The Chlamydial Plasmid-Encoded Protein pgp3 Is Secreted into the Cytosol of Chlamydia-Infected Cells

Cell culture and chlamydial infection.HeLa cells (human cervical carcinoma epithelial cells; ATCC catalog no. CCL2) and the following chlamydial organisms were used in the current study: C. trachomatis serovars A, D, and L2; C. muridarum strain Nigg; C. psittaci strain 6BC; and C. pneumoniae strain AR39. The chlamydial organisms were propagated, purified, aliquoted, and stored as described previously (6). For infection, HeLa cells grown in either 24-well plates with coverslips or tissue flasks containing Dulbecco modified Eagle medium (GIBCO BRL, Rockville, MD) with 10% fetal calf serum (GIBCO BRL) at 37°C in an incubator supplied with 5% CO₂ were inoculated with chlamydial organisms at an multiplicity of infection of 0.5 (or as indicated for individual experiments) as described previously (6). The infected cultures were processed at different time points after infection for either immunofluorescence assays or Western blot analyses as described below.

Chlamydial gene cloning, fusion protein expression, and antibody production.The eight ORFs carried by the pCHL1 plasmid (10) from C. trachomatis serovar D organisms were cloned into pGEX vectors (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The forward primers used for cloning each of the eight pORFs were as follows: pORF1, 5′-CCGGAATTC(restriction site)-(overlapping region)ATGGGCTCGATGGCTTTCCA-3′; pORF2, 5′-CGCGGATCC-ATGGGTAAAGGGATTTTATCTTT-3′; pORF3, 5′-CGCGGATCC-ATGAAAACTCGTTCCGAAATAG-3′; pORF4, 5′-CGCGGATCC-ATGGTAAATTATAGTAACTGCCA-3′; pORF5, 5′-CGCGGATCC-ATGGGAAATTCTGGTTTTTATTTG-3′; pORF6, 5′-CGCGGATCC-ATGCAAAATAAAAGAAAAGTGAGG-3′ pORF7, 5′-CGCGGATCC-GTGGGATGCAACTTGGCCC-3′; and pORF8, 5′-CGCGGATCC-GTGAACAAACTAAAAAAAGAAGC-3′. The reverse primers were as follows: pORF1, 5′-TTTTCCTTTTGCGGCCGC(restriction site)-(overlapping region)TCACAATATTGTGGGTGTTTGT-3′; pORF2, 5′-TTTTCCTTTTGCGGCCGC-CTATATTAGAGCCATCTTCTTTG-3′; pORF3, 5′-TTTTCCTTTTGCGGCCGC-TTACCATACTTTTTTAATAGCGG-3′; pORF4, 5′-TTTTCCTTTTGCGGCCGC-TTAATTAATAGATTCTTGTTCTAATT-3′; pORF5, 5′-TTTTCCTTTTGCGGCCGC-TTAAGCGTTTGTTTGAGGTATTA-3′; pORF6, 5′-TTTTCCTTTTGCGGCCGC-CTATTCAGCCTTGGAAAACATG-3′; pORF7, 5′-TTTTCCTTTTGCGGCCGC-TCACGTTGTCCTCTGAGAGT-3′; and pORF8, 5′-TTTTCCTTTTGCGGCCGC-TCAGCTCTTTTGCTTAAAAAGTT-3′. The cloned pORFs were expressed as fusion proteins with glutathione S-transferase (GST) fused to the N terminus of the chlamydial proteins as previously described (55). Expression of the fusion proteins was induced with isopropyl-β-d-thiogalactoside (IPTG) (Invitrogen, Carlsbad, CA), and the fusion proteins were extracted by lysing the bacteria via sonication in Triton X-100 lysis buffer (1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 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 antibodies (PAbs) (72) and monoclonal antibodies (MAbs) (71, 75). The fusion protein-specific antibodies were then used to localize the endogenous proteins in chlamydial organism-infected cells via an indirect immunofluorescence assay (35, 70, 75). To purify pgp3 for macrophage stimulation experiments, the GST-pgp3 fusion protein was absorbed onto the glutathione beads, and, after thorough washing, the pgp3 protein was cleaved off the beads with a precision protease from Pharmacia following the manufacturer's instruction. Since the precision protease was supplied in the form of a GST fusion protein, it bound to the glutathione beads while cleaving off the pgp3. Therefore, only the pgp3 protein was released into the supernatant. After further steps of cleaning and concentrating using additional glutathione beads and Centricon (Millipore, Billerica, MA), the protein was ready to use in the macrophage stimulation experiments. As a negative control for the macrophage stimulation experiments, we also similarly cloned, expressed, and purified Cpn0324, a Chlamydia pneumoniae genome-encoded hypothetical protein with a molecular mass of 43 kDa designated LcrE and predicted to be a regulatory molecule for the type III pathway (http://stdgen.northwestern.edu/ ). In addition, the pCHL1 ORF5 and -8 and CPAFct (CPAF from C. trachomatis serovar D) 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 fluorescent 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, Carlsbad, CA). At 24 hours after transfection, the RFP chlamydial fusion proteins were visualized via either the fusion tag RFP or the mouse anti-chlamydial protein antibody labeling. In other experiments, some of the pDsRed-transfected cultures were subsequently infected with chlamydial organisms 12 h after transfection, and the effect of the RFP fusion protein expression on the chlamydial infection was evaluated 30 h after infection. All plasmid clones carrying chlamydial genes used in the current study were confirmed via DNA sequencing using a service from the DNA core at the University of Texas Health Science Center at San Antonio.

Immunofluorescence assay.HeLa cells grown on coverslips were fixed with 2% paraformaldehyde (Sigma, St. Luis, MO) dissolved in phosphate-buffered saline 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 stain (blue; Sigma) was used to visualize nuclear DNA. A rabbit anti-chlamydial organism antibody (R1L2, raised with C. trachomatis L2 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 immunoglobulin G (IgG) secondary antibody conjugated with Cy2 (green; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used to visualize chlamydial inclusions. The various mouse antibodies plus a goat anti-mouse IgG conjugated with Cy3 (red; Jackson ImmunoResearch) were used to visualize the corresponding antigens. The mouse antibodies included PAbs made against the pCHL1 ORF GST fusion proteins (current study) and against GST-CT119 (IncA) (data not shown) and MAbs 2H4 (IgG2a) and 4E6 (IgG1) against GST-pORF5 (pgp3) (current study) and 100a against the CPAFct C terminus (72). In some cases, the primary antibodies were preabsorbed with either the corresponding or heterologous fusion proteins immobilized onto glutathione-conjugated agarose beads (Pharmacia) prior to staining. The preabsorption was carried out by incubating the antibodies with bead-immobilized antigens for 1 h at room temperature or overnight at 4°C followed by pelleting the beads. The remaining supernatants were used for immunostaining. For costaining between pgp3 (MAb2H4, IgG2a) and CPAF (MAb100a, IgG1), a goat anti-mouse IgG2a-Cy3 conjugate (red) in combination with a goat anti-mouse IgG1-Cy2 conjugate (green) was used. For the transfected cell samples, the RFP chlamydial fusion proteins were visualized via the fusion tag RFP (red) or by costaining with a mouse antibody. Some RFP-pgp3- and RFP-IncA-transfected cell samples were also subjected to subsequent chlamydial infection in order to evaluate the effect of the RFP fusion protein expression on chlamydial infection (22). In these experiments, the fusion proteins were visualized by RFP (red) and the chlamydial organisms were labeled with a combination of rabbit anti-chlamydial organism antibody and goat anti-rabbit IgG-Cy2 conjugate (green). The chlamydial infection rates were calculated for RFP-positive and -negative cell populations (from the same coverslips), respectively.

Cell samples after the appropriate immunolabeling 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 (69, 72). 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 multiple colors. An Olympus FluoView laser confocal microscope (Olympus) was used to further analyze the costained samples at the University of Texas Health Science Center at San Antonio institutional core facility as described previously (33, 34). All microscopic images were processed using the Adobe Photoshop program (Adobe Systems, San Jose, CA).

Western blot assay.The Western blot assay was carried out as described elsewhere (70, 72, 76). Briefly, either the total cell lysate, cell fraction, purified EB/RB, or fusion protein samples were solubilized in 2% sodium dodecyl sulfate (SDS) sample buffer and loaded onto SDS-polyacrylamide gel wells. The whole-cell samples were fractionated into cytosolic (S100) and nuclear fractions as previously described (19, 72). In some cases, cytosolic preparations from chlamydia-infected cells (L2S100) were precipitated with protein G-agarose beads (Pharmacia) bound with either the anti-pgp3 (MAb clone 2H4) or anti-CPAF (clone 100a) antibodies, and the precipitates were resolved in SDS gels for Western blotting. After electrophoresis, the resolved protein bands were transferred to nitrocellulose membranes and the membrane blots were detected with primary antibodies, including mouse PAbs against the eight plasmid fusion proteins (current study) and GST-CT813 fusion protein (CT813 is a known inclusion membrane protein [Inc]) (6)] and mouse MAbs 100a against the CPAF C terminus (72), 2H4 against pgp3, MC22 against chlamydial major outer membrane protein (MOMP) (72), and W27 against mammalian HSP70 (catalog no. SC-24; Santa Cruz Biotech). The primary antibody binding was probed with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson Immunologicals, West Grove, PA) and visualized with an enhanced chemiluminescence kit (Santa Cruz Biotech).

ELISA.Mouse Raw 264.7 (TIB71; ATCC) macrophages were cultured in RPMI 1640 with 10% fetal calf serum at 2 × 10⁵ cells/well in 24-well plates. The purified proteins were added to the cultures as stimuli at the final concentrations indicated in the figures for 24 h. Escherichia coli lipopolysaccharide (LPS) (Sigma) was used as a positive control stimulus. In some experiments, the stimuli were pretreated with heat (boiling for 10 min), polymyxin B (Sigma) (mixing with antigens at a final concentration of 10 μg/ml and incubating at 37°C for 2 h), or antibody depletion prior to the stimulation. The antibody depletion was carried out by mixing the pgp3 protein solution (1 μg/ml) with protein G-agarose bead-immobilized MC22 (specific to chlamydial MOMP) or 2H4 (specific to pgp3) MAb at 4°C overnight. After the agarose beads were spun down, the remaining supernatants were used for stimulation. The culture supernatants were harvested for cytokine measurements using commercially available enzyme-linked immunosorbent assay (ELISA) kits. Both the mouse MIP-2 (mouse homolog of interleukin-8, DY452) and tumor necrosis factor alpha (TNF-α) (MTA00) ELISA kits were from R&D Systems, Inc. (Minneapolis, MN). The ELISA was carried out following the instructions provided by the manufacturer or as described elsewhere (76). Briefly, 96-well ELISA microplates (Nunc, Rochester, NY) were coated with a capture antibody, and after blocking, the cytokine samples or standards were added to the coated plates, followed by a biotin-conjugated detection antibody. The antibody binding was measured with horseradish peroxidase-conjugated avidin plus a soluble colorimetric substrate [2,2′-azino-di(3-ethylbenzthiazoline)sulfonic acid (ABTS)]. The absorbance was read at 405 nm using a microplate reader (Molecular Devices Corporation, Sunnyvale, CA). Sometimes, the culture samples were diluted prior to the ELISA measurements in order to keep the absorbance readings within the linear range. The cytokine concentrations were calculated based on the absorbance values, cytokine standards, and sample dilution factors. The cytokine levels were expressed as pg per ml of culture supernatants. The data presented here came from three independent experiments.

Statistical analysis.A two-tailed-Student t test was used to analyze all data in the current study.

Production of antibodies against chlamydial plasmid-encoded proteins.To determine the locations of the plasmid-encoded proteins during chlamydial infection, we used a fusion protein approach to raise antibodies for all eight plasmid ORFs. Two different annotations of the pORFs are compared in Table 1, and the annotation described by Thomas et al. (65) was used in the current study. The eight pORFs were successfully expressed as GST fusion proteins (Fig. 1A). Although degradation fragments were always detectable, a significant amount of full-length fusion protein was produced for all eight ORFs. These fusion proteins were used to immunize mice and generate PAbs. The anti-fusion protein antibodies recognized the corresponding fusion proteins without obvious cross-reactivity with heterologous fusion proteins at the antibody concentrations used in the Western blot assay (Fig. 1B). Despite the fact that all fusion proteins contained a common GST fusion tag, the anti-fusion protein antibodies failed to recognize GST in unrelated fusion proteins when the antibodies were properly diluted. These observations suggest that the fusion protein immunogens predominantly induced antibodies specifically recognizing the corresponding chlamydial plasmid proteins, possibly due to the relatively high immunogenicity of the prokaryotic proteins compared to the common GST tag. We then used these anti-fusion protein antibodies to label the corresponding endogenous proteins in Chlamydia-infected cells (Fig. 1C). All antibodies detected specific signals, suggesting that all ORFs carried by the chlamydial cryptic plasmid were expressed during chlamydial infection. More importantly, the anti-GST-ORF5 (also designated pgp3) fusion protein antibody detected a dominant signal in the cytosol of the chlamydia-infected cells (Fig. 1C, panels i and m). We further raised MAbs against the GST-pgp3 fusion protein. Both the anti-GST-pgp3 PAbs and MAbs detected strong signals in the cytosol with a pattern similar to that of anti-CPAF but not anti-IncA antibodies (Fig. 1D). These PAbs and MAbs were further evaluated for their binding specificities. When these and other control antibodies were used to react with cells transfected with the recombinant pDsRed plasmids coding for RFP-pgp3, RFP-CPAFct, or RFP-pORF8, we found that the antibodies recognized only cells expressing the corresponding antigens and not those expressing heterologous antigens, although all antigens were successfully expressed as RFP fusion proteins (Fig. 1E). Furthermore, the ability of these antibodies to recognize endogenous proteins can be blocked only with the corresponding and not with the unrelated GST fusion proteins (Fig. 1F). The above-described experiments together have convincingly demonstrated that the anti-pgp3 antibodies specifically detected cytosolic signals in Chlamydia-infected cells.

• Open in new tab
• Download powerpoint

• Open in new tab
• Download powerpoint

• Open in new tab
• Download powerpoint

FIG. 1.

Production of antibodies against chlamydial plasmid-encoded proteins. (A) The eight ORFs encoded by pCHL1 from C. trachomatis serovar D organisms were expressed as GST fusion proteins, designated GST-pORF1 to 8. The fusion proteins were resolved in an SDS gel and protein bands visualized using Coomassie brilliant blue staining. Although degradation bands were always detected, all samples contained obvious full-length fusion proteins as indicated on the right. The pORF5-encoded protein is also called pgp3. (B) The SDS gel-resolved protein bands (as described for panel A) were blotted onto a nitrocellulose membrane for reaction with mouse sera (each raised with the corresponding GST-pORF fusion protein). The primary-antibody-recognized protein bands were visualized using an enzyme-conjugated secondary antibody and enhanced chemiluminescence. All eight anti-fusion protein antibodies recognized the corresponding full-length fusion proteins without significant cross-reactivity with heterologous fusion proteins. (C) The eight mouse anti-fusion protein antibodies were also reacted with HeLa cells infected with C. trachomatis L2 organisms. The mouse antibody bindings were visualized with a Cy3-conjugated goat anti-mouse secondary antibody (red), the chlamydial organisms were visualized with a combination of rabbit anti-chlamydial organisms and a goat anti-rabbit-Cy2 conjugate (green), and the nuclear DNA was visualized with Hoechst DNA dye (blue). It is clear that the anti-GST-pORF5 (pgp3) detected a strong cytosolic signal (panels i and m, arrows), while the other seven antibodies recognized the chlamydial organisms inside the inclusions. (D) The immunostaining was carried out as described for panel C. Both the anti-GST-pORF5 (pgp3) PAb (panels a and f) and MAbs (clones 2H4 [panels b and g] and 4E6 [c and h]) detected strong signals in the chlamydia-infected cell cytosol with a pattern similar to that of the anti-CPAF (panels d and i) but not the anti-IncA (panels e and j) antibody. (E) Genes coding for pORF5 (pgp3) and pORF8 (both from pCHL1) and CPAFct (from C. trachomatis serovar D) were cloned into pDsRed vector and expressed as RFP fusion proteins (red) in HeLa cells. The transfected cells were stained with antibodies against pgp3, CPAF, and pORF8 as indicated on the left. All antibodies recognized only HeLa cells expressing the corresponding antigens (yellow) (panels a, d, g, k, and o) without cross-reacting with the heterologous antigens. (F). Antibodies against pgp3, CPAF, or pORF8 as listed on the left were reacted with chlamydia-infected cells as described for panel C with or without preabsorption using GST-pgp3, GST-CPAFct, and GST-pORF8 fusion proteins. The antibody binding to the endogenous chlamydial proteins was removed by preabsorption with the corresponding (panels b, f, j, o, and t) but not the heterologous (panels c, d, g, h, k, l, n, p, r, and s) fusion proteins.

pgp3 is a unique secretion protein during chlamydial infection.Having verified the antibody binding specificity, we used these antibodies to further confirm the cytosolic localization of pgp3 and to probe the relationship between the chlamydial plasmid-encoded pgp3 and genome-encoded CPAF. The chlamydia-infected cells were fractionated into cytosolic and nuclear fractions and probed for the presence of pgp3 and other control proteins in a Western blot assay (Fig. 2A). Most pgp3 proteins were detected in the cytosolic fraction (HeLa-L2S100) and only a small amount of pgp3 in the nuclear fraction (HeLa-L2pellet) or purified RBs or EBs. The cytosolic fraction was not contaminated by either chlamydial inclusions or chlamydial organisms, since neither CT813 (an inclusion membrane protein) nor MOMP (an abundant organism outer membrane protein) was detected in the cytosol fraction. CT813 was detected only in the whole infected cell lysate and infected cell nuclear fraction, while MOMP was detected in the purified organisms in addition to the inclusion-containing samples. The cell fractionation and Western blot detection results clearly supported the microscopic observation that most pgp3-specific fluorescent signals were in the cytosol of the chlamydia-infected cells, with a small amount present inside the inclusions (Fig. 1C, panels i & m; Fig. 1D, panels a to c and f to h; and Fig. 1F, panels a to l). Interestingly, CPAF was detected only in the cytosolic fraction (Fig. 2A, panel b), which was also consistent with the microscopic observation that the CPAF signal was only found in the cytoplasm of cells containing mature inclusions (Fig. 1D, panels d and i, and 1F, panels m to p). The difference between the distribution patterns of pgp3 and CPAF in chlamydial organism-infected cells may suggest different secretion pathways or functions of the two despite both being in the host cell cytosol. We further analyzed the relationship between cytosolic pgp3 and CPAF using a costaining approach (Fig. 2B). Although pgp3 appeared to partially overlap with CPAF under a conventional fluorescence microscope (panels a to d), the two rarely overlapped with each other when being carefully examined using confocal microscopy (panels e to h and h1). The lack of association between pgp3 and CPAF was further confirmed using a coimmunoprecipitation assay (data not shown), in which neither pgp3 nor CPAF was coprecipitated by antibodies against the other although both were pulled down by the corresponding antibodies. Together these results have demonstrated that pgp3 is a newly identified secretion protein that is different from the known secretion protein CPAF, suggesting that pgp3 may fulfill a different function from CPAF during chlamydial infection.

• Open in new tab
• Download powerpoint

FIG. 2.

pgp3 is a unique secretion protein. (A) HeLa cells infected with C. trachomatis L2 organisms were fractionated into cytosolic (HeLa-L2S100) and nuclear (HeLa-L2 pellet) fractions. The cellular fractions along with total cell lysates and purified RBs and EBs as listed at the top were resolved in SDS gels, and the resolved protein bands were blotted onto a nitrocellulose membrane for reaction with antibodies listed on the left. All antibodies detected their corresponding proteins in the HeLa-L2 whole-cell lysate sample (lane 2) (as indicated on the right), and both pgp3 and CPAF were mainly enriched in the cytosolic fraction (lane 4 in panels a and b). However, pgp3 but not CPAF also appeared in the purified RB and EB preparations, although in small quantities (lanes 5 and 6 in panel a). (B) HeLa cells infected with C. trachomatis L2 organisms were costained with antibodies for pgp3 (clone 2H4 plus Cy3-conjugated goat anti-mouse IgG2a) (red) (panels b and f), CPAF (clone 100a plus Cy2-conjugated goat anti-mouse IgG1) (green) (panels a and e), and the Hoechst dye for DNA (blue) (panels c and g) and observed under both conventional fluorescence (panels a to d) and confocal (panels e to h1) microscopes. Note that pgp3 does not overlap with CPAF (panels h and h1).

Time course expression of pgp3 during chlamydial infection.We first monitored the expression of pgp3 in comparison with that of CPAF, CT813 (Inc), and MOMP in a Western blot assay (Fig. 3A). The pgp3 protein was first detected at 24 h after infection, and the anti-pgp3 PAb detected only pgp3 without cross-reacting with any other proteins from chlamydia-infected whole cell lysates (Fig. 3A, panel a). The three chlamydial genome-encoded proteins CPAF, CT813, and MOMP were also first detected at 24 h postinfection. All chlamydial proteins, whether encoded by a plasmid or the genome, increased their levels as infection progressed to 36 h and stayed high up to 48 h. When the protein expression was examined using the immunofluorescence assay under the microscope, we found that both pgp3 and CPAF were expressed as early as 12 h after infection (Fig. 3B, panels c [c1 and c2] and h [h1 and h2]), and cytosolic secretion of these two proteins was obvious by 24 h after infection (panels d and i). Once secreted, both proteins stayed in the cytosol for up to 48 h after infection, and some pgp3, but not CPAF, signals remained in the inclusions during the late stages of infection (panels e, f, k, and l).

• Open in new tab
• Download powerpoint

FIG. 3.

Time course of expression of pgp3. (A) HeLa cells infected with C. trachomatis L2 organisms were processed for Western blot detection of chlamydial proteins, including pgp3 and CPAF, and mammalian HSP70 (as a loading control) with antibodies listed at the left. All antibodies specifically detected the corresponding proteins (as indicated at the right) at or after 24 h postinfection, with the exception that the mammalian HSP70 was detected in all samples. (B) The cell samples infected as described for panel A were also used in an immunofluorescence assay as described for Fig. 1C for detection of pgp3 (panels a to f) and CPAF (panels g to l). Both pgp3 and CPAF were first detected at 12 h (panels c, c1, c2, i, i1, and i2) and were found in the host cell cytosol at or after 24 h postinfection. Note that a portion of pgp3 but not CPAF remained inside the inclusions even at late stage of the infection.

The pgp3 protein is secreted by all chlamydial species that carry the cryptic plasmid.We first checked whether pgp3 is encoded in all chlamydial plasmids and analyzed homology of the pgp3 amino acid sequences from different plasmids (Table 2). The sequences of pgp3 encoded by plasmids isolated from different strains of C. trachomatis are highly conserved, with more than 95% identity. However, the pgp3 sequence identity varies between 50 and 80% among different chlamydial species, with the highest identity (83%) between C. caviae and C. psittaci and the lowest (43%) between C. pneumoniae N16 and C. suis. The pgp3 amino acid sequence variation largely parallels with the genome sequence variation between the different chlamydial species, suggesting that the plasmid and pgp3 have adapted to their corresponding chlamydial host strain environments. We then used the antibody against pgp3 from C. trachomatis serovar D to probe the expression of pgp3 in various chlamydial strains and species (Fig. 4). In a Western blot, the anti-pgp3 antibody recognized pgp3 in cells infected with all C. trachomatis, C. muridarum, and C. psittaci organisms, which are known to carry the cryptic plasmid, but failed to detect anything in cells without infection or in cells infected with C. pneumoniae human isolate AR39, which is known to lack the cryptic plasmid (Fig. 4A). We then used this antibody to localize pgp3 in cells infected with these chlamydial organisms. pgp3 was detected in the cytosol of HeLa cells infected with the chlamydial serovars and species that express pgp3 (Fig. 4B), suggesting that pgp3 secretion may be maintained by selection pressure imposed by the chlamydial organisms that carry the cryptic plasmid.

• Open in new tab
• Download powerpoint

FIG. 4.

pgp3 is secreted into host cell cytosol by chlamydial species that carry the chlamydial plasmid. HeLa cells infected with C. trachomatis serovars D, L2, and A (40 h postinfection); C. muridarum (24 h); C. psittaci (30 h); and C. pneumoniae (72 h) were processed for Western blotting (A) or immunofluorescence staining (B) as described in for Fig. 3A and 1C, respectively. The anti-GST-pgp3 fusion protein antibody specifically detected the pgp3 protein band in cells infected with the plasmid-containing chlamydial organisms (A, panel a), and this antibody detected significant signals (B, red) in the cytosol of cells infected with chlamydial organisms that express pgp3 (panels a to h, j, and k) but not the C. pneumoniae organisms deficient in pgp3 expression (panels i and l).

Prior expression of pgp3 in host cell cytoplasm does not affect the subsequent chlamydial infection.We next evaluated the potential effect of pgp3 on chlamydial infection by using a transfection approach (Fig. 5). At 12 hours after transfection with plasmids coding for RFP alone or various RFP fusion proteins, the HeLa cell cultures were infected with chlamydial organisms. At 30 hours after infection, the infection rates for RFP-positive cells (cells that were positively transfected) and RFP-negative cells (cells that were not transfected with pDsRed plasmids) from the same cultures were calculated separately. We found no significant difference in the chlamydial infection rates of these two cell populations in either the cultures transfected with the pDsRedC1 vector alone or those transfected with the pDsRedC1-pgp3 plasmid (P > 0.05), demonstrating that prior expression of RFP or RFP-pgp3 fusion proteins in HeLa cells did not alter the susceptibility of the transfected cells to the subsequent chlamydial infection. As a positive control, RFP-IncA fusion protein expression significantly reduced the infection rate in the RFP-positive cells (P < 0.05). These observations suggest that the cytosolic pgp3 may not directly alter the chlamydial intracellular replication but may participate in the chlamydial interactions with host cells during chlamydial infection.

• Open in new tab
• Download powerpoint

FIG. 5.

Effect of RFP-pgp3 fusion protein expression on chlamydial infection. HeLa cells grown on coverslips in 24-well plates were transfected with pDsRed vector alone or the recombinant plasmid pDsRed-IncA or -pgp3, and at 12 h after transfection the cultures were infected with C. trachomatis serovar D organisms. At 30 h after the infection, the cell samples were processed for immunostaining with a rabbit anti-chlamydial organism antibody plus a goat anti-rabbit IgG conjugated with Cy2 (green) and a DNA dye (blue). The RFP fusion proteins were visualized via RFP (red). (A) Representative images of the cultures transfected with pDsRed (panel a), pDsRed-IncA (b), or pDsRed-pgp3 (c). (B) Quantitative results, for which 100 RFP-positive cells and 100 RFP-negative cells were counted from each coverslip culture that was transfected with the corresponding plasmids as listed along the x axis. The rates of chlamydial infection in these two different cell populations from the same cultures were calculated separately are and displayed along the y axis. The data come from three independent experiments. A statistically significant difference in infection rate (P < 0.05, two-tailed t test) between the RFP-positive and -negative cells in the cultures transfected with pDsRedC1-CT119 (IncA) was observed. No other cultures showed any significant differences. Error bars indicate standard deviations.

Purified pgp3 protein induces inflammatory cytokines in mouse macrophages.Since the Chlamydia-induced host inflammatory responses chlamydial greatly contribute to pathogenesis, we purified pgp3 protein and assessed the ability of the purified pgp3 protein to induce cytokine production in mouse Raw macrophages (Fig. 6). The pgp3 protein was purified by cleaving off the pgp3 portion from the GST-pgp3 fusion proteins immobilized onto the glutathione-agarose beads (Fig. 6A). We found that the purified pgp3 induced both TNF-α and MIP-2 (interleukin-8) production in the Raw macrophage cultures in a dose-dependent manner (Fig. 6B). Since the pgp3 was purified by cleavage from the agarose bead-immobilized GST-pgp3 fusion proteins, we expected very little contamination by bacterial LPS. This is because LPS that did not stick to the beads was washed away prior to the cleavage, while LPS that did stick to the beads remained on the beads after the cleavage. However, to further exclude the potential contribution of LPS to the pgp3-induced cytokines, we pretreated a parallel set of pgp3 preparations with 10 μg/ml polymyxin B, an antibiotic known to bind to LPS and block LPS function, and the polymyxin B was maintained in the macrophage culture throughout the experiments. These treatments effectively prevented LPS from inducing cytokines (P < 0.01) but failed to affect the pgp3-induced production of cytokines, including TNF-α (Fig. 6B, panel a) and MIP-2 (Fig. 6B, panel b). A negative control chlamydial protein (prepared in the same way as pgp3) showed no obvious stimulation of the macrophages. We further used heat treatment to exclude the potential LPS contribution. The fact that LPS is heat stable and proteins are generally heat labile has been utilized to distinguish the roles of proteins and LPS in cytokine induction (2). Both pgp3 and LPS samples were boiled for 10 min before being added to the cultures. As expected, the heat treatment completely blocked the TNF-α induction by pgp3 protein (P < 0.01) but had little effect on LPS stimulation of TNF-α. More importantly, the ability of pgp3 to stimulate TNF-α production was removed by depletion with the pgp3-specific MAb 2H4 but not the chlamydial MOMP-specific MAb MC22. The observations described above together have convincingly demonstrated that the purified pgp3 protein specifically induced cytokine production.

• Open in new tab
• Download powerpoint

FIG. 6.

Induction of inflammatory cytokines by pgp3. (A) pgp3 was cleaved off from the agarose bead-immobilized GST-pgp3 fusion protein (lane 1) using a GST precision protease. The pGEX-6p expression vector encodes a precision protease site between GST and pgp3. After the cleavage, the supernatant (lane 2) was harvested and the beads were sequentially washed three times (lanes 3, 4, and 5). The remaining bead sample after digestion and washing was loaded in lane 6. The protein bands shown in panel a were visualized with Coomassie blue dye staining, while panel b shows the Western blot results probed with a mouse anti-pgp3 polyclonal antibody. The amount of protein per lane for panel b was scaled down to 1/1,000 in order to be able to visualize clear bands in the Western blot. It is worth noting that the Coomassie blue dye visualized the GST precision protease bound to the glutathione on the beads (panel a, lane 6). However, no signal was detected in the same sample with the anti-pgp3 antibody (panel b, lane 6) despite the large amount of GST remaining bound to the beads (panel a, lane 6). (B) The cleaved pgp3 protein fractions were harvested and pooled together for stimulating mouse Raw 246.7 macrophages at final concentrations ranging from 1 to 10 μg/ml as indicated. As a negative control, the chlamydial protein Cpn0324 was similarly prepared and used at 1 and 10 μg/ml. LPS at a final concentration of 10 ng/ml was used as a positive control. A parallel set of pgp3, LPS, and Cpn0324 samples were pretreated with 10 μg/ml of polymyxin B for 30 min at 37°C, and the polymyxin B was maintained in the cultures throughout the incubation. At 24 hours after stimulation, the culture supernatants were harvested for cytokine measurements using ELISA and the concentrations of cytokines were determined. It is clear that both pgp3 and LPS induced significant amounts of TNF-α (panel a) and MIP-2 (panel b). However, polymyxin B inhibited only LPS-induced cytokines (P < 0.01, two-tailed Student t test) without affecting the cytokine production induced by pgp3. The data are from three independent experiments. Error bars indicate standard deviations. (C) Parallel samples of pgp3 and LPS were boiled for 10 min (bars 1 to 4) or depleted with the MOMP-specific MAb MC22 (bar 5) or the pgp3-specific MAb 2H4 (bar 6) prior to being added to the macrophage cultures. Note that the heat treatment almost completely blocked TNF-α induction by pgp3 without affecting LPS-induced TNF-α production, and the pgp3-specific MAb significantly depleted the TNF-α stimulation ability of pgp3. The data are from three independent experiments. Error bars indicate standard deviations.