Oral Presentation 14th Lorne Infection and Immunity 2024

Exogenous arginine enhances the diagnosis of 'undetectable' polymyxin-dependent Acinetobacter baumannii (#62)

Meiling Han 1 2 , Yasser Alssadi 1 2 , Jinxin Zhao 1 2 , Yan Zhu 1 2 , JING LU 1 2 , Xukai Jiang 1 2 , Rhys Dunstan 1 2 , Anton Le Brun 3 , Christopher Barlow 1 2 , Phillip Bergen 1 2 , Hsin-Hui Shen 1 2 , Trevor Lithgow 1 2 , Darren Creek 4 5 , Tony Velkov 1 , Jian Li 1
  1. Faculty of Medicine, Nursing & Health Sciences, Monash University, Melbourne, VIC, Australia
  2. Monash University, Clayton, Melbourne, VIC, Australia
  3. Australian Nuclear Science and Technology Organisation, Sydney, NSW, Australia
  4. Monash University, Parkville, VIC, Australia
  5. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia

Polymyxins are last-line antibiotics against the top-priority pathogen Acinetobacter baumannii, yet polymyxin-dependent resistant isolates have emerged. These unique strains are unculturable on agar plates without polymyxins, escaping the current clinical diagnosis procedure and causing treatment problem. In the present study, we integrated multi-omics (incl. genomics, transcriptomics and metabolomics) to examine the mechanism of polymyxin-dependent growth of A. baumannii and discovered that arginine metabolism is a critical factor. Specifically, the arginine degradation pathway was significantly altered in polymyxin-dependent strains compared to wild-type strains, with critical metabolites (e.g., L-arginine and L-glutamate) severely depleted and the expression of astABCDE operon significantly increased. Our stable isotope labelling study with 13C6-L-arginine as the sole carbon source demonstrated significantly higher uptake of L-arginine and faster generation of L-glutamate in polymyxin-dependent strain AB5075D compared to the wild-type strain AB5075S, indicating increased metabolic activity due to arginine supplementation which led to suppressed polymyxin dependence. Deletion of the first gene astA in the arginine degradation pathway of AB5075D produced similar effects to arginine supplementation, namely substantial growth enhancement and decreased polymyxin dependence, with the downstream genes astB/D/E and related metabolites (e.g., N2-succinyl-L-arginine, N2-succinyl-L-ornithine and L-ornithine) significantly depleted. Our membrane lipidomics results showed decreased phosphatidylglycerol (PG) proportion and increased phosphatidylethanolamine (PE) proportion in the outer membrane (OM) of the astA deletion mutant AB5075DDastA compared to its parent AB5075D. The downregulation of pldA (encoding an OM phospholipase) and decreased abundance of lysoPE and sn-glycerol-PE due to astA deletion supported the increased PE level in the OM of AB5075DDastA. Our molecular dynamics simulations and neutron reflectometry studies showed that the above membrane lipid change decreased the interaction with polymyxins. Overall, this study elucidates the molecular mechanism of how arginine metabolism impacts polymyxin-dependence in A. baumannii and provides critical knowledge to improve the diagnosis and treatment of ‘undetectable’ polymyxin-dependent A. baumannii in patients.