Proteolysis is an essential and ubiquitous post-translational protein modification (PTM) for the normal functioning of many biological systems. In prokaryotes, the scale of proteolytic regulation of the proteome is poorly characterised. The first systems-wide analysis of proteolytic cleavage in the bacterium Mycoplasma hyopneumoniae, a minimal genome porcine pathogen, was published in 2017. This landmark study demonstrated an unprecedented level of proteolytic processing targeting surface proteins with a role in interactions between M. hyopneumoniae and receptors on the surface of target host cells. Mycoplasma species are often highly host-adapted and known to infect vertebrates, invertebrates and plants, utilising specific individual characteristics and virulence factors.
To expand our knowledge of proteolytic processing, we examined the systems-wide processing events in the human respiratory pathogen Mycoplasma pneumoniae using an N-terminome approach. This method used dimethyl labelling on the protein level and mass spectrometry to identify cleavage sites in 391 proteins. These data indicate that 56% of the predicted proteome is affected by proteolytic processing, producing many novel proteoforms from a genome of 688 ORFs. Biotinylation and trypsin shaving of cell surface proteins was used to identify 160 proteins located on the surface of M. pneumoniae and over 80% of these (134 proteins) are targeted by proteolysis. Affinity chromatography was used to characterise putative host-pathogen protein interactions, demonstrating that surface proteins which undergo more extensive processing were correlated with a greater variety of interactions with host molecules. Notably, these data mirror the findings reported in M. hyopneumoniae, as well as a recent study of Spiroplasma citri, which demonstrates that proteolytic processing may be a fundamental mechanism required to generate proteoforms on the surface of the Mollicutes. These findings have significant implications for our understanding of protein maturation and the development of vaccines against these important pathogens.