Characterization of hypothetical proteins Cpn0146, 0147, 0284 & 0285 that are predicted to be in the Chlamydia pneumoniae inclusion membrane
© Luo et al; licensee BioMed Central Ltd. 2007
Received: 09 January 2007
Accepted: 15 May 2007
Published: 15 May 2007
Although more than 100 Chlamydia pneumoniae hypothetical proteins have been predicted to be inclusion membrane proteins, only a few have been experimentally demonstrated to be in the inclusion membrane. Using antibodies raised with fusion proteins, we characterized four such hypothetical proteins encoded by two gene clusters (Cpn0146-147 and Cpn0284-285) in the C. pneumoniae genome.
Cpn0146 and 0147 were detected in the inclusion membrane while Cpn0284 and 0285 inside inclusion and mainly associated with reticulate bodies although all four proteins contain an N-terminal bi-lobed hydrophobic region, a signature motif assigned to inclusion membrane proteins. These four hypothetical proteins were only detected in cells infected with C. pneumoniae but not other chlamydial species, with Cpn0147 at 6 hours and Cpn0146, 0284 & 0285 at 24 hours after infection. Cpn0146 & 147 but not Cpn0284 and 285 co-localized with a host cell endoplasmic reticulum marker, a property known to be possessed by some chlamydial inclusion membrane proteins, when expressed in the host cell cytosol via transgenes. However, the endoplasmic reticulum localization of the C. pneumoniae inclusion membrane proteins did not result in inhibition of the subsequent C. pneumoniae infection.
The hypothetical proteins Cpn0146 & 0147 were localized in the C. pneumoniae inclusion membrane while Cpn0284 & 0285 within the inclusion although all four were predicted to be Inc proteins, suggesting the need to experimentally characterize the predicted Inc proteins.
The obligate intracellular chlamydial pathogens include the species Chlamydia trachomatis (C. trachomatis; ) and C. pneumoniae  that mainly infect humans and C. muridarum (formerly known as C. trachomatis mouse pneumonitis agent, designated as MoPn, ref: ), C. caviae , C. psittaci (38), C. abortus  and C. felis  that are mainly animal pathogens. The species C. pneumoniae, C. caviae, C. psittaci, C. abortus &C. felis are also grouped as an independent genus termed Chlamydophilae based on their genetic relatedness . The C. pneumoniae organisms infect the human respiratory system, not only causing respiratory pathologies but also exacerbating pathologies in other organs such as the vascular wall [7–10]. The C. caviae GPIC organisms can infect both the ocular and urogenital tissues in guinea-pig, which has been used as a model system for studying the pathogenesis of Chlamydia-induced diseases . The C. psittaci 6BC organisms cause avian chlamydiosis that can lead to serious health problems for humans who are in close contact with the infected birds . Both the C. abortus &C. felis organisms can affect the health of various domesticated animal species [4, 13, 14]. Despite the profound difference in host range, tissue tropism, disease process, all chlamydial species share similar genome sequences [1–5] and possess a common intracellular growth cycle with distinct biphasic stages .
Chlamydial organisms have adapted an obligate intravacuolar growth life style with a two-phase cycle [16, 17]. The infection starts with endocytosis of an infectious elementary body (EB) into a host cell, followed by rapid differentiation of the EB into a non-infectious but metabolically active reticulate body (RB). After the RB undergoes numerous rounds of replication, the progeny RBs can differentiate back into EBs before exiting to infect the adjacent cells. Chlamydial organisms accomplish all their biosynthesis and particle assembly within the cytoplasmic vacuole (designated as inclusion). The chlamydial inclusions not only support chlamydial replication but also protect the replicating organisms from host defense mechanisms such as lysosomal fusion [15, 18]. At the same time, Chlamydia must import nutrients and metabolic intermediates from host cells into the inclusions [19, 20]. However, the molecular mechanisms by which Chlamydia organisms interact with host cells are largely unknown. The fact that Chlamydia-encoded proteins are found in the inclusion membrane (designated as Inc; ) suggests that the Inc proteins may participate in the chlamydial interactions with host cells [22, 23]. Therefore, searching for and characterization of novel inclusion membrane proteins may provide important information for understanding chlamydial pathogenic mechanisms.
Various approaches have been utilized to identify chlamydial Inc proteins, including direct antibody detection [21, 24–27], accessibility to host cell cytoplasm immune proteasome processing [28, 29], secretion by heterologous type III secretion systems [30, 31] and common structural feature-based computer predictions [32, 33]. Although a total of 104 hypothetical proteins encoded in C. pneumoniae genome were predicted to be Inc proteins by computer programs [32, 33], only a few were proven to be in the inclusion membrane of the C. pneumoniae-infected cells by direct antibody labeling . Since not all Inc proteins can be identified by computer prediction and not all predicted Inc proteins are localized in the inclusion membrane of chlamydial organism-infected cells [29, 32], it is critical to use experimental approaches to confirm the localization of the putative Incs and to further characterize the Inc proteins. In the current study, we detected the hypothetical proteins Cpn0146 & 0147 in the C. pneumoniae inclusion membrane and Cpn0284 & 0285 within the inclusion although all four were predicted to be Inc proteins [32, 33]. Furthermore, Cpn0146 & 0147 but not Cpn0284 & 0285 co-localized with a host cell endoplasmic reticulum (ER) marker when expressed via transgenes although the ER co-localization did not significantly affect the subsequent C. pneumoniae infection.
1. Localization of Cpn0146 and 0147 in the inclusion membrane and Cpn0284 and Cpn0285 within the inclusion of C. pneumoniae-infected cells
2. Specificity of the anti-chlamydial fusion protein antibodies
3. The hypothetical proteins Cpn0146, 0147, 0284 & 0285 are unique to the C. pneumoniae species
4. Time course expression of Cpn0146, 0147, 0284 & 0285
5. Localization of Cpn0146 & 0147 but not Cpn 0284 & 0285 in the host cell endoplasmic reticulum
In the current study, we have provided the first experimental evidence demonstrating that the hypothetical proteins Cpn0146 & 147 are Inc proteins while Cpn0284 and 0285 are associated with RBs inside the inclusions although all 4 proteins were predicted to be in the inclusion membrane based on their N-terminal bi-lobed hydrophobic motifs [32, 33]. Although the antibodies used for localizing these four proteins were raised with fusion proteins, we have convincingly demonstrated that the anti-fusion protein antibodies specifically recognized the corresponding endogenous antigens during C. pneumoniae infection. It is interesting that Cpn0147 was detected as early as 6 hours while IncA was only detected 12 hours and Cpn0146, 0284 & 0285 24 hours after infection. These differential protein expression patterns suggest that Cpn0147 and IncA are early genes while Cpn0146, 0284 &0285 late genes although further monitoring transcripts is required for providing evidence to support the conclusion. Although Cpn0146 and 0147 are encoded in the same gene cluster and oriented in the same direction in the C. pneumoniae genome, Cpn0147 is expressed much earlier than Cpn0146, suggesting that the expression of these two genes are regulated independently. Regardless of the difference in the timing of expression, both Cpn0146 and 0147 proteins remained in the inclusion membrane once detected. However, the RB proteins Cpn0284 and 0285 fluctuated along the infection cycle with strong signals in small inclusions and no or very low signals in large inclusions between 24 and 96 hours and reappeared in large inclusions by 120 hours after infection, suggesting that Cpn0284 & 0285 are RB-specific proteins although more biochemical assays are required for confirming this conclusion.
Since the identification of the first chlamydial inclusion membrane protein , various features have been assigned to chlamydial inclusion membrane proteins, including localization in the inclusion membrane, accessibility to host immunoproteasomal processing, secretability by various heterologous type III secretion systems, bi-lobed hydrophobic motifs, ER-like distribution and the ability to render host cell resistance to subsequent chlamydial infection when pre-expressed in host cell cytosol. Here, we systematically analyzed four predicted Inc proteins encoded by two separate gene clusters in the C. pneumoniae genome. We found that Cpn0146 & 0147 but not Cpn0284 & 0285 were localized in the inclusion membrane during C. pneumoniae infection and co-localized with ER when expressed via transgenes, which suggests that the ability of C. pneumoniae proteins to co-localize with ER is a more precise predictor for inclusion membrane localization than the bi-lobed hydrophobic motifs. However, more studies are required for strengthening such a correlation.
Despite the tremendous efforts that have been made in the past decade in identifying and characterizing chlamydial Inc proteins, the precise functions of the Inc proteins are largely unknown. Among the numerous Inc proteins identified in C. trachomatis and C. caviae, some Inc proteins (such as IncA) have been shown to participate in vesicle fusion [42–44] while others (such as IncG) to directly interact with host cell molecules [45–47] during chlamydial infection. Delevoye et al  has recently correlated the oligomerization and ER colocalization of the C. trachomati and C. caviae IncA proteins with their abilities to prevent subsequent organism infection and to disrupt the organism developmental cycle of existing infection and further mapped the functional region to the IncA C-terminal fragment that contains putative leucine zipper domains. Although C. pneumoniae IncA also contains the C-terminal putative leucine zipper domains  and has the ability to localize to ER (current study, Fig. 5), it failed to affect the subsequent C. penumoniae organism infection (Fig. 6), suggesting that ER localization is not sufficient for inhibiting chlamydial infection. The fact that none of the C. pneumoniae Inc proteins tested so far affected the subsequent C. pneumoniae infection suggests that Inc proteins from C. pneumoniae may exert their functions in different modes due to the unique growth properties of C. pneumnoniae organisms. We are in the process of developing novel approaches for further characterizing the C. pneumoniae Inc proteins.
1. Cell culture and chlamydial infection
HeLa 229 cell (ATCC, Manassas, VA 20108) monolayers were infected with C. pneumoniae AR39, Mul or 2043 strains (kindly provided by Dr. Harlan Caldwell, RML, NIAID, NIH, Hamilton, Montana; Both Mul & 2043 strains are recent clinical isolates), C. caviae GPIC, C. psittaci 6BC, C. muridarum (also known as MoPn) or C. trachomatis serovar D or L2 organisms at an MOI of 0.5 in DMEM (Invitrogen, Carlsbad, CA) with 10% fetal calf serum (FCS; Atlanta Biologicals, Lawrenceville, GA) and with or without 2 μg/ml of cycloheximide (Sigma, St. Luis, MO) for 6 to 120 hours (as indicated in individual experiments). The infected cultures grown on coverslips were processed for various immunoassays.
2. Prokaryotic expression of C. penumoniae proteins and antibody production
The open reading frames (ORFs) coding for hypothetical proteins Cpn0146, 0147, 0284 and 0285 from the C. pneumoniae genome  were cloned in full-length into pGEX vectors (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) using the C. pneumoniae AR39 organism genomic DNA as template. We used the ORF designations described for the CWL029 genome sequence when we started the C. pneumoniae gene cloning/fusion protein project. In order to maintain consistence, we are still using the Cpn designations in the current study although the DNA template is from AR39 strain organisms. Please note that Cpn0146 is designated as CP0627, Cpn0147 as CP0626, Cpn0284 as CP0474 and Cpn0285 as CP0473 in the AR39 genome sequence. The amino acid sequences of these 4 proteins are identical between CWL029 and AR39. The C. pneumoniae proteins were expressed as fusion proteins with glutathione-s-transferase (GST) fused to the N-terminus of the chlamydial proteins as previously described . Expression of the fusion proteins was induced with isopropyl-beta-D-thiogalactoside (IPTG; Invitrogen) and the fusion proteins were extracted by lysing the bacteria via sonication in a Triton-X100 lysis buffer (1%TritonX-100, 1 mM PMSF, 75 units/ml of Aprotinin, 20 μM Leupeptin and 1.6 μM Pepstatin). After a high-speed centrifugation to remove debris, the fusion protein-containing supernatants were purified using glutathione-conjugated agarose beads (Pharmacia) and the purified proteins were used to immunize mice for producing both polyclonal antisera (pAb; ref:  and monoclonal antibodies (mAb; ref: . The fusion protein-specific antibodies were then used to localize the endogenous proteins in C. pneumoniae-infected cells via an indirect immunofluorescence assay [50, 51].
3. Eukaryotic expression of C. pneumoniae proteins
The C. pneumoniae ORFs Cpn0146, 0147, 0284 &0285 were also cloned into the pDsRed Monomer C1 mammalian expression vector (BD Biosciences Clontech, San Jose, CA) and expressed as fusion proteins with a Red fluorescence protein (RFP) fused to the N-terminus. The recombinant plasmids were transfected into HeLa cells using the lipofectamine 2000 transfection reagent following the protocol recommended by the manufacture (Invitrogen). The RFP chlamydial fusion proteins were visualized via either the fusion tag RFP or the mouse anti-chlamydial protein antibody labeling 24 hours after transfection or as indcated in individual experiments. To assess the effects of the RFP-Cpn fusion proteins on the subsequent chlamydial infection, the transfected cells were infected with C. pneumoniae AR39 organisms. The infected cultures grown on coverslips were processed for visualization of the transfection and infection via an immunofluorescence assay. Cells expressing RFP were counted and % of RFP+ cells that contain chlamydial inclusions was calculated. In each experiment, ~100 RFP+ cells were counted from 5 to 10 random views and three separate experiments were carried out. The mean values were compared between the sample expressing RFP alone and samples expressing RFP-Cpn fusion proteins using a two-tailed Student t test. The results were expressed as means plus/minus standard errors. As a positive control, a recombinant pDsRed plasmid encoding the RFP-GPICIncA fusion protein was similarly transfected into HeLa cells followed by the C. caviae GPIC organism infection. The rates of GPIC infection in RFP+ cells were acquired and analyzed as described above.
4. Immunofluorescence assay
HeLa cells grown on coverslips were fixed with 2% paraformaldehyde (Sigma) dissolved in PBS for 30 min at room temperature, followed by permeabilization with 1% saponin (Sigma) for an additional 30 min. After washing and blocking, the cell samples were subjected to antibody and chemical staining. Hoechst (blue, Sigma) was used to visualize nuclear DNA. A rabbit anti-chlamydial organism antibody (R12AR39, raised with C. pneumoniae AR39 organisms, unpublished data) or anti-CT395 (raised with the CT395 fusion protein; CT395 is a GrpE-related chaperonin with >70% amino acid sequence identity among all chlamydial species; unpublished data) plus a goat anti-rabbit IgG secondary antibody conjugated with Cy2 (green; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used to visualize chlamydial inclusions. The mouse antibodies including both polyclonal antisera (pAbs) and monoclonal antibodies (mAbs) raised against various reference proteins and the C. pneumoniae GST fusion proteins plus a goat anti-mouse IgG conjugated with Cy3 (red; Jackson ImmunoResearch) were used to visualize the corresponding antigens. In some cases, the primary antibodies were pre-absorbed with either the corresponding or heterologous fusion proteins immobilized onto agarose beads (Pharmacia) prior to staining cell samples. The pre-absorption approach was carried by incubating the antibodies with bead-immobilized antigens for 1 h at room temperature (RT) or overnight at 4°C followed by pelleting the beads. The remaining supernatants were used for immunostaining. For the transfected cell samples, the RFP chlamydial fusion proteins were visualized via the fusion tag RFP (red) and by co-staining with a mouse antibody plus a Cy2 conjugate (green). For determining the subcellular location of the RFP fusion proteins, a rabbit anti-Calnexin antibody (Cat# SPA-860, Stressgen Bioreagents Corp., Ann Arbor, MI) in combination with a Cy2-conjugated goat anti-Rabbit IgG (green) was used to label the host cell endoplasmic reticulum.
The cell samples after the appropriate immuno-labeling were used for image analysis and acquisition with an Olympus AX-70 fluorescence microscope equipped with multiple filter sets (Olympus, Melville, NY) as described previously [52, 53]. Briefly, the multi-color-labeled samples were exposed under a given filter set at a time and the single color images were acquired using a Hamamatsu digital camera. The single color images were then superimposed with the software SimplePCI to display multi-colors. An Olympus FluoView™ Laser Confocal Microscope (Olympus) was used to further analyze the co-stained samples at the UTHSCSA institutional core facility. All microscopic images were processed using the Adobe Photoshop program (Adobe Systems, San Jose, CA).
5. Western blot assay
The Western blot assay was carried out as described elsewhere [51, 54]. Briefly, the chlamydial GST fusion proteins were solublized in 2% SDS sample buffer and loaded to SDS polyacrylamide gel wells. After electrophoresis, the proteins were transferred to nitrocellulose membranes and the blots were detected with primary antibodies. The primary antibody binding was probed with an HRP (horse radish peroxidase)-conjugated secondary antibody and visualized with an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). In some cases, the primary antibodies were also subjected to pre-absorption as described above prior to reacting with the nitrocellulose membrane.
This work was supported in part by grants (to G. Zhong) from the US National Institutes of Health.
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