Mycoplasma hyopneumoniae is a primary colonising agent and economically-devastating porcine respiratory pathogen that is controlled by partially-effective bacterin vaccine formulations and widespread antibiotic therapy. Adherence and colonisation is in part mediated by the P97 and P102 adhesin families that directly interact with the host extracellular matrix, via glycosaminoglycans, fibronectin and the fibrinolytic protein, plasminogen. Post-translational proteolytic cleavage is essential to the production of functional, mature proteoforms on the surface of this ubiquitous pathogen. We aim to determine whether proteolytic cleavage is restricted to the adhesin families using a high-throughput, proteome-wide methodology.

Using protein dimethyl labelling, reversed-charge enrichment of N-termini and ‘shotgun’ mass spectrometry, the N-terminal sequences of mature M. hyopneumoniae proteins were characterised. Our data interrogated protein start sites and identified the precise location of protein cleavage in functionally diverse proteins. These data were combined with surfaceome studies, affinity chromatography using diverse host molecules as bait and mass mapping by SDS-PAGE to confirm the existence of proteoforms and to investigate their putative functions.

Our approach confirmed the existence of multiple cleavage events, previously characterised in the adhesin families, validating our approach. Additionally, we identified internal cleavage events in lipoprotein families and proteins with well-studied canonical functions in the cytosol. The majority of the proteins targeted by endoproteolysis were identified in our surfaceome studies. Furthermore, our chromatography studies suggest that these proteins are multifunctional, with implications for the function of moonlighting proteins on the surface of M. hyopneumoniae. Consistent with this hypothesis, homologues of these ‘moonlighting’ proteins have been identified as virulence factors in other pathogenic bacteria but the data reported here is the first description of them being targets of endoproteolysis. The sequence motifs associated with the characterised cleavage events in this study are similar to those already described in studies of the adhesin families suggesting that the same protease(s) process functionally-diverse proteins on the cell surface. These observations have widespread ramifications for microbial pathogenesis and vaccine design.