HeLa 229 cells (No. TCHu 20) and Vero cells (No. GNO10) were purchased from the National Collection of Authenticated Cell Cultures (NCACC; Beijing, China). HEp-2 cells (ATCC, No. CCL-23) and DF-1 cells (ATCC, No. CRL-12203) were maintained in 8% foetal bovine serum (FBS; Gibco, Beijing, China)/Dulbecco’s modified Eagle’s medium (DMEM) (Solarbio Life Science Ltd., Beijing, China), and Chinese hamster ovary (CHO-K1; No. SCSP-507) cells were purchased from NCACC and cultured in Ham’s F-12K (Invitrogen, Beijing, China) supplemented with 10% FBS. All cell lines were cultured at 37°C with a 5% CO₂ flow. C. psittaci 6BC (GenBank: CP002549.1) was kindly donated by Professor Yimo Wu (University of South China, Hunan, China) and cultivated, purified and titrated as previously described (23). C. trachomatis L2 (Institute for Functional Microbial Genomics, Heinrich-Heine-Universität, Düsseldorf, Germany) were propagated in HEp-2 cells as described (15).

The following antibodies were used in this study: anti-GAPDH (MA5-15738), anti-6×His (MA1-21315), anti-HA (26183) and anti-flag (MA1-91878) mouse monoclonal antibodies (mAbs) and goat anti-mouse IgG H+L (HRP) secondary antibody (31430), which were commercially obtained from Thermo Fisher Scientific (Beijing, China), and anti-EGFR (sc-373746), and anti-phospho-EGFR (1068) (sc-81488) and anti-Grb2 (sc-8034) mAbs, which were purchased from Santa Cruz Biotechnology (Santa Cruz, Shanghai, China). The remaining anti-EGFR rabbit mAbs (4267; CST, Beijing, China) and goat anti-rabbit IgG H&L (HRP) secondary antibody (ab6721; Abcam, Beijing, China) were also commercially available products. Anti-GroEL rabbit pAbs, anti-MOMP mouse mAbs and anti-S1 rabbit pAbs were from Heinrich-Heine-Universität and described previously (11).Anti-MOMP mouse mAbs were prepared by immunizing mice with recombinant MOMP from C. psittaci 6BC in our laboratory as described previously (19). Anti-Pmp17G rabbit polyclonal antibodies (pAbs) were prepared in rabbits inoculated with recombinant Pmp17G protein that was expressed and purified from E. coli BL21 cells (TransGen Biotech Ltd., Beijing, China). The antibodies were purified using HiTrap™ rProtein AF (GE, Beijing, China). The specificities of the MOMP monoclonal antibodies and polyclonal antibodies against Pmp17G were identified with ELISAs as described previously (19).

The pmp17G gene was amplified from EBs of C. psittaci 6BC and then subcloned into a pKM255 shuttle vector(Heinrich-Heine-Universität, Düsseldorf, Germany), a pET28a prokaryotic vector or a pCMV-C-HA eukaryotic vector (Beyotime Biotech Ltd., Shanghai, China), respectively. Targeted ectodomain of Pmp17G protein was listed in Figure S2 . cDNA samples of EGFR from HeLa 229 cells were amplified by RT-PCR and then subcloned into a pCMV-C-flag vector (Beyotime Biotech Ltd, Shanghai, China), named pCMV-EGFR-N-flag. Four restriction enzymes and T4 DNA ligase were obtained from a commercial company (New England Biolabs, Beijing, China). PCR was performed using Q5 High-Fidelity DNA polymerase (New England Biolabs, Beijing, China). PCR primers were listed in Table S1 . Both recombinant Pmp17G protein and MOMP protein were induced by isopropyl-β-D-thiogalactopyranoside (IPTG; Beyotime Biotech Ltd., Shanghai, China) in 500 ml of cultured E. coli BL21 cells. After purification with Ni-NTA Resin(88221, Thermo Fisher Scientific, Beijing, China), the recombinant MOMP protein and Pmp17G were confirmed by SDS-PAGE and Western blots as described previously (19).

First, HeLa 229, Vero and DF-1 cells were grown in 24-well plates for 24 h. Then, the culture media were removed, and the cells were washed 3 times with cold PBS and then incubated with 200 µl of Pmp17G (200 µg/ml) to cover the surface of the cells. Subsequently, the cultures were incubated at 37°C with 5% CO₂ flow and monitored at 15, 30, 60, 90, and 120 min. Finally, the cell cultures were washed 5 times with PBS, and then, the cells were lysed in 50 µl of RIPA buffer with phenylmethylsulfonyl fluoride (PMSF; Beyotime Biotech Ltd, Shanghai, China). The lysed solutions were centrifuged at 12,000 g for 10 min, and then, the supernatants were boiled with loading buffer. Finally, 10 µl of the cell lysate was identified by SDS-PAGE and Western blots.

Chlamydial transformation was performed as described previously (24). Briefly, the pKM255::pmp17G plasmid (Institute for Functional Microbial Genomics, Heinrich-Heine-Universität, Düsseldorf, Germany), was extracted from a Dam- and Dcm-methylase-deficient strain of E. coli (GM-48), using a plasmid mega kit (Qiagen, Hilden, Germany). Penicillin (1µg/µl) was used for the selection of transformed C. trachomatis L2. A mixture of C. trachomatis L2 EBs and pKM255::Pmp17G was incubated in calcium chloride buffer (10 mM Tris, 50 mM calcium chloride, pH 7.4) for 30 min at room temperature. Subsequently, C. trachomatis L2 was mixed with pKM255::pmp17G and incubated with HEp-2 cells in calcium chloride buffer for 1 h at room temperature. The total calcium chloride mixture was discarded and replaced with fresh 10% FBS/DMEM containing 2 µg/ml cycloheximide. After 24 hpi, penicillin (1 µg/µl) was added and further incubated for 36 h at 37°C under 5% CO₂. Afterwards, the infected epithelial cells were scraped and lysed with glass beads for next passages untill inclusions were observed obviously. Positive expression of Pmp17G in C. trachomatis L2 was identified to bind anti-flag antibody by immunofluorescence (IF). Furthermore, location of the Pmp17G was analyzed using different detergent extractions as described previously (11).

For overexpressing proteins, HeLa 229 cells were transfected with plasmids containing the EGFR gene and/or pmp17G gene using Lipofectamine™ 3000 (Invitrogen, Beijing, China) according to the manufacturer’s protocol. As for immunoprecipitation(IP) of surface complexes, HeLa 229 cells were grown to 90% confluency, and the cell cultures were washed with PBS and incubated with 200 µg/ml purified Pmp17G in FBS-free DMEM at 37°C for 2 h. Twenty-four hours after transfection or 2 h after incubation, cell cultures were rinsed 3 times with PBS and then lysed in IP lysis buffer (containing 1% NP-40, 1% Triton X-100, 20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM Na₃VO₄, 5 mM NaF, and 2 mM EDTA) and protease inhibitor cocktail (Beyotime Biotech Ltd, Shanghai, China) at 4°C for 30 min. Then, cell lysates were precleared with standard IgG (rabbit sera) (Solarbio Life Science, Beijing, China) together with protein A/G PLUS-agarose (Santa Cruz, Shanghai, China) at 4°C for 30 min. Then, the precleared cell lysates were used for IP reactions. Co-IP reactions were carried out by mixing anti-EGFR mAbs or anti-Pmp17G pAbs at 4°C for 6 h and then washing the lysates 3 times with IP buffer. Finally, the target proteins were identified by Western blots using HA-tagged or flag-tagged antibodies.

HeLa 229 cells were incubated with Pmp17G (200 µg/ml) at 37°C for 2 h. Afterwards, the unbound proteins were discarded after washing 5 times with PBS, and the cells were infected with C. psittaci 6BC at an MOI of 5 in medium containing 8% FBS/DMEM and 2 µg/ml cycloheximide (Sigma-Aldrich, Shanghai, China) and then incubated at 37°C for 36-48 h. To confirm blocking infection, C. psittaci 6BC EBs were diluted with SPG solution (containing 0.25 M sucrose, 10 mM sodium phosphate, and 5 mM L-glutamic acid, pH 7.2-7.5) to a final concentration of 1.0×10⁶ inclusion-forming units (IFUs)/ml. The diluted EBs were incubated with purified anti-Pmp17G pAbs at 50 µg/ml, and the cultured EBs were allowed to react at room temperature for 2 h using a shaking machine. Later, HeLa 229 cells were infected with the above cultures at 37°C for 36-48 h. IFUs were quantified using a monoclonal antibody against MOMP and IF microscopy. The adhesion studies and internalization assays were carried out using Pmp17G-coated fluorescent beads as described previously (25).

To identify the effect of EGFR on C. psittaci 6BC infection, HeLa 229 cells were pretreated with different concentrations of cetuximab (Sigma-Aldrich, Shanghai, China), an anti-EGFR antibody, at 37°C for 2 h. Then, the unbound antibodies were removed from the reaction followed by washing 5 times with PBS, and finally, cell cultures were infected with C. psittaci 6BC at an MOI of 5 and incubated at 37°C for 36-48 h. In another experiment, EGFR in HeLa 229 cells was knocked down via transfection with EGFR siRNA (Santa Cruz, Shanghai, China) according to the manufacturer’s protocol, and the knockdown efficiency was confirmed by Western blots. Then, the transfected HeLa 229 cells were exposed to C. psittaci 6BC at an MOI of 5 at 37°C for 36-48 h. To construct overexpressed EGFR in CHO-K1 cells, the cells were transfected with pCMV-EGFR-N-flag at 37°C for 24 h. Subsequently, the overexpressed cells were infected at an MOI of 5 at 37°C for 36-48 h. IFUs were determined as described above.

HeLa 229 cells were cultivated with 8% FBS at 37°C for 24 h and then replaced with FBS-free medium for 12 h. Subsequently, cell cultures were stimulated with different concentrations of Pmp17G (from 25 to 500 µg/ml) or C. psittaci 6BC (MOI from 0.1 to 100) at 37°C for 2 h. Then, the cell cultures were washed 5 times with cold PBS, and the cells were lysed in 50 µl of IP lysis buffer with protease and phosphatase inhibitor cocktail. The lysed solutions were centrifuged at 12,000 g for 10 min, and then, the supernatants were boiled with 1x loading buffer for 10 min. Later, 10 µl of the cell lysates were identified by SDS-PAGE and Western blots. The supernatants were tested for EGFR, pEGFR₁₀₆₈, and Grb2 expression using anti-EGFR, anti-pEGFR₁₀₆₈, anti-Grb2 and anti-GAPDH antibodies, respectively. The optimal concentrations of Pmp17G and EBs of C. psittaci 6BC were applied in a time-dependent manner. HeLa cells were infected with 200 µg/ml Pmp17G or C. psittaci 6BC at an MOI of 5 for different durations (from 0, 5, 10, 15, 30 to 60 min). Then, the cell cultures were treated as described above. To carry out Co-IP with EGFR at Try1068 and Grb2, HeLa cells were infected with 200 µg/ml Pmp17G or C. psittaci 6BC at an MOI of 5 for 30 min. The cell cultures were treated IP lysis buffer containing a protease and phosphatase inhibitor cocktail. Finally, the supernatants were subjected to IP with anti-EGFR antibodies as explained above.

Statistical analyses were carried out by analysis of variance (ANOVA). Multiple comparisons and differences were analyzed using the least-significant difference (LSD) method. A p-value (two-tailed) of 0.05 or less was considered to indicate significance. Analyses were carried out using GraphPad Prism software (GraphPad Software Inc., CA, USA) and Image J (National Institutes of Health, Maryland, USA).

In the present study, strong Pmp17G adhesion was found to HeLa 229 cells, and secondary higher attaching capability was observed in Vero cells than DF-1 cells did. Regarding the time course, early adhesion was observed at 15 min, and an increasing attachment was correlated with prolonged incubation and the strongest signal was detected at 120 min, while secondary adhesion occurred at 60 min in Vero cells and 120 min in DF-1 cells, respectively ( Figure 1A ). Subsequently, 3 different host cells were incubated with Pmp17G, and the relative C. psittaci infection of HeLa 229 cells, DF-1 cells and Vero cells was reduced to 72.15%, 70.04% and 76.97%, respectively, which was higher than that of the heparin-positive group. However, no difference was found among the 3 host cells prior to treatment with recombinant Pmp17G ( Figure 1B ), indicating that Pmp17G might attach to multiple host cells and involve in tissue tropism, contributing to the wide spectrum of C. psittaci infection in comparison with C. trachomatis (15) and C. pneumoniae (17).

Regarding the only one pmpG gene of C. trachomatis, the role of ectopic expression of C. psittaci-specific Pmp17G in C. trachomatis will be a fundamental model to elucidate the functions of the duplication of pmpGs in C. psittaci. Interestingly, expression of C. psittaci-specific Pmp17G in C. trachomatis L2 was observed using indirect immunofluorescence (IF) at 24-hour post infection (hpi) ( Figure 2A ). Moreover, locations of C. psittaci-specific PmpG17 were found both on outer membrane and cytoplasm of EBs by Western blots. Meanwhile, GroEL was located both on outer membrane surface and cytoplasm, MOMP as outer membrane protein and S1 protein located in cytoplasm of C. trachomatis L2 were used as the control groups ( Figure 2B ). To warrant above observation, Hep-2 cells were treated with Pmp17G or Ctad1 and infected with ectopic expression of C. trachomatis L2 (pKM255::pmp17G).Interestingly, Pmp17G displayed strong blocking infection compared to Ctad1 post infection of C. trachomatis L2 (pKM255::pmp17G) (p<0.05) ( Figure 2C ).

Pmp17G was incubated with HeLa 229 cells for 2 h, and the attached fractions in the pull-down elution using Ni-NTA were identified and analyzed by MALDI-MS ( Figure S1 ). Although no difference was found in the SDS-PAGE assay with Coomassie staining ( Figure S1A ), an increasing intensity was observed in the Pmp17G-linked DTSSP compared to Pmp17G alone ( Figure S1B ), and higher intensity of Pmp17G-related EGFR was found in the Pmp17-linked group compared to the Pmp17G alone group ( Figure S1C ). To confirm that Pmp17G directly interacted with EGFR in host cells, Co-immunoprecipitation (Co-IP) assays were carried out with anti-His mAbs or anti-EGFR mAbs, and Pmp17 binding to EGFR was confirmed by Western blots ( Figures 3A, B ). Then, overexpression of Pmp17G and EGFR was used to validate the interaction. Finally, positive band of Pmp17G-recognized EGFR was identified with anti-HA antibody ( Figure 3C ) and anti-flag antibody ( Figure 3D ), indicating that EGFR is a receptor of Pmp17G.

To further confirm the roles of EGFR during C. psittaci infection, serial doses of cetuximab were used to block EGFR in host cells. After inoculation with C. psittaci 6BC, 20 μg/ml cetuximab was optimized for blocking Chlamydia infection, and a dose-dependent effect was found in a neutralization test ( Figure 4A ). After treatment with EGFR siRNA, the relative infection was reduced dramatically compared to that of the control siRNA and the mock groups ( Figure 4B ). Moreover, the relative infection was increased significantly in the EGFR overexpression group compared to that in the CHO-K1 cell lines or CHO-K1 cells transfected with the control vector (p<0.01) ( Figure 4C ), indicating that EGFR plays a major role in early C. psittaci infection.

After C. psittaci 6BC was pretreated with anti-Pmp17G antibody or anti-MOMP antibody, a significant difference was found among the IgG (mouse) group, IgG(rabbit) group, the MOMP antibody-treated group and the Pmp17G antibody-treated group, suggesting that the Pmp17G antibody reduced dramatically the extent of the Chlamydia infection ( Figure 5A ). To monitor Pmp17G-mediated adhesive capability, an increase in the FITC-A subset was observed in the Pmp17G-coated group compared to the positive MOMP-coated group (p<0.05) or the negative BSA-coated group (p<0.01). To elucidate the role of EGFR, HeLa 229 cells were pretreated with cetuximab and then incubated with Pmp17G-coated green fluorescent beads. Bead adhesion to host cells was inhibited significantly in the cetuximab/Pmp17G group compared to the Pmp17G group (p<0.05) ( Figure 5B ). More interestingly, only Pmp17G-coated beads exhibited intracellular localization and 20.7% internalized beads were determined while BSA-labelled beads presented solely on cellular surfaces ( Figures 5C, D ), suggesting that Pmp17G is not only an adhesin but also an invasin during C.psittaci infection.

After HeLa 229 cells were inoculated with different doses of C. psittaci 6BC, phosphorylation of EGFR at site 1068 was activated in a dose-dependent manner with C. psittaci inoculation ranging from 0.1 to 1.0 MOI, while a gradual reduction in EGFR phosphorylation was observed with C. psittaci 6BC inoculation ranged from 5.0 to 100.0 MOI. Compared to pY1068-EGFR, no difference of EGFR expression was found after infection with different doses of C. psittaci. In contrast, Grb2 expression was downregulated in a dose-dependent manner ( Figure 6A ). Upon Pmp17G treatment, the expression of EGFR, pY1068-EGFR and Grb2 displayed similar trends ( Figure 6B ). The time-dependent effect was assessed, and the results showed that increased EGFR phosphorylation was observed with prolonged time, while a negative correlation was found between time points and Grb2 expression after exposure to EBs ( Figure 6C ). Upon Pmp17G treatment, EGFR phosphorylation increased gradually with prolonged time. However, the expression of EGFR and Grb2 displayed no difference after Pmp17G treatment ( Figure 6D ). To determine whether Grb2 was translocated from the cellular reticulum to EGFR phosphorylation at site 1068 in the plasma membrane, we carried out Co-IP to determine whether EGFR binds to Grb2. Subsequently, positive bands were observed in both the C. psittaci group and the Pmp17G treated group ( Figure 6E ), indicating that EGFR phosphorylation might recruit Grb2 and trigger C. psittaci internalization.