Late endocytic multivesicular bodies intersect the chlamydial inclusion in the absence of CD63

Abstract

Chlamydiae are obligate intracellular bacterial pathogens that replicate solely within a membrane-bound vacuole termed an inclusion. Within the confines of the inclusion, the replicating bacteria acquire amino acids, nucleotides, and other precursors from the host cell. Trafficking from CD63-positive multivesicular bodies to the inclusion was previously identified as a novel interaction that provided essential precursors for the maintenance of a productive intracellular infection. The present study analyzes the direct delivery of resident protein and lipid constituents of multivesicular bodies to the intracellular chlamydiae. The manipulation of this trafficking pathway with an inhibitor of multivesicular body transport and the delivery of exogenous antibodies altered protein and cholesterol acquisition and delayed the maturation of the chlamydial inclusion. Although inhibitor studies and ultrastructural analyses confirmed a novel interaction between CD63-positive multivesicular bodies and the intracellular chlamydiae, neutralization with small interfering RNAs and anti-CD63 Fab fragments revealed that CD63 itself was not required for this association. These studies confirm CD63 as a constituent in multivesicular body-to-inclusion transport; however, other requisite components of these host cell compartments must control the delivery of key nutrients that are essential to intracellular bacterial development.

FIG. 1.

CD63 and LBPA localize to the chlamydial inclusion by immunofluorescence and immunoelectron microscopy. HEp-2 cells were infected with C. trachomatis E for 24 and 48 h, immunolabeled with the indicated MVB-specific antibodies, and analyzed by confocal and electron microscopy. (Upper) Optical sections (0.5 μm thick) of infected cells immunolabeled with anti-CD63-Alexa Fluor 488 and anti-LBPA-Alexa Fluor 568. The TOPRO-3 labeling of the equivalent confocal slice identified intracellular bacteria and host cell nuclei (merged image). Arrowheads identify chlamydial inclusions. The white line in the merged image indicates the position of the profile line used for the analysis of intensity distribution. Scale bar, 20 μm. (Lower) The graph on the left shows that the intensity distribution profiles confirmed that CD63 (green line) and LBPA (red line) had high intensity values peripheral to the inclusion and also displayed intensity levels above background at the site of the TOPRO-3-positive (blue line) chlamydial inclusion. The profile through the host cell nuclei indicates the background intensity levels of CD63 and LBPA. The images on the right show cells that were infected for 24 and 48 h and immunolabeled with anti-LBPA (12 nm colloidal gold) and anti-CD63 (20-nm colloidal gold) antibodies, revealing the presence of these two constituents adjacent to the chlamydial inclusion within compartments that morphologically resembled MVBs (arrowheads). CD63 and LBPA also were evident along the inclusion membrane and in small vesicles within the inclusion lumen (arrows and enlarged in insert). C, Chlamydia. Scale bar, 0.5 μm.

FIG. 2.

Inhibitor of MVBs prevents CD63 transport to the chlamydial inclusion. HEp-2 cells were infected with C. trachomatis serovar E, cultured in the presence of 10 μM U18666A for 48 h, and compared to untreated cells that had been infected for 48 h. Infected cells were immunolabeled with anti-CD63 antibody (18-nm colloidal gold) and analyzed by electron microscopy. CD63 was present within compartments that morphologically resembled MVBs (arrowheads), which appeared enlarged in U18666A-treated cells. CD63 also was evident along the inclusion membrane and in small vesicles within the inclusion lumen (arrows and enlarged in inserts) in untreated control cells. CD63 was not identified within the inclusion of U18666A-treated cells. C, Chlamydia. Scale bar, 0.5 μm. The graph on the right shows the reduction in the recovery of infectious Chlamydia observed in cells cultured in the presence of U18666A, as assessed at 48 h postinfection. Data are presented as the mean infection-forming units of triplicate cultures ± SD.

FIG. 3.

Inhibitor of MVBs alters the distribution of cholesterol in Chlamydia-infected cells. HEp-2 cells were infected with C. trachomatis serovar E, cultured in the presence of 10 μM U18666A for 48 h, and compared to untreated infected cells. Infected cells were immunolabeled with anti-CD63 antibody (Alexa Fluor 488) and filipin and analyzed by fluorescence microscopy. TOPRO-3 labeling identified intracellular bacteria and host cell nuclei (merged image). (Upper) Untreated control cells at 18 h postinfection revealed the incorporation of cholesterol into the membrane of inclusions (arrows and insert) equivalent in size to those of the inclusions of cells after 48 h of inhibitor treatment. (Middle) Untreated control cells at 48 h postinfection showed the incorporation of cholesterol into the inclusion membrane (arrows and insert). (Lower) Treatment with U18666A results in the disruption of chlamydial development and the abundant accumulation of cholesterol in CD63-positive compartments (arrowheads and insert, lower right) with the lack of the incorporation of cholesterol into the chlamydial inclusion (arrows and insert, center). Scale bar, 20 μm.

FIG. 4.

siCD63 prevents the acquisition of CD63 by the chlamydial inclusion. At 48 h postinfection, HEp-2 cells transfected with siControl and siCD63 RNAs were immunolabeled with the indicated MVB-specific antibodies and analyzed by confocal and electron microscopy. (Upper) The images on the left show 0.5-μm-thick optical sections of infected cells immunolabeled with anti-CD63-Alexa Fluor 488 and anti-LBPA-Alexa Fluor 568. The TOPRO-3 labeling of the equivalent confocal slice identified intracellular bacteria and host cell nuclei (merged image). Arrows indicate transfected cells with decreased levels of CD63. The asterisk indicates an internal untransfected CD63-positive control cell. The white line in the merged image indicates the position of the profile line used for the analysis of intensity distribution. Scale bar, 20 μm. The graphs on the right show that intensity distribution profiles confirmed that CD63 (green line) and LBPA (red line) had intensity levels above background in a CD63-positive cell. In cells in which CD63 expression was reduced (arrows in the images on the left), profiles revealed a reduction of CD63 within the inclusions to background levels with no effect on the levels of LBPA. (Lower) Infected cells immunolabeled with anti-LBPA (12-nm colloidal gold) and anti-CD63 (20-nm colloidal gold) antibodies revealed the presence of CD63 (circled in green) and LBPA within the inclusion lumen (arrows and enlarged in insert) in cells transfected with the siControl RNA. In cells transfected with siCD63 RNA, the level of CD63 present in MVBs was reduced with only LBPA within the inclusion lumen. C, Chlamydia. Scale bar, 0.5 μm.

FIG. 5.

siCD63 does not alter the distribution of cholesterol in Chlamydia-infected cells. At 48 h postinfection, HEp-2 cells transfected with siCD63 RNA were immunolabeled with the indicated anti-CD63 antibody (Alexa Fluor 488) and filipin and analyzed by fluorescence microscopy. TOPRO-3 labeling identified intracellular bacteria and host cell nuclei (merged image). No difference in the incorporation of cholesterol was evident in cells in which CD63 expression was reduced (insert) compared to that of control cells within the same field. Arrows indicate chlamydial inclusions. An asterisk indicates an internal untransfected CD63-positive control cell. Scale bar, 20 μm. The graph on the right shows that there was no significant effect of siCD63 RNA on the recovery of infectious Chlamydia, as assessed at 48 h postinfection. Data are presented as the mean infection-forming units of triplicate cultures ± SD.

FIG. 6.

siCD63 prevents the acquisition of internalized exogenous anti-CD63 antibody by the chlamydial inclusion. HEp-2 cells transfected with siControl and siCD63 RNAs were infected with C. trachomatis serovar E and cultured in the presence of exogenous (Exog) anti-CD63 from 24 to 48 h postinfection. (Left) Infected cells labeled with Lysotracker Red and then fixed and immunolabeled with anti-mouse Alexa Fluor 488. The TOPRO-3 labeling of the equivalent confocal slice identified intracellular bacteria and host cell nuclei (merged image). Optical sections (0.5 μm thick) of cells transfected with siControl RNA revealed that exogenously added anti-CD63 antibody trafficked to and accumulated in TOPRO-3-positive chlamydial inclusions and resulted in reduced inclusion size (arrows). In cells transfected with siCD63 RNA and with the subsequent downregulation of CD63 expression (not shown), the exogenous anti-CD63 was excluded from the chlamydial inclusion (arrowheads) and accumulated in lysosomes (Lysotracker Red positive) with no alteration of inclusion development. The white line in the merged image indicates the position of the profile line used for the analysis of intensity distribution. Scale bar, 20 μm. (Right) Intensity distribution profiles confirmed that internalized anti-CD63 antibody (green line) displayed intensity levels above background at the site of the TOPRO-3-positive (blue line) chlamydial inclusion in siControl cells. In siCD63-infected cells, high intensity values for anti-CD63 (green line) localized with Lysotracker Red (red line) peripheral to the inclusion and host cell nuclei but displayed background intensity levels at the site of the chlamydial inclusion. The profile of the host cell nuclei indicates the background intensity levels of CD63 and Lysotracker Red.

FIG. 7.

Internalized exogenous (Exog) anti-CD63 Fab fragments localize to the chlamydial inclusion without the disruption of intracellular chlamydial development. (Left) A nonreducing SDS-PAGE gel of whole anti-CD63 Ig (150 kDa) and purified anti-CD63 Fab fragments (50 kDa). (Middle) HEp-2 cells infected with C. trachomatis E and cultured in the presence of the indicated exogenous antibody from 24 to 48 h postinfection. Infected cells were labeled with Lysotracker Red and then fixed and immunolabeled with fluorescein-conjugated anti-mouse IgG Fab fragment-specific antibody. The TOPRO-3 labeling of the equivalent confocal slice identified intracellular bacteria and host cell nuclei (merged image). Optical sections (0.8 μm thick) revealed that exogenously added control anti-LAMP-1 antibody was excluded from the chlamydial inclusion (arrowheads) and accumulated in lysosomes (Lysotracker Red positive). The exogenously added anti-CD63 antibody trafficked to perinuclear endocytic vesicles that generally did not colocalize with Lysotracker Red but accumulated in TOPRO-3-positive chlamydial inclusions (arrows) and resulted in reduced inclusion size. The exogenously added anti-CD63 Fab fragments trafficked to perinuclear endocytic vesicles (generally Lysotracker Red negative) and accumulated in TOPRO-3-positive chlamydial inclusions (arrows) with no alteration in inclusion size. The white line in the merged image indicates the position of the profile line used for the analysis of intensity distribution. Scale bar, 20 μm. (Right) Intensity distribution profiles of cells treated with control antibody showed high intensity values for anti-LAMP-1 (green line) localized with Lysotracker Red (red line) peripheral to the inclusion and host cell nuclei but displayed background intensity levels at the site of the chlamydial inclusion. In cells treated with anti-CD63 antibody or Fab fragments, intensity distribution profiles confirmed that internalized antibody (green line) displayed intensity levels above background at the site of the TOPRO-3-positive (blue line) chlamydial inclusion. The profile of the host cell nuclei indicates the background intensity levels of CD63 and Lysotracker Red. Ctrl, control.

FIG. 8.

Internalized exogenous (Exog) anti-CD63 antibody alters the distribution of cholesterol in Chlamydia-infected cells. HEp-2 cells infected with C. trachomatis serovar E and cultured in the presence of the indicated exogenous antibody from 24 to 48 h postinfection were fixed, labeled with anti-mouse Alexa Fluor 488 and filipin, and analyzed by fluorescence microscopy. TOPRO-3 labeling identified intracellular bacteria and host cell nuclei (merged image). (Upper) In cells cultured with exogenously added control anti-LAMP-1 antibody, filipin labeling showed the incorporation of cholesterol into Chlamydia and the inclusion membrane (arrows and insert). (Lower) Internalization of exogenous anti-CD63 antibody results in the disruption of chlamydial development with the lack of incorporation of cholesterol into the chlamydial inclusion (arrows and insert). Scale bar, 20 μm. Ctrl, control.