Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights as an adhesin in Mycoplasma hyorhinis adhesion to epithelial cells as well as a plasminogen receptor mediating extracellular matrix degradation

Abstract

Mycoplasma hyorhinis infects pigs causing polyserositis and polyarthritis, and has also been reported in a variety of human tumor tissues. The occurrence of disease is often linked with the systemic invasion of the pathogen. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), one of the key enzymes of glycolysis, was reported as a surface multifunctional molecule in several bacteria. Here, we investigated whether GAPDH could manifest binary functions; as an adhesin to promote colonization as well as a plasminogen receptor functioning in extracellular matrix (ECM) degradation to promote systemic invasion. The surface localization of GAPDH was observed in M. hyorhinis with flow cytometry and colony blot analysis. Recombinant GAPDH (rGAPDH) was found to be able to bind porcine-derived PK-15 and human-derived NCI-H292 cells. The incubation with anti-GAPDH antibody significantly decreased the adherence of M. hyorhinis to both cell lines. To investigate its function in recruiting plasminogen, firstly, the interaction between rGAPDH and plasminogen was demonstrated by ELISA and Far-Western blot assay. The activation of the rGAPDH-bound plasminogen into plasmin was proved by using a chromogenic substrate, and furtherly confirmed to degrade extracellular matrix by using a reconstituted ECM. Finally, the ability of rGAPDH to bind different ECM components was demonstrated, including fibronectin, laminin, collagen type IV and vitronectin. Collectively, our data imply GAPDH as an important adhesion factor of M. hyrohinis and a receptor for hijacking host plasminogen to degrade ECM. The multifunction of GAPDH to bind both plasminogen and ECM components is believed to increase the targeting of proteolysis and facilitate the dissemination of M. hyorhinis.

Keywords: Adhesion; Extracellular matrix; GAPDH; M. hyorhinis; Plasminogen.

Figure 1

Expression and purification of the rGAPDH. A SDS-PAGE analysis of the rGAPDH. B Western blotting with anti-His-tag monoclonal antibody. M, protein molecular weight marker, lane 1 and 2, whole cell lysate of E. coli BL21 carrying recombinant vector pET-28a-gapdh before and after induction by IPTG overnight; lane 3, purified rGAPDH through Ni-chelating affinity chromatography.

Figure 2

Surface localization of GAPDH in M. hyorhinis. A Detection of GAPDH by flow cytometry. M. hyorhinis were incubated with anti-rGAPDH serum or negative serum respectively. M. hyorhinis treated with PBS was used as the blank control. The level of mean fluorescence intensity (MFI) of M. hyorhinis incubated with anti-rGAPDH serum is expressed as the percentage of the bacteria incubated with negative serum after deduction of the background (**P < 0.01). Data are expressed as mean ± SD of at least three experiments with samples in triplicate. B Detection of surface GAPDH by colony blot analysis. Reaction of M. hyorhinis colonies transferred to PVDF membrane was probed with anti-rGAPDH serum or negative serum.

Figure 3

Cytoadhesion of the rGAPDH protein detected by indirect immunofluorescence assay. The rGAPDH (A, C) or BSA (B, D) was incubated with PK-15 (A, B) or NCI-H292 (C, D) cells, respectively. Bound protein was detected with anti-rGAPDH serum and Cy3-conjugated secondary antibody (red). Nuclei were stained with Honchest 33,342 (blue).

Figure 4

Microtiter plate adhesion assay (MPAA) for the binding of the rGAPDH protein to cell membrane proteins. Microtiter plates were coated with membrane protein of PK-15 or NCI-H292 cells. Increasing concentrations of rGAPDH protein were added to individual wells (A: PK-15; C: NCI-H292). Bound rGAPDH was detected with anti-His-tag monoclonal antibody compared with the wells with no added protein. The adhesion of 50 μg/mL of rGAPDH to the cells (B: PK-15; D: NCI-H292) was inhibited by anti-rGAPDH serum but not by the preimmune serum. Data are expressed as mean ± SD of at least three experiments with samples in triplicate. ** indicate P < 0.01.

Figure 5

Adhesion inhibition assay of anti-rGAPDH antibody. M. hyorhinis were incubated with anti-rGAPDH serum or negative serum before adding into the cell culture plate. The number of mycoplasma adhering to the cells was expressed as CCU/mL. Data are expressed as mean ± SD of at least three experiments with samples in triplicate. **P < 0.01. A PK cell; B NCI-H292 cell.

Figure 6

Binding activity of the rGAPDH protein to plasminogen and the activation of the rGAPDH-bound plasminogen to plasmin. A ELISA was performed to characterize the ability of immobilized rGAPDH binding plasminogen. B Far Western blot analysis of binding activity of rGAPDH to plasminogen. Bound plasminogen was determined by anti-plasminogen antibody. BSA was chosen as negative control for non-specific binding. C Plasminogen bound to the coated rGAPDH in microtiters plate was activated to plasmin by tPA. The presence of lysine analogue, ε-ACA decreased the binding of plasminogen to rGAPDH. Activity of plasmin was evaluated by adding chromogenic substrate, and OD405nm was measured. Data are expressed as mean ± SD of at least three experiments with samples in triplicate. **P < 0.01.

Figure 7

Electron microscopic visualization of the degradation of Matrigel reconstituted basement membrane. The rGAPDH-harboring polystyrene beads (A, B) or the BSA-harboring beads (C, D) were incubated with plasminogen and tPA, and then added on the 3-μm filters in Transwell cell culture chamber inserts previously coated with Matrigel reconstituted basement membrane. After an incubation of 40 h, the filters were fixed with 2.5% glutaraldehyde, and examined in scanning electron microscope. B and D are the enlarged view of a part of A and C, respectively.

Figure 8

Binding of the rGAPDH protein to different ECM components in ELISA experiment. Microtiters plate was coated with Matrigel, fibronectin, collagen or laminin solution. Various concentrations of rGAPDH or BSA were added and detected by anti-His-tag monoclonal antibody. For detecting the binding to vitronectin, microtiters plate was coated with rGAPDH or BSA. Various concentrations of vitronectin were added and detected by anti-vitronectin monoclonal antibody. *P < 0.05, **P < 0.01, compared with the negative control (BSA).