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First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02733

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Trafficking from CD63-positive late endocytic multivesicular bodies is essential for intracellular development of Chlamydia trachomatis

Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (111K): [in a new window]   Fig. 1. Constituents of late endocytic multivesicular compartments localized to the chlamydial inclusion by indirect immunofluorescence. HEp-2 cells were infected with C. trachomatis E for 36 hours, immunlabeled with the indicated MVB-specific antibodies and analyzed by confocal microscopy. (Upper panels) 0.5-µm-thick optical sections of infected cells, immunolabeled with antibodies against CD63, MLN64, LBPA and LAMP-1, followed by the appropriate secondary antibody conjugated to Alexa Fluor 594. (Middle panels) TOPRO-3 labeling of the equivalent confocal slice to reveal intracellular bacteria and host-cell nuclei. (Lower panels) Merged images. Arrows indicate representative chlamydial inclusions, arrowheads indicate representative nuclei. CD63, MLN64 and LBPA localized to the TOPRO-3-positive chlamydial inclusions. Bar, 20 µm.

View larger version (78K): [in a new window]   Fig. 2. CD63 accumulated within the chlamydial inclusion. HEp-2 cells were infected with C. trachomatis E for 48 hours and analyzed by indirect immunofluorescence. The cytosolic face of the inclusion membrane was immunolabeled with anti-IncA antibody (anti-rabbit Alexa Fluor 488, see Results for details) and infected cells were then labeled with anti-CD63 antibody (anti-mouse Alexa Fluor 594). TOPRO-3-labeling was used to identify both intracellular bacteria and the host cell nuclei. (Upper panels) A 0.5-µm-thick optical section shows labeled CD63 localized to TOPRO-3-positive intracellular bacteria within the confines of the IncA-positive inclusion membrane. The white line in the merged image indicates the position of orthogonal analysis. Bar, 20 µm. (Lower panels) An orthogonal view of the infected cell (height of combined Z-sections 9.2 µm,) confirmed CD63 localization to TOPRO-3-labeled intracellular bacteria within the depths of the chlamydial inclusion as defined by IncA-labeling.

View larger version (125K): [in a new window]   Fig. 3. Immunoelectron microscopy analysis of CD63 association with the chlamydial inclusion. HEp-2 cells were infected with C. trachomatis E for 48 hours and analyzed by cryo immunoelectron microscopy. Infected cells immunolabeled with anti-CD63 antibody (anti-mouse-18 nm colloidal gold) revealed CD63 within the chlamydial inclusion and in compartments adjacent to the inclusion that morphologically resembled MVBs (arrows). CD63-labeling was also evident along the inclusion membrane (upper panel, arrowhead; enlarged in right panel) and in small vesicles within the inclusion lumen (lower panel arrowheads; enlarged in right panel). C, Chlamydia; m; mitochondria. Bar, 0.5 µm. For quantification of anti-CD63-labeling, the density of colloidal-gold particles was determined per cross-sectional area of the compartments indicated (particles/µm2). The density was also expressed as fold over background, based on the average background-labeling in mitochondria and nuclei.

View larger version (152K): [in a new window]   Fig. 4. CD63-HA localizes to the chlamydial inclusion in transfected cells. HEp-2 cells were transfected with CD63-HA for 24 hours, then infected with C. trachomatis E for 48 hours and analyzed by indirect immunofluorescence and cryo immunoelectron microscopy. (Left panels) 0.5-µm-thick optical sections of infected cells immunolabeled with anti-HA antibody (anti-rabbit Alexa Fluor 488). TOPRO-3-labeling of the equivalent confocal slice was used to reveal intracellular bacteria and host-cell nuclei. Anti-HA antibody localized CD63-HA to TOPRO-3-positive intracellular bacteria in transfected cells (arrow) with no anti-HA antibody labeling associated with inclusions of adjacent untransfected cells (arrowhead). Bar, 20 µm. (Right panels) Transfected cells immunolabeled with anti-HA antibody (anti-rabbit-18 nm colloidal gold) revealed CD63-HA within the chlamydial inclusion and in compartments adjacent to the inclusion that morphologically resembled MVBs (arrows). Labeled CD63 was also evident in small vesicles within the inclusion lumen (lower panel, arrowhead; enlarged in insert). C, Chlamydia. The lack of label in mitochondria (m) served as an internal control for background labeling. Bar, 0.5 µm.

View larger version (44K): [in a new window]   Fig. 5. Internalized exogenous anti-CD63 antibody localized to the chlamydial inclusion. HEp-2 cells were infected with C. trachomatis E and cultured in the presence of exogenous anti-CD63 and anti-LAMP-1 antibodies (left panel), or anti-CD63 antibody and FITC-labeled dextran (right panel) from 24 hours to 48 hours post infection. Infected cells were then fixed and immunolabeled with anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 (left panel), or anti-mouse Alexa Fluor 594 only (right panel). TOPRO-3-labeling was used to identify intracellular bacteria and the host-cell nuclei. 0.5-µm-thick optical sections revealed that exogenously added anti-CD63 antibody trafficked to and accumulated in TOPRO-3-positive chlamydial inclusions (arrowheads). Concurrent addition of exogenous anti-LAMP-1 antibody and dextran-FITC to the culture medium resulted in their uptake into endocytic compartments of the host cell, but exclusion from the chlamydial inclusion. The arrow in the merged image indicates the position of the profile line used for analysis of intensity distribution. Bar, 20 µm. (Lower panels) Intensity distribution profiles confirmed the presence of anti-CD63 antibody (red line) within the TOPRO-3-positive inclusion (blue line). Anti-LAMP-1 antibody (green line, upper profile) and dextran-FITC (green line, lower profile) had high-intensity values peripheral to the inclusion and host-cell nuclei, but displayed background-intensity levels at the site of the chlamydial inclusion.

View larger version (72K): [in a new window]   Fig. 6. Internalized exogenous anti-CD63 antibody disrupted intracellular chlamydial development. HEp-2 cells were infected with C. trachomatis E and cultured in the presence of exogenous anti-CD63 from 24 hours to 48 hours post infection. (Left panels) TOPRO-3-labeling was used to analyze morphological changes in inclusion development. TOPRO-3 labeled intracellular bacteria and the host-cell nuclei, with the chlamydial inclusions colorized blue (Photoshop 7.0) in obtained black and white images. 0.5-µm-thick optical sections revealed that the addition of exogenous anti-CD63 antibody resulted in reduced inclusion-size relative to untreated controls. (Center panels) Electron micrographs of the intracellular bacteria revealed primarily EBs in the untreated control cells, but predominantly RBs delayed in re-differentiation to infectious EBs in infected cells treated with exogeneous anti-CD63. Arrowheads indicate representative EBs. (Right panel) A reduction in recovery of infectious Chlamydia was observed in cells cultured in the presence of anti-CD63 antibody. Data are presented as the mean infectious-forming units of triplicate cultures ± s.e.m.

View larger version (92K): [in a new window]   Fig. 7. Inhibitors of MVBs disrupted inclusion maturation. HEp-2 cells were infected with C. trachomatis E; 1 hour post infection, the inhibitors 3-methyladenine, LY-294002 and U18666A were added to the culture medium at the indicated concentrations. (Upper panel) Fluorescence analysis of the effect of inhibitors on inclusion-size and -maturation assessed at 48 hours post infection. Cells were immunolabeled with the chlamydial-specific anti-hsp60 antibody (anti-mouse Alexa Fluor 594) and 0.5-µm-thick confocal slices revealed significantly reduced inclusion size in the presence of inhibitors. Bar, 20 µm. (Center panel) Electron micrographs of the intracellular bacteria revealed primarily electron-dense EBs in the untreated control cells, but also small inclusions containing RBs in infected cells treated with inhibitors. Bar, 0.5 µm. (Lower panel) A reduction in recovery of infectious Chlamydia was observed in cells cultured in the presence of inhibitors as assessed at 48 hours post infection. Data are presented as mean infectious-forming units of triplicate cultures ± s.e.m.

View larger version (114K): [in a new window]   Fig. 8. Inhibitors of MVBs disrupted trafficking of sphingomyelin to the chlamydial inclusion. HEp-2 cells infected with C. trachomatis were cultured in the absence or presence of 5 mM 3-methyladenine or 10 µM U18666A. Post infection, at the times indicated, cells were labeled with NBD-ceramide and analyzed 1 hour after back-exchange. Infected cells were fixed, immunonlabeled with anti-golgin-97 or anti-CD63 antibody (anti-mouse Alexa Fluor 594), and 0.5-µm-thick optical sections were acquired by confocal microscopy. (Left panels) Control cells, 18 and 48 hours post infection. The NBD probe was incorporated in the Golgi complex and TOPRO-3-positive chlamydial inclusions. (Right panels) Treatment of infected cells with 3-methyladenine or U18666A resulted in the incorporation of fluorescent probe into swollen CD63-positive compartments but was excluded from TOPRO-3-positive chlamydial inclusions. Arrowheads indicate representative swollen NBD- and CD63-positive compartments. Arrows indicate chlamydial inclusions. Insert in lower left panel shows NBD-labeled Chlamydia (untreated, 18 hours post infection) in inclusions of equivalent size to 3-methyladenine- and U18666A-treated inclusions (48 hours post infection) that lacked NBD incorporation. Bar, 20 µm.

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