Glaesserella parasuis serotype 4 exploits fibronectin via RlpA for tracheal colonization following porcine circovirus type 2 infection

Abstract

Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin β1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-β signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.

Fig 1. PCV2 infection promotes the adhesion of GPS4 in STEC.

Adhesion of GPS4 to STEC. After PCV2 infection at 24, 36, and 48h, the cells were infected with GPS4 for another 2 h, and the number of adherent bacteria was counted. The CFUs of GPS4 in PCV2-uninfected STEC cells are considered to be 1. Results are shown as means ± SDs of three independent experiments. Statistical analysis was performed using two-way ANOVA. ***, P <0.001; ****, P < 0.0001; ns, not significant.

Fig 2. PCV2 infection induces Fn expression on tracheal epithelial cells.

After PCV2 infection for 36 h, swine tracheal epithelial cell surface proteins were isolated and identified by mass spectrometry (A and B). (A) LC-MS/MS analysis result for Fn. (B) LC-MS/MS spectrum for the Fn peptide. (C) Lysates of STEC cells infected with PCV2 (1 MOI) for 36 h were analyzed by Western blot. Image J was used to analyze the density of protein bands, and GAPDH was used as an internal reference. (D) mRNA levels of Fn in STEC cells infected with PCV2 for 24, 36, and 48h were analyzed by RT-qPCR. Data are shown as means ± SDs of three independent experiments. Statistical analysis was performed using an unpaired t-test (C) and two-way ANOVA (D). *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Fig 3. PCV2 infection-induced Fn promotes GPS4 adherence.

In order to preliminarily verify whether PCV2 infection-induced Fn is associated with GPS4 adhesion, Fn was interfered with with siRNA. (A) Un-transfected and Fn siRNA-transfected STEC cells, Western blot analysis of Fn protein levels. Image J was used to analyze the density of protein bands, and GAPDH was used as an internal reference. (B) The total mRNA level of Fn was analyzed by RT-qPCR. (C) PCV2 infection un-transfected and Fn siRNA-transfected STEC cells for 36 h, GPS4 infection for another 2 h, adhesion assay was performed. To further examine whether PCV2-induced Fn promotes GPS4 adhesion, CRISPR/Cas9/sgRNA technology was performed to target the exon of the Fn gene in STEC cells specifically. (D) Western blot detects the expression of Fn in STEC infected with lentivirus, and GAPDH was used as an internal reference. (E) Adherence assays of GPS4 in STEC and STEC/Fn^- cells inoculated with PCV2 (MOI = 1) or DMEM for 36 h before GPS4 (MOI = 100) infection for 2 h. The colony forming units (CFUs) of GPS4 in PCV2-uninfected STEC cells are considered to be 1. (F) Colonization of GPS4 in STEC cells inoculated with PCV2 (MOI = 1) or DMEM for 36 h before treatment with rabbit polyclonal anti-Fn antibody or rabbit monoclonal anti-immunoglobulin G (IgG; 10 μg/ml) for 3 h, followed by GPS4 (MOI = 100) infection for another 2 h. (G) Colonization of GPS4 in STEC cells inoculated with PCV2 (MOI = 1) for 36 h before treatment with rabbit anti-Integrin β1 antibody or rabbit monoclonal anti-immunoglobulin G (IgG; 10 μg/ml) for 3 h, followed by GPS4 (MOI = 100) infection for another 2 h. The CFUs of GPS4 in PCV2-uninfected STEC cells are considered to be 1. Data are shown as mean ± SDs of three experiments, and Image J software was used to analyze the intensity of protein bands (A) and GAPDH as an internal reference. Statistical analysis was performed using t-test (A, B, and C), two-way ANOVA (E and F) and one-way ANOVA (G). *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

Fig 4. GPS4 rare lipoprotein A (rlpA) is a determinant of bacterial adherence via the Fn receptor.

(A) Schematic representation of the cellular Fibronectin monomer [51]. (B) Western blot identification results of eukaryotic expression of N-terminal Fn type I and type II domains (FnN19). (C) LC-MS/MS analysis results for RlpA. (D) LC-MS/MS spectrum for the RlpA peptide. (E) Growth curve determination of wild-type strain and ΔrlpA deletion strain. (F) Effects of deletion of rlpA on GPS4 adherence. The CFUs of GPS4 in WT-infected STEC cells are considered to be 1. (G) Effect of Fn knockout on RlpA deletion strain adherence. The CFUs of △rlpA-GPS4 in STEC cells are considered to be 1. Data are shown as mean ± SDs of three experiments (E, F and G). Statistical analysis was performed using two-way ANOVA (E, F and G). **, P < 0.01; ****, P < 0.0001; ns, not significant.

Fig 5. RlpA directly interacts with host Fibronectin.

(A) Affinity purification of rRlpA-GST pulls down HEK293T cell lysate transfected with 7×His-FnN19-pcDNA3.1⁺, and empty GST-tag as control, GST agarose beads capture protein complexes, and the samples were detected with anti-GST and anti-His antibodies, respectively. (B) Pull-down assay confirmed the direct interaction between RlpA and Fn. RlpA-pEGFP-C3 and 7×His-FnN19-pcDNA3.1⁺ were cotransfected into HEK293T cells. The cell lysate was captured by Ni-NTA Agarose Resin 6FF beads, washed, and eluted in the sample buffer. Fractions were probed with anti-GFP and anti-His antibodies. (C) GST pull-down assay determined the interaction between RlpA and Fn in vitro. Affinity purification of rRlpA-GST pulls down rFn protein, and empty GST-tag as control, GST agarose beads capture protein complexes, and the samples were detected with anti-GST and anti-Fn antibodies, respectively. (D) Surface plasmon resonance experiment showing the dose-dependent binding profile of rRlpA (0.016–0.500 μM) over immobilized Fn. RU, response units. (E) Colocalization of the RlpA and Fn proteins was analyzed using immunofluorescence microscopy. STEC cells were cotransfected with RlpA-pEGFP-C3 and 7×His-FnN19-pcDNA3.1⁺ and labeled with Mouse anti-His-tag antibodies, followed by goat anti-mouse IgG conjugated with Alexa Fluor 647 secondary antibodies. The cells were then observed using immunofluorescence microscopy. Green represents RlpA, red represents Fn, and blue represents the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm; colocalization assay suggests that RlpA is almost completely coincident with Fn. The data shown are presented as the mean ± SD of the values obtained in three independent experiments.

Fig 6. PCV2 infection-induced Fn promotes GPS4 adhesion via the TGF-β signaling pathway.

STEC cells were infected with 1 MOI PCV2 virus for 36 h. (A) Smad2 phosphorylation was analyzed by Western blot. (B) The cell lysates were analyzed by Western blot for the expression of Fn using the specific antibodies in the absence or presence of 25 μM SB431542 (a TGF-β receptor kinase inhibitor). Image J software was used to analyze the intensity of protein bands, and GAPDH was used as an internal reference (A and B). (C) mRNA levels of Fn in STEC cells infected with PCV2 for 36 h in the absence or presence of 25 μM SB431542 were analyzed by RT-qPCR. (D) Colonization of GPS4 in STEC cells pretreated with 25 μM SB431542 or dimethyl sulfoxide (DMSO) for 2 h before inoculation with PCV2 (MOI = 1) or DMEM for 36 h, followed by GPS4 (MOI = 100) infection for 2 h. The CFUs of GPS4 in DMSO-treated and PCV2-infected STEC cells are considered to be 1. The data shown are presented as the mean ± SD of the values obtained in three independent experiments. Statistical analysis was performed using t-test (A) and one-way ANOVA (B, C, and D). **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

Fig 7. Schematic summary of PCV2 infection promotes GPS4 tracheal colonization.

PCV2 infection promoted GPS4 colonization by enhancing Fn expression via the activated TGF-β signaling pathway. Importantly, GPS4 outer membrane protein RlpA directly interacts with PCV2-induced Fn to promote GPS4 tracheal colonization. SB431542 inhibitor treatment significantly inhibited GPS4 colonization in STECs after PCV2 infection.