Poster Presentation AUS-oMicS 2025

The characterisation of Anti-Microbial Resistance through examination of the Interactomic profile of Staphylococcus aureus SH1000. (121210)

Aiden Brennan 1 , Matthew O'Rourke 1 2 , Matt Padula 1
  1. University of Technology Sydney, Sydney, NEW SOUTH WALES, Australia
  2. Centenary Centre for Inflammation, Sydney, New South Wales, Australia

Anti-Microbial Resistance (AMR) is a well-established global issue, with proteomics playing a crucial role in its research [1]. However, there is a significant lack of in-depth information on the binding of antibiotics, and hence a lack of deeper understanding of the mechanisms of action of antibiotics, as well as their effect on Protein-Protein Interactions (PPIs). Binding interactions between ligands (such as antibiotics) and proteoforms, and the effect of these interactions on the AMR profile of bacteria are largely unexplored in bottom-up proteomics workflows, which focus primarily on changes in the abundance of ORF products. Numerous methodologies have been established to study the interactome in other biological systems, including thermal proteome profiling [2] and limited proteolysis mass spectrometry (Lip-MS) [3]; however, their use in AMR research remains limited. In the present study, both methods were applied to the analysis of Staphylococcus aureus SH1000 after treatment with antibiotics, elucidating a number of conformational changes and PPIs across the proteome. Combined with abundance information from a peptide-centric workflow, a proteome-wide interactomic profile for SH1000 was established. Of specific interest, AmaP showed decreased thermal stability and abundance in response to treatment with ampicillin.  Changes to the structure of this protein have been shown to potentially be involved in an increased stress response and antibiotic resistance in S. aureus through changes to its binding pattern with the protein Asp23 [4].  Additionally, several known ampicillin binding sites, such as penicillin-binding proteins, showed both an increase in thermal stability and a significant increase in abundance. Proteins showing stability changes were then correlated with Lip-MS data to gain further information on these conformational changes. Further work into the interactomics of drug-protein binding in clinical strains is critical to continue growing our understanding of antimicrobial resistance in bacteria and the mechanisms of the drugs used to treat bacterial infection.

  1. Torres-Sangiao, E., et al., Proteomic Approaches to Unravel Mechanisms of Antibiotic Resistance and Immune Evasion of Bacterial Pathogens. Front Med (Lausanne), 2022. 9: p. 850374.
  2. Savitski, M.M., et al., Tracking cancer drugs in living cells by thermal profiling of the proteome. Science, 2014. 346(6205): p. 1255784.
  3. Schopper, S., et al., Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry. Nat Protoc, 2017. 12(11): p. 2391-2410.
  4. Muller, M., et al., Deletion of membrane-associated Asp23 leads to upregulation of cell wall stress genes in Staphylococcus aureus. Mol Microbiol, 2014. 93(6): p. 1259-68.