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. 2012 May;14(5):656-68.
doi: 10.1111/j.1462-5822.2012.01747.x. Epub 2012 Feb 15.

Chlamydia trachomatis hijacks intra-Golgi COG complex-dependent vesicle trafficking pathway

I D Pokrovskaya  1 , J W Szwedo, A Goodwin, T V Lupashina, U M Nagarajan, V V Lupashin
Affiliations

Chlamydia trachomatis hijacks intra-Golgi COG complex-dependent vesicle trafficking pathway

I D Pokrovskaya et al. Cell Microbiol. 2012 May.

Abstract

Chlamydia spp. are obligate intracellular bacteria that replicate inside the host cell in a bacterial modified unique compartment called the inclusion. As other intracellular pathogens, chlamydiae exploit host membrane trafficking pathways to prevent lysosomal fusion and to acquire energy and nutrients essential for their survival and replication. The Conserved Oligomeric Golgi (COG) complex is a ubiquitously expressed membrane-associated protein complex that functions in a retrograde intra-Golgi trafficking through associations with coiled-coil tethers, SNAREs, Rabs and COPI proteins. Several COG complex-interacting proteins, including Rab1, Rab6, Rab14 and Syntaxin 6 are implicated in chlamydial development. In this study, we analysed the recruitment of the COG complex and GS15-positive COG complex-dependent vesicles to Chlamydia trachomatis inclusion and their participation in chlamydial growth. Immunofluorescent analysis revealed that both GFP-tagged and endogenous COG complex subunits associated with inclusions in a serovar-independent manner by 8 h post infection and were maintained throughout the entire developmental cycle. Golgi v-SNARE GS15 was associated with inclusions 24 h post infection, but was absent on the mid-cycle (8 h) inclusions, indicating that this Golgi SNARE is directed to inclusions after COG complex recruitment. Silencing of COG8 and GS15 by siRNA significantly decreased infectious yield of chlamydiae. Further, membranous structures likely derived from lysed bacteria were observed inside inclusions by electron microscopy in cells depleted of COG8 or GS15. Our results showed that C. trachomatis hijacks the COG complex to redirect the population of Golgi-derived retrograde vesicles to inclusions. These vesicles likely deliver nutrients that are required for bacterial development and replication.

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Conflict of interest statement

Conflict of interest statement

None declared.

Figures

Fig. 1
Fig. 1. Recruitment of fluorescently tagged COG3 and COG8 to the chlamydial inclusions
Stable YFP-COG3 (A,C) and COG8-GFP (B,D) HeLa cells infected with Chlamydia trachomatis L2 (MOI 1.0) were fixed either 8 h (A,B) or 24 h (C,D) after infection. Cells were stained for chlamydial LPS using conjugated mAbs LPS (HiLyte Fluor -555) for 8 h infection or IncA (HiLyte Fluor -555) for 24 h infection, Giantin (HiLyte Fluor-649), and DAPI. Control uninfected YFP-COG3 (E) and COG8-GFP (F) cells were stained for COG8, COG6, Giantin and DAPI as indicated. Slides were visualized using a Zeiss LSM 510 Meta Laser confocal microscope. Arrows indicate the membrane of chlamydial inclusions. The merge images are shown on the right. Scale bar, 10 µm.
Fig. 2
Fig. 2. Endogenous COG8 is associated with C. trachomatis inclusions
HeLa cells infected with C. trachomatis serovar L2 (A,B) or serovar D (C) (MOI 1.0) were fixed either 8 h (A) or 24 h (B, C) post-infection and analyzed by confocal microscopy. In (C) cells were treated with chloramphenicol 3 h post-infection to block bacterial protein synthesis. Cells were stained for chlamydial LPS (HiLyte Fluor-555) and COG8 (HiLyte Fluor-488) (A), GM130 (HiLyte Fluor-555), COG8 (HiLyte Fluor-488) and DAPI (B), or LPS (HiLyte Fluor-555), COG8 (HiLyte Fluor-488) and DAPI (C). The merge images are shown on the right. The intensity profiles showed COG8 (green) specifically localized in a close proximity to inclusion membrane surrounding actively growing bacteria (LPS (red) in (A) and DAPI (blue) in (B)). Arrows indicate the membrane of chlamydial inclusions. Scale bar, 10 µm.
Fig. 3
Fig. 3. Colocalization of SNARE protein GS15 with C. trachomatis
HeLa cells (A–D) or HeLa cells stably transfected with GFP-GS15 (E) infected with C. trachomatis L2 (B,C,E) or D (A,D) for 24 h were fixed and labeled with antibodies and DAPI as indicated. In (D) cells were treated with chloramphenicol 3 h after infection to block bacterial protein synthesis. The merge images are shown on the right. In (C) four Z sections of merged GM130/GS15/DAPI staining are shown. Arrows indicate the membrane of chlamydial inclusions. Scale bar, 10 µm.
Fig. 4
Fig. 4. Recruitment of GS15 and COG complex subunits to inclusions formed by C. trachomatis serovar D and C. muridarum
HeLa cells infected with C. trachomatis serovar D (A and B) or C. muridarum (C) for 24 h were fixed and labeled with antibodies against COG8 and GS15 (A), Giantin and COG3 (B), GS15 and COG3 (C) and DAPI. The merge images are shown on the right. Arrows indicate the membrane of chlamydial inclusions. Scale bar, 10 µm.
Fig. 5
Fig. 5. COG complex subunits localized to chlamydial inclusions before GS15
Stably transfected GFP-GS15 HeLa cells infected with C. trachomatis serovar L2 (A–C) or serovar D (D–E) were fixed either 8 h (A, B) or 24 h (C, D, E) after infection. In (E) cells were treated with chloramphenicol 3 h after infection to block bacterial protein synthesis. Cells were stained for chlamydial LPS (HiLyte Fluor-555) and COG8 (HiLyte Fluor-647) (A, E), DAPI and COG8 (HiLyte Fluor-647) (C), or chlamydial LPS (HiLyte Fluor-555) and COG6 (HiLyte Fluor-647) (B,D). The merge images are shown on the right. Arrows indicate the membrane of chlamydial inclusions. Scale bar, 10 µm.
Fig. 6
Fig. 6. Localization of GS15 to chlamydial inclusion is abrogated in COG3 KD cells
HeLa cells stably expressing GFP-GS15 (A, B), or YFP-COG3 (C) were treated with control siRNA (A), COG3 siRNA (B) or GS15 siRNA (C) for 72 h and then infected with C. trachomatis serovar D and fixed 24 h post infection. Cells were stained for COG6 (HiLyte Fluor-555) and for DAPI (A,B) or IncA (HiLyte Fluor-488) (C). The merge images are shown on the right. Arrows indicate the membrane of chlamydial inclusions. Scale bar, 10 µm.
Fig. 7
Fig. 7. GS15, COG8 and Rab6 are important factors for C. trachomatis development
Western blot of control, GS15KD, COG8 KD and Rab6 KD HeLa lysates showing the efficiency of KD (A). COG8, GS15 and Rab6 were silenced by transfection of specific siRNAs. Cells were then fixed and stained with GM130 antibody and DAPI. Note that Golgi complex morphology has not been changed dramatically in knock-down cells (B). Down-regulation of COG8, Rab6 and GS15 affects infectious chlamydial yields significantly. HeLa cells were transfected with COG8siRNA, Rab6siRNA, GS15siRNA or control siRNA and infected 48h later with C. trachomatis, serovar D at 0.01 and 0.1 MOI. 48 h post infection, cells were harvested and infectious EBs obtained were determined by infecting a fresh McCoy cells layer and IFU determined. *p<0.05, **p<0.01, *** p<0.001 by one-Way ANOVA and post-hoc Tukey multiple comparison between control and specific KDs (C).
Fig. 8
Fig. 8. Ultrastructural analysis of chlamydial inclusions
HeLa cells were transfected with control siRNA (A), COG8 siRNA (B), or GS15 siRNA (C) and infected with C.trachomatis serovar D 72 h (MOI=5) after transfection. Cells were fixed 24 h after infection and processed for TEM. Representative inclusions (out of ~20 random sections) are shown at 6500× magnification (Scale bar, 1 µm) and at 25000× magnification (Last image of each panel, Scale bar, 0.2 µm). Note that inclusions in COG8 KD and GS15 KD cells contained large amount of membranous material and bacteria ghosts (arrows) in addition to reticular bodies (RB). Al least some membranous material inside the inclusion in COG8KD cells is originated from the lysed RBs (D, arrowheads). Significantly more membranous material was observed in inclusions of COG8 KD (24/26 inclusions) and GS15 KD cells (14/16 inclusions) compared to control (5/20 inclusions). P<0.001 (control vs COG8 KD) and P=0.002 (control vs. GS15 KD) by Fisher exact test.
Fig. 9
Fig. 9. Model for the recruitment of the COG complex and COG-interacting proteins to chlamydial inclusions
Solid arrows represent known protein-protein interactions. Dotted arrows represent recruitment to chlamydial inclusions. The model shows that recruitment of COG complex is essential for GS15 recruitment to the inclusion.
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References

    1. Bruinsma P, Spelbrink RG, Nothwehr SF. Retrograde transport of the mannosyltransferase Och1p to the early Golgi requires a component of the COG transport complex. J Biol Chem. 2004;279:39814–39823. - PubMed
    1. Caldwell HD, Kromhout J, Schachter J. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect Immun. 1981;31:1161–1176. - PMC - PubMed
    1. Capmany A, Damiani MT. Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication. PLoS One. 2011;5:e14084. - PMC - PubMed
    1. Carabeo RA, Mead DJ, Hackstadt T. Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion. Proc Natl Acad Sci U S A. 2003;100:6771–6776. - PMC - PubMed
    1. Cocchiaro JL, Kumar Y, Fischer ER, Hackstadt T, Valdivia RH. Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole. Proc Natl Acad Sci U S A. 2008;105:9379–9384. - PMC - PubMed

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