Poster Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

Bacterial moonlighting proteins in Mycoplasma hyopneumoniae: A pathogenic role for glycolytic enzymes (#164)

Marcelo Moreno 1 2 , Jess L. Tacchi 1 2 , Benjamin B.A. Raymond 1 2 , Michael Widjaja 1 2 , Iain J. Berry 1 2 , Matt P. Padula 1 2 , Steven P. Djordjevic 1 2
  1. the ithree institute, Sydney, NSW, Australia
  2. UTS, Sydney, NSW, Australia

Mycoplasma species are strictly parasitic bacteria from the Mollicutes class that are thought to have undergone extensive genomic reduction from a low G + C Firmicute ancestor. As such, Mycoplasma spp. lack a TCA cycle and genes for cell wall, amino acid, cholesterol and nucleotide biosynthesis. Mycoplasma hyopneumoniae (Mhp) is an economically significant pathogen that destroys mucociliary function in the respiratory tract of pigs. Our surfaceome studies show that most cilium adhesin family members (15 in total) and many lipoproteins are accessible on the cell surface, and are targets of processing events.

As has been found in other surfaceome studies of Gram positive pathogens and commensal bacteria, we have identified proteins that do not contain any known signaling mechanisms for their secretion. Among these surface proteins are metabolic enzymes and ribosomal proteins. We argue that if these “cytosolic” proteins carry cleavage recognition motifs then they may also be processed to form functional protein fragments which may aid in pathogenesis. To assist with the identification of cleavage fragments, we applied a series of affinity chromatography assays using heparin, actin, fibronectin and other host proteins as bait. Protein cleavage fragments were mapped by a combination of 1D and 2D SDS-PAGE and LC-MS/MS. Precise cleavage sites were identified by dimethyl-labelling of neo-N-termini and LC-MS/MS.

These enrichment strategies enabled us to map the cleavage fragments of a number of surface-associated “cytosolic” proteins, confirming that they are available to the post-translational processing machinery. This lends weight to the hypothesis that protein moonlighting is a strategy that has evolved to increase protein function in  genome-reduced bacteria. Evidence that moonlighting proteins are targets of processing events will be presented.