Mycoplasma species are strictly parasitic bacteria that have undergone extensive genomic reduction and therefore biochemical efficiency is essential for their survival. They require their host for the essential supply of lipids, amino acids and nucleotides for growth. The pig pathogen Mycoplasma hyopneumoniae infects the respiratory system of pigs and causes significant agricultural losses. One hypothesis is to explain their success as chronic pathogens suggests that the Mollicutes have evolved multifunctional proteins as a means to increase protein function without expanding gene content.

Multifunctional proteins were first described in 1995 and have since been found in all biological kingdoms. Bacterial moonlighting proteins have been found to participate in key host-pathogen interactions and primarily function as adhesins, metabolic enzymes, ribosomal proteins, chaperones, and proteases. Moonlighting proteins can be detected in cellular fractions other than those stated by initial annotation and online databases, and by identifying the secondary functions of known proteins. The extracellular function of these proteins expressed by bacteria are predominantly roles in host interaction, primarily binding to extracellular matrix or host cell surface molecules, 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.

Specific surface-exposed proteins from M. hyopneumoniae identified by cell-surface proteomic analyses were selected as possible moonlighting proteins because of their annotations and bioinformatics predictions to be cytosolic. 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 host molecule binding motifs specific for heparin and therefore potential binding sites on these putative moonlighting proteins can be identified.