Mycoplasma species are strictly parasitic bacteria that have undergone extensive genomic reduction and therefore biochemical efficiency is essential for their survival. By taking advantage of the available host nutrients, and assigning more than one function to each of many proteins involved in classical housekeeping roles, Mycoplasma species have competitively benefited from their reduced genome.

Multifunctional proteins were first described in 1995 and have since been found in all biological kingdoms. One approach for identifying moonlighting proteins is by detecting them in cellular fractions other than those stated by initial annotation and online databases. Many bacterial moonlighting proteins have been found to have additional roles in host interaction, primarily binding to extracellular matrix or host cell surface antigens, but also in modulating host immune function. Studies have shown that glycolytic enzymes which are conserved in both the pathogen and the host can introduce autoimmunity in the host organism. Other previous studies successfully immunised mice to surface-exposed glycolytic pathway enzymes from Streptococcus pneumoniae which provided cross-strain protection to virulent strains of the pathogen.

In this project, the specific surface-exposed proteins from M. hyopneumoniae were investigated as possible moonlighting proteins that may aid the pathogen in binding to host cells and connective tissues. The method employed to select the proteins for this project was by identifying proteins annotated to be intracellular but demonstrated in our lab to be located on the surface of M. hyopneumoniae cells by cell surface proteomic analysis. The natively purified recombinant proteins were characterised for heparin, plasminogen and fibronectin binding using microscale thermophoresis, blotting techniques and heparin affinity chromatography to elucidate their functional role as moonlighting proteins. With the use of protein modelling programs and bioinformatics software, the antigen-binding motifs specific for host molecules and therefore potential binding sites on these putative moonlighting proteins can be identified.