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. 2022 Jan 31:12:818487.
doi: 10.3389/fimmu.2021.818487. eCollection 2021.

Polymorphic Membrane Protein 17G of Chlamydia psittaci Mediated the Binding and Invasion of Bacteria to Host Cells by Interacting and Activating EGFR of the Host

Xiaohui Li  1 , Zonghui Zuo  2   3 , Yihui Wang  1 , Johannes H Hegemann  3 , Cheng He  1
Affiliations

Polymorphic Membrane Protein 17G of Chlamydia psittaci Mediated the Binding and Invasion of Bacteria to Host Cells by Interacting and Activating EGFR of the Host

Xiaohui Li et al. Front Immunol. .

Abstract

Chlamydia psittaci (C. psittaci) is an obligate intracellular, gram-negative bacterium, and mainly causes systemic disease in psittacine birds, domestic poultry, and wild fowl. The pathogen is threating to human beings due to closely contacted to employees in poultry industry. The polymorphic membrane proteins (Pmps) enriched in C. psittaci includes six subtypes (A, B/C, D, E/F, G/I and H). Compared to that of the 1 pmpG gene in Chlamydia trachomatis (C. trachomatis), the diverse pmpG gene-coding proteins of C. psittaci remain elusive. In the present study, polymorphic membrane protein 17G (Pmp17G) of C. psittaci mediated adhesion to different host cells. More importantly, expression of Pmp17G in C. trachomatis upregulated infections to host cells. Afterwards, crosstalk between Pmp17G and EGFR was screened and identified by MALDI-MS and Co-IP. Subsequently, EGFR overexpression in CHO-K1 cells and EGFR knockout in HeLa 229 cells were assessed to determine whether Pmp17G directly correlated with EGFR during Chlamydial adhesion. Finally, the EGFR phosphorylation was recognized by Grb2, triggering chlamydial invasion. Based on above evidence, Pmp17G possesses adhesive property that serves as an adhesin and activate intracellular bacterial internalization by recognizing EGFR during C. psittaci infection.

Keywords: C. psittaci; EGFR; Pmp17G; adhesion; invasion.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Pmp17G mediated adhesion to host cells, and recombinant Pmp17G blocked C. psittaci infection. (A) HeLa 229 cells, Vero cells and DF-1 cells were incubated with 200 μg/ml Pmp17G, while inactivated EBs at an MOI of 5 were used as a positive control for different durations. Positive bands were reacted with anti-His mAbs or anti-MOMP antibody, and GAPDH was the loading control. (B) HeLa 229 cells, Vero cells and DF-1 cells were pretreated with 200 μg/ml of Pmp17G and 500 μg/ml heparin, as the positive control for 2 h, and then exposed to C. psittaci 6BC at an MOI of 5 for 36-48 h Inclusion-forming units (IFUs) were quantified by indirect immunofluorescence (IIF). Relative Infection (%)=(IFUs of the treated group/IFUs of the PBS group)×100. Statistical analysis was performed by one-way ANOVA, and the data from 3 independent experiments were expressed as the means ± standard deviations (SD). Asterisks indicate statistical significance: **p < 0.01.
Figure 2
Figure 2
Expression of C.psittaci-specific PmpG17 upregulated C. trachomatis infection. (A) Identification of C. psittaci-specific PmpG17 in C. trachomatis L2 strain by indirect immunofluorescence at 24 hpi. (B) Expressions of C psittaci-specific PmpG17 were located both on outer membrane and cytoplasm of elementary bodies by Western blots. GroEL, a heat-shock protein located both on outer membrane surface and cytoplasm, MOMP, an outer membrane protein, and S1 protein, an intracellular protein of C. trachomatis L2 as the control group. Elementary bodies of C. trachomatis L2 (pMK255::pmp17G) were treated with PBS, 1% Triton-X 100, 2% Sarkosyl or N-40 with DTT, respectively. Pellet (P) and supernatant (S) fractions were prepared by centrifugation and incubated with anti-S1, anti-MOMP, anti-GroEL1 and anti-flag antibodies, respectively. (C) HEp-2 cells were preincubated with 200 μg/ml of recombinant Pmp17G or Ctad1 as a positive control. Afterwards, above cell cultures were inoculated with C. trachomatis L2 (pMK255::pmp17G) at an MOI of 5 for 24 hours. IFUs were quantified as above described. Relative infection (%)=(IFUs of the treated group/IFUs of the PBS group)×100. Statistical analysis was performed by one-way ANOVA, and the data from 3 independent experiments were expressed as the means ± SD. Asterisks indicate statistical significance: *p < 0.05.
Figure 3
Figure 3
Crosstalk of Pmp17G with EGFR. (A) Pmp17G was incubated with HeLa 229 cells for 2 h, immunoprecipitated with anti-His mAbs, and then reacted with anti-EGFR mAbs and anti-His mAbs. (B) After incubation with HeLa 229 cells, IP was assayed with anti-EGFR mAbs, and then, Pmp17G-recognized EGFR was identified with anti-His mAbs and anti-EGFR mAbs using Western blots. (C) Co-expression of Pmp17G and EGFR in HeLa 229 cells. HeLa 229 cells were transfected with pCMV-Pmp17G-HA and pCMV-EGFR-N-flag plasmids, reacted with anti-HA antibody for IP, and then incubated with anti-flag and anti-EGFR mAbs by Western blots. (D) Co-expression of Pmp17G and EGFR in HeLa 229 cells was evaluated by Co-IP. HeLa 229 cells were transfected with pCMV-Pmp17G-HA and pCMV-EGFR-N-flag plasmids, immunoprecipitated with anti-flag antibody, and then identified with anti-flag and anti-EGFR mAbs using Western blots.
Figure 4
Figure 4
EGFR was essential for C. psittaci infection. (A) HeLa 229 cells were treated with cetuximab (10 μg/ml and 20 μg/ml) for 2 h and then inoculated with C. psittaci 6BC (MOI=5). Relative infection was calculated on the basis of inclusion bodies, and Relative infection (%)=(IFUs of the treated group/IFUs of the control group) ×100. (B) HeLa cells were transfected with EGFR siRNA (siEGFR) or control siRNA (Ctrl). Twenty-four hours after the 2nd treatment with siRNA, the cell cultures were exposed to C psittaci 6BC (MOI=5) and, concurrently lysed to determine efficiency of siRNA by Western blots. The intensity of the bands was quantified using ImageJ. GAPDH was used as the loading group. Intensity was determined as follows: Intensity(%)=(EGFR/GAPDH) ×100. In addition, inclusion bodies were counted and relative infection was determined as follows: Relative infection (%)=(IFUs of the siRNA-treated group/IFUs of the mock group) ×100. (C) CHO-K1 cells were transfected with exotic EGFR-flag, and ectopic expression of EGFR was analysed by Western blots. Twenty-four hours post-transfection, cell cultures were infected with C. psittaci 6BC (MOI=5). At 36-48 hpi, inclusions were measured by IIF. Relative infection was calculated using the formula: Relative infection(%)= (IFUs of the CHO-K1 group/IFUs of the HeLa 229 group) × 100. Statistical analysis was performed by one-way ANOVA, and the data were expressed as the means ± SD. Asterisks indicate statistical significance: *p < 0.05, and **p < 0.01.
Figure 5
Figure 5
Pmp17G contributed to adhesion and invasion during C. psittaci infection. (A) C. psittaci 6BC was treated with anti-Pmp17G antibody or anti-MOMP antibody while IgG(mouse), IgG(rabbit) and PBS were control groups. Then, HeLa 229 cells were infected with these preparations for 36-48 h Relative infection (%)=(IFUs of the antibody-treated group/IFUs of control group) ×100. (B) Pmp17G adhesive capability to HeLa 229 cells. HeLa 229 cells were incubated with BSA-, MOMP- or Pmp17G-coated green fluorescent beads for 2 h at 37°C. Then, cell cultures were treated with trypsin (non-EDTA) for 3 min and washed 3 times with PBS, and the cells were resuspended in PBS. Adhesion of FITC-A subsets was measured using flow cytometry and proportion of FITC-A subsets was determined by dividing the number of FITC-labelled cells by the total number of cells. Three replicates of each measurement were performed. (C) Pmp17G invasion capability into HeLa 229 cells. HeLa 229 cells were incubated with BSA-, or Pmp17G-coated green fluorescent beads for 6 h at 37°C. Subsequently, the cell cultures were washed 5 times with PBS and fixed with 0.4% paraformaldehyde. Later, the extracellular binding beads were stained with 6×His-tag antibody (blue). Internalized beads displayed green. Cell structures were stained with Evans blue (red). (D) Internalized bead(%)=(Numbers of internalized beads/all the binding beads) ×100. Statistical analysis was performed by one-way ANOVA, and the data were expressed as the means ± SD. Asterisks indicate statistical significance: *p < 0.05, and **p < 0.01.
Figure 6
Figure 6
Pmp17G stimulated the phosphorylation of EGFR and downregulated Grb2 expression. (A) HeLa 229 cells were infected with serial doses of C. psittaci (MOI from 0.1 to 100), and the expression of EGFR, pY1068-EGFR and Grb2 was identified using Western blots. (B) HeLa 229 cells were treated with serial doses of Pmp17G (from 10 μg/ml to 500 μg/ml), and the expression of these proteins was determined using Western blots. (C) HeLa 229 cells were infected with C. psittaci (MOI=1.0), and the expression of the aforementioned proteins was identified at different time points (at 5, 15, 30, 60, 90, 120, and 180 min) by Western blots. (D) HeLa 229 cells were treated with 200 μg/ml of Pmp17G, and the expression levels of these proteins were determined at different time points. GAPDH was used as the loading control. (E) Grb2 recognized pY1068-EGFR by Co-IP assay. HeLa cells were treated with C. psittaci 6BC (MOI=1.0) or 200 μg/ml of Pmp17G, and IP was performed with anti-EGFR mAbs for 0.5 h at 4°C. Then, IP compounds were incubated with anti-EGFR, anti-pY1068-EGFR or anti-Grb2 antibodies, respectively and identified by Western blot assay. β-Tubulin was used as the loading control.
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