generate an inclusion originating from a single bacterium (B–C). The infected cells were labeled with antibodies against CERT (CERT, green) (A–C) and the Golgi maker p115 (p115, red) (A, C) or the inclusion membrane protein IncA (IncA, red) (B). (D) HeLa cells expressing a VAPB-GFP fusion protein (VAPB-GFP, green) and infected with C. trachomatis for 24 h, were labeled with antibodies against CERT (CERT, red). The host cell nuclei and the bacterial DNA were labeled with the DNA dye Hoechst (DNA, blue) (A–D). The merge images are shown on the right. Scale Bar, 10 µm.

Figure 2. ER tubules are in close apposition with C. trachomatis inclusion membrane.

(A–C) Electron micrographs of HeLa cells infected with C. trachomatis for 24 h. (A) Low magnification micrograph of the section of an entire inclusion. The black boxes numbered 1 to 4 indicate parts of the inclusion that make close contact with the ER tubules. (B) Higher magnification micrographs corresponding to the black boxes numbered 1 to 4 in (A). (C) Close up of the black boxes numbered 5 and 6 in (B) showing the close apposition (∼10 nm) of the ER tubules with the inclusion membrane. N, Nucleus. RB, Chlamydia Reticulate Body. IB, Chlamydia Intermediate Body. White arrowheads, ER tubules. Scale bar, 5 µm (A), 500 nm (B), 250 nm (C).

Figure 3. CERT- and VAPB-positive ER tubules are in close apposition with C. trachomatis inclusion.

(A–B) Cryo-immunogold electron micrographs of HeLa cells expressing CERT-GFP (A) or VAPB-GFP (B) fusion proteins and infected with C. trachomatis for 24 h. The sections were labeled with anti-GFP antibodies coupled to 10 nm gold particles. RB, Chlamydia Reticulate Body. Black arrowheads, Inclusion membrane. White arrowheads, ER tubule. Scale Bar, 250 nm.

Figure 4. The PH domain mediates the recruitment of CERT to the inclusion.

HeLa cells expressing CERT-GFP fusion proteins (green), containing either full-length CERT (FL-CERT) (A) or the PH domain of CERT only (CERT-PH) (B) or CERT deleted of its PH domain (CERT ΔPH) (C), and infected with C. trachomatis for 24 h, were labeled with the inclusion membrane protein IncA (IncA, red). The DNA dye Hoechst labeled the host cell nuclei and the bacterial DNA (DNA, blue). The merge images are shown on the right. Scale Bar, 10 µm.

Figure 5. CERT recruitment to the inclusion is ARF1-independent.

HeLa cells expressing Arf1-GFP (ARF1, green) were infected for 24 h with C. trachomatis in the absence (upper panels) or in the presence of 1 µg/ml BFA (lower panels) and labeled using antibodies against CERT (red). The DNA dye Hoechst labeled both the nuclei and C. trachomatis (DNA, blue). The merge images are shown on the right. Scale Bar, 10 µm. C. trachomatis infection, performed in the presence of BFA, led to the formation of smaller inclusions as previously reported by Hackstadt et. al. .

Figure 6. The Chlamydia inclusion protein, IncD, interacts with the PH domain of CERT.

(A) Schematic representation of the incDEFG operon and the IncD protein. (B) Lysates from HEK293 cells co-expressing 3xFLAG-IncD and the indicated GFP-CERT fusion proteins were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Left Panel, 2% Total) and the immunoprecipitated proteins (Right Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot with antibodies against GFP (Top Panels) and FLAG (Bottom Panels). (C) Lysates from HEK293 cells co-expressing 3xFLAG-IncD, 3xFLAG-IncE or 3xFLAG-IncF and GFP-CERT fusion proteins were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Right Panel, 2% Total) and the immunoprecipitated proteins (Left Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot with antibodies against GFP (Top Panels) and FLAG (Bottom Panels). (D) Lysates from HEK293 cells co-expressing 3xFLAG-IncD and the indicated GFP or YFP fusion proteins were immunoprecipitated with anti-FLAG M2 beads. A portion of the cell lysate (Right Panel, 2% Total) and the immunoprecipitated proteins (Left Panel, IP) were separated by SDS-PAGE and analyzed by immunoblot with antibodies against GFP (Top Panels) and FLAG (Bottom Panels). (E) The indicated GST fusion proteins, immobilized onto glutathione sepharose were incubated in the presence of the indicated purified MBP fusion proteins. The protein complexes bound to the resin were separated by SDS-PAGE and analyzed by immunoblot with antibodies against MBP (Top Panels) and GST (Bottom Panels). The inputs for each purified protein are shown in the right panels (Input).

Figure 7. IncD co-localizes with CERT onto C. trachomatis inclusion membrane.

(A–B) HeLa cells infected with C. trachomatis for 24 h were fixed and permeabilized using 4% PFA and Saponin, respectively, (PFA-Sap., top panels) or Methanol (MetOH, bottom panels) and labeled with antibodies against the inclusion membrane protein IncD (IncD, green) (A–B) and the inclusion membrane protein IncA (IncA, red) (A) or CERT (CERT, red) (B). The host cell nuclei and the bacterial DNA were labeled with the DNA dye Hoechst (DNA, blue). The merge images are shown on the right. The arrowheads indicate an area of the inclusion membrane enriched in CERT and IncD. Scale Bar, 10 µm.

Figure 8. CERT and VAPA/B depletion impairs C. trachomatis inclusion development.

HeLa cells were transfected with control siRNA (GFPsi) or two different pool of CERT siRNA (CERTsi1 and CERTsi2) or a pool of siRNA against VAPA and VAPB (VAPA&Bsi) for 3 days and infected with C. trachomatis for 32 h. (A) Immunofluorescence images to illustrate the difference in inclusion size between control (GFPsi) and CERT- (CERTsi1) or VAPA/B- (VAPA&Bsi) depleted cells. The cells were fixed and labeled with an antibody against C. trachomatis (Chlamydia, green). The DNA dye Hoechst labeled the host cell nuclei and the bacterial DNA (DNA, red). The merge images are shown on the right. Scale Bar, 10 µm. (B) For each condition, the surface area of 300 inclusions was determined. Each point represents data from a single inclusion. The grey lines indicate the mean values from the data for each condition. The difference between control and CERT or VAPA&B siRNA was statistically significant; ***P<0.0001 (Student's t test). (C) The number of infectious bacteria measured as IFUs/ml was determined 48 h p.i.. Data show the mean and standard deviation of triplicates of a representative experiment.

Figure 9. Schematic representation of ER-Chlamydia inclusion membrane contact sites.

ER-Inclusion membrane contact sites are formed upon Chlamydia infection. These sites are enriched for the ceramide transfer protein CERT. CERT contacts the ER by binding to VAPA/VAPB and interacts with IncD onto the inclusion membrane, through its PH domain. The close apposition of ER tubules to the inclusion membrane may produce a dynamic environment specialized in non-vesicular trafficking of lipids, such as ceramide, leading to metabolism and signaling events that will ensure proper bacterial development.