Localization of the hypothetical protein Cpn0585 in the inclusion membrane of Chlamydia pneumoniae-infected cells

Abstract

Cpn0585, encoded by a hypothetical open reading frame in Chlamydia pneumoniae genome, was detected in the inclusion membrane during C. pneumoniae infection using both polyclonal and monoclonal antibodies raised with Cpn0585 fusion protein. The anti-Cpn0585 antibodies specifically recognized the endogenous Cpn0585 without cross-reacting with IncA (a known inclusion membrane protein of C. pneumoniae) or other control antigens. A homologue of Cpn0585 in the C. caviae species (encoded by the ORF CCA00156) was also localized in the inclusion membrane of the C. caviae-infected cells. The Cpn0585 protein became detectable 24h while CCA00156 as early as 8h after infection. Once expressed, both proteins remained in the inclusion membrane throughout the rest of infection course.

Fig. 1. Detection of Cpn0585 in the inclusion membrane of C. pneumoniae-infected cells

HeLa cells were infected with C. pneumoniae AR39 organisms at an MOI of 0.5 in the presence of 2μg/ml of cycloheximide for 72 hours. The infected cultures grown on coverslips were processed for the following immunostainings: (A) Cpn0585 was probed with a mouse antiserum (pAb, panel a) and monoclonal antibodies (mAb clone 3D11 with a heavy chain isotype of IgM, panel b; 12F2, IgM, panel c), all of which were raised with the GST-Cpn0585 fusion protein and visualized with a Cy3-conjugated goat anti-mouse IgG (red). A rabbit anti-AR39 antiserum (R12AR39) together with a Cy2-conjugated goat anti-rabbit IgG (green) was used to visualize the C. pneumoniae organisms and Hoechst to visualize DNA. (B) The AR39 organism-infected cell samples were co-stained with the anti-Cpn0585 mAb 3D11 (green) and DNA Hoechst dye (blue) in combination of antibodies recognizing other C. pneumoniae reference proteins, including CPAFcp (EB3.1, IgG1), IncA (2B12.1, IgG1), MOMP (GZD1E8, IgG1) and HSP60 (BC7.1, IgG1; all in red). The co-stained samples were also observed under a confocal microscope (images not shown) and the antibody specificities were confirmed using various approaches (data not shown).

Fig. 2. Localization of CCA00156 in the inclusion membrane of GPIC-infected cells

HeLa cells were infected with C. caviae GPIC organisms at an MOI of 0.5 in the presence of 2μg/ml of cycloheximide for 40 hours. The infected cultures grown on coverslips were processed for immunostainings with mouse antibodies against CCA00156 (pAb, panels a–d), GPIC-IncA (pAb, e–h), GPIC-CPAF (pAb, i–l) and GPIC HSP60 (BC7.1, m–p), all of which were visualized with a Cy3-conjugated goat anti-mouse IgG (red). A rabbit anti-GPIC antiserum together with a Cy2-conjugated goat anti-rabbit IgG (green) was used to visualize the C. caviae organisms and Hoechst to visualize DNA. The images were acquired using a conventional fluorescence microscope. The antibody specificities were further confirmed using pre-absorption (data not shown).

Fig. 3. Monitoring the expression of Cpn0585 (A) and CCA00156 (B) proteins during chlamydial infections

HeLa cells infected with C. pneumoniae AR39 (A) or C. caviae GPIC (B) organisms for various periods of time as indicated on top of the figures and the culture samples were subjected to immunostaining with anti-Cpn0585 or anti-Cpn IncA (A, red) and anti-CCA00156 or anti-GPIC IncA (B, red). The rabbit antibodies against AR39 (A) or GPIC (B) were used to visualize the organisms (green) and Hoechst dye for DNA (blue). The images were acquired using the conventional fluorescence microscope. Note that Cpn0585 protein was first detected 24 hours (A, panel d) and Cpn IncA 12 hours (A, panels k & k1) after infection with C. pneumoniae while both CCA00156 and GPIC IncA were detected at 8 hours after infection with C. caviae (B; panels d, d1, k, k1). White arrows indicate that the inclusion membrane protein labeling (red) appears to surround the organism labeling (green) in panels d & k1 (A) and d1 & k1 (B).