Chlamydia pneumoniae GroEL1 protein is cell surface associated and required for infection of HEp-2 cells

Abstract

Chlamydia pneumoniae is an important obligate intracellular pathogen that replicates within an inclusion in the eukaryotic cell. The initial event of a chlamydial infection is the adherence to and subsequent uptake of the infectious elementary bodies (EBs) by the human cell. These processes require yet-unidentified bacterial and eukaryotic surface proteins. The GroEL1 protein, which exhibits a very strong antigenicity and in vitro can activate various eukaryotic cells, is a potential pathogenicity factor. We localized the protein during the infection process and found it in the inclusion but outside the chlamydial particles. GroEL1 was also localized on the surface of EBs, and the protein could be washed off the EBs. Latex beads coated with recombinantly produced GroEL1 (rGroEL1) bound in a dose-dependent manner to HEp-2 cells. Likewise, GroEL1, when expressed and displayed on the yeast cell surface, mediated adhesion to HEp-2 cells. Interestingly, the homologous GroEL2 and GroEL3 proteins showed no adhesive properties. Incubation of primary umbilical vein endothelial cells with soluble GroEL1 and GroEL1-coated latex beads activated the translocation of the general transcription factor NF-kappaB into the nucleus. Finally, preincubation of HEp-2 cells with rGroEL1 significantly reduced subsequent infection with C. pneumoniae, although adhesion of infectious bacteria to eukaryotic cells was not affected. Taken together, these data support a role for extracellular GroEL1 in the establishment of the chlamydial infection.

FIG. 1.

Localization of chlamydial GroEL1 within the chlamydial inclusion. HEp-2 cells infected with C. pneumoniae at an MOI of 0.5 for 60 h were fixed either with methanol to visualize both intra- and extrachlamydial proteins or with formaldehyde plus 0.5% Triton X-100 to visualize extrachlamydial proteins (5). Indirect immunofluorescence microscopy was performed using antibodies against intrachlamydial DnaK (A and C) or C. trachomatis ribosomal S1 protein (B and D) and surface-localized chlamydial OmcB (C and D) and chlamydial GroEL1 (A and B). Bar, 10 μm.

FIG. 2.

Surface localization of C. pneumoniae GroEL1. (A) Chlamydial GroEL1 protein could be detected on Percoll gradient-purified EBs without (w/o) fixation as well as after methanol fixation using indirect immunofluorescence microscopy. Antibodies directed against the intrachlamydial DnaK protein could detect the antigen only after methanol fixation. Bar, 1 μm. (B) Percoll gradient-purified EBs were washed twice with PBS; the input, wash fractions 1 and 2, and pellet were separated by SDS-PAGE and analyzed by Western blot analysis using antibodies against chlamydial OmpA, DnaK, and GroEL1. The wash fractions loaded contained three times as much protein as the pellet samples. Data are representative of several separate experiments.

FIG. 3.

Adhesion of rGroEL1-coated latex beads to HEp-2 cells. (A) Coomassie blue-stained SDS-PAGE of the affinity-purified rGroEL1 protein after expression in E. coli (lane C) and analysis of purified rGroEL1 by Western blot analysis using an antibody against the N-terminal His tag (lane WB; the arrow marks the rGroEL1 protein band). Lane M, molecular mass markers. (B) Latex beads were coated with BSA or His-tagged recombinant proteins as indicated (protein concentration during coating, 200 μg/ml). Coated beads were incubated with HEp-2 cells. Adhesion of protein-coated latex beads was visualized by phase-contrast microscopy (magnification, ×63). Arrows indicate latex beads attached to HEp-2 cells. Bar, 10 μm. (C) Dose-dependent adhesion of rGroEL1-coated beads to HEp-2 cells. Beads were coated with different rGroEL1 protein concentrations. The control beads (BSA, Inv, and E. coli GroEL) were always coated with a protein concentration of 200 μg/ml. Results of the adhesion experiments are given as bound beads per HEp-2 cell (n = 1,000 HEp-2 cells; number of experiments = 4). Data shown are means ± standard deviations.

FIG. 4.

Yeast cells expressing Aga2-GroEL1 on the yeast cell surface adhere to human cells. (A) Untransformed yeast cells or yeast cells expressing Aga2, Aga2-Inv, Aga2-OmcB, or Aga2-GroEL1 from a plasmid were incubated with 1 × 10⁵ HEp-2 cells, and the number of yeast cells associated with HEp-2 cells was determined microscopically. Shown are typical photomicrographs. Bar, 10 μm. (B) Diagrammatic representation of the number of yeast cells expressing Aga2, Aga2-Inv, Aga2-OmcB, and Aga2-GroEL-1 adhering to 1,000 HEp-2 cells (number of experiments = 4). Data shown are means ± standard deviations.

FIG. 5.

Assay for binding of C. pneumoniae rGroEL2- or rGroEL3-coated latex beads to HEp-2 cells. Latex beads were coated with the proteins indicated (protein concentration during coating, 200 μg/ml) and incubated with HEp-2 cells as described in Materials and Methods. Results of the binding experiments are given as bound beads per single HEp-2 cell (n = 1,000 HEp-2 cells; number of experiments = 4). Data shown are means ± standard deviations.

FIG. 6.

C. pneumoniae rGroEL1 activates NF-κB translocation in HUVECs. (A) HUVECs were infected with C. pneumoniae or as control with PBS for 2 h. NF-κB localization was determined by indirect immunofluorescence microscopy as described in Materials and Methods. (B) HUVECs were stimulated with 20 μg/ml rGroEL1 or with BSA for 5 h. NF-κB localization was determined as described above. (C) HUVECs were stimulated with rGroEL1-coated latex beads or BSA-coated latex beads for 5 h. NF-κB localization was determined as described above. (D) Quantification of NF-κB activation in the experiments shown in panels A, B, and C. Bars represent the ratio of NF-κB translocation into the nucleus to the total number of HUVECs cells. Each value represents the mean from three experiments ± the standard deviation.

FIG. 7.

Soluble chlamydial rGroEL1 protein does not interfere with EB attachment to human cells but reduces subsequent infection by C. pneumoniae. (A) HEp-2 cells were incubated with increasing amounts (20 μg/ml to 640 μg/ml) of rGroEL1 for 2 h prior to infection with purified C. pneumoniae EBs. At 60 h postinfection, cells were fixed and analyzed by indirect immunofluorescence microscopy as described in Materials and Methods. HEp-2 cells pretreated with PBS or 200 μg/ml BSA prior to infection with C. pneumoniae served as controls. The number of chlamydial inclusions was determined, and the value found for the PBS control was set to 100% (n = 20 microscopic fields; number of experiments = 4). (B) Binding of purified, viable, CFSE-stained C. pneumoniae EBs to HEp-2 cells was detected by flow cytometric analysis. In control experiments, the attachment of C. pneumoniae EBs to HEp-2 cells incubated with PBS, 200 μg/ml BSA, or 500 μg/ml heparin was monitored and compared with binding rates in samples to which 20 μg/ml or 200 μg/ml rGroEL1 protein had been added (number of experiments = 4). Error bars indicate standard deviations.