Hijacking of Host Plasminogen by Mesomycoplasma (Mycoplasma) hyopneumoniae via GAPDH: an Important Virulence Mechanism To Promote Adhesion and Extracellular Matrix Degradation.

Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world's swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae.

Development of an indirect competitive enzyme linked immunosorbent assay for the quantitative detection of Mycoplasma hyopneumoniae during the vaccine production process.

Inactivated Mycoplasma hyopneumoniae vaccine is used extensively to control M. hyopneumoniae infection worldwide. Quantification techniques are essential in the process of standardizing and validating vaccines. In this study, we developed and optimized an indirect competitive enzyme linked immunosorbent assay (ic-ELISA) for the rapid quantification of M. hyopneumoniae antigen during vaccine production. Briefly, whole M. hyopneumoniae antigen was coated onto microtiter plates, and a polyclonal antibody against M. hyopneumoniae recombinant elongation factor thermo unstable (EF-Tu) protein was prepared and added with the samples to be tested. The methods were optimized and showed significant reproducibility, with coefficients of variation of 4.01% and 6.14% for the intra-and inter-assays, respectively. Quantification of M. hyopneumoniae cultures at different growth stages using the ic-ELISA test showed a similar curve to that of the traditional color changing units (CCU) assay, with a delay in the time when the amount reached the peak and started to fall. In the inactivated vaccine production process, the cultures could be harvested later than that for the live vaccine, at about 12 h after the end of the logarithmic growth phase. Different batches of cultures were measured for their relative potency value compared with the in-house reference vaccine, which was used to determine whether the cultures met the antigen amount requirements for vaccine preparation. The curves of the CCU titer and ic-ELISA titer in the logarithmic phase correlated strongly and a linear regression equation was established to calculate the CCU values rapidly using the ic-ELISA results. In conclusion, an ic-ELISA method was established to rapidly assess the amount of antigen in an M. hyopneumoniae culture during the vaccine production process.