Poster Presentation 25th Annual Lorne Proteomics Symposium 2020

Defining the interactome of the immunity protein GccH - what can it tell us about glycocin F induced bacteriostasis?   (#133)

Gillian E Norris 1 2 , Sean W Bisset 1 2 , Marc A Bailie 2 , Trevor S Loo 2 , Mark L Patchett 2
  1. Maurice Wilkins Centre for Biodiscovery, Auckland, New Zealand
  2. School of Fundamental Sciences, Massey University, Palmerston North, New Zealand

Glycocin F (GccF) is a diglycosylated bacteriocin produced by Lactobacillus plantarum KW30, which has two N-acetylglucosamine (GlcNAc) moieties, one of which (linked to a loop serine) is essential for activity, while the other, a more unusually cysteine linked GlcNAc substantially enhances activity (50 fold). Nanomolar concentrations of GccF induce rapid bacteriostasis (within minutes) of susceptible pathogenic bacteria such as Enterococcus faecalis and E. faecium. Previous research has implicated the transmembrane domain of the GlcNAc-specific phosphotransferase system (PTS) transporter, PTS18CBA, in bacteriostasis, but exactly how it is involved is not yet known. Recent work in our lab identified the immunity protein used to protect producer cells from the effects of GccF.  GccH, a 118 amino acid protein that has no homology to any other protein in the data base apart from one whose gene sequence lies immediately adjacent to that encoding the PTS (38 % identity over 42 amino acids).  To help us understand the mechanism of action of GccF, we investigated the potential binding partners of this immunity protein. To do this, we expressed HA-tagged GccH in L. plantarum cells and carried out anti-HA pulldowns on GccF-treated and untreated cells. Proteins were extracted from the cells in the presence of detergent to ensure that membrane proteins were solubilised  On-bead digest of the immunoprecipitated proteins was carried out, and the peptides separated using nano LCMSMS. Peptides were identified with Proteome Discoverer2.2 and proteins interacting with GccH included the GlcNAc-specific PTS transporter and GlcNAc-6-phosphate deaminase, the first enzyme that interacts with imported GlcNAc. The significance of these and related results will be discussed.