Oral Presentation BACPATH 2022

Daptomycin resistance in Staphylococcus aureus mediated by multiple peptide resistance factor mprF (#26)

Jhih-Hang Jiang 1 2 , Sigrid Lange 1 , Wei Gao 3 , Liam Donovan 1 , Romain Guérillot 3 , Anton Le Brun 4 , Bart A Eijkelkamp 5 , Thusitha Rupasinghe 6 , Malcolm J McConville 6 7 , Benjamin P Howden 8 , Hsin-Hui Shen 9 10 , Anton Y Peleg 1 2
  1. Infection Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia
  2. Department of Infectious Diseases, The Alfred Hospital, Melbourne, VIC, Australia
  3. Microbiology and Immunology, the University of Melbourne, Parkville, Victoria, Australia
  4. Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology Organisation, Kirrawee DC, NSW, Australia
  5. College of Science and Engineering, Flinders University, Adelaide, South Australia, Australia
  6. Metabolomics Australia, Bio21 Institute of Molecular Science and Biotechnology, Parkville, Victoria, Australia
  7. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia
  8. Melbourne Diagnostic Unit, University of Melbourne, Parkville, VIC, Australia
  9. Department of Materials Science and Engineering, Faculty of Engineering, Monash University, Clayton, VIC, Australia
  10. Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

Staphylococcus aureus remains to be one of the most concerning bacterial pathogens, with its capability to adapt host immune systems and antibiotic attacks. Treatment of severe infections caused by methicillin-resistant S. aureus (MRSA) relies on last-line antibiotics, including daptomycin. Daptomycin targets bacterial cell membrane for its bactericidal effects, with a mechanism thought to be similar to host cationic antimicrobial peptides (CAMPs). The rise of daptomycin resistance (DAP-R) is alarming and is often associated with persistent and complicated infections. Nonsynonymous mutations in multiple peptide resistance factor (mprF) have been highly associated with DAP-R. MprF is a bifunctional enzyme to synthesize lysyl-phosphatidylglycerol (L-PG) and translocate L-PG from the inner leaflet to the outer leaflet of cell membranes. S. aureus relies on MprF to defend attacks from host CAMPs. How these mprF mutations lead to DAP-R is not entirely clear. Here, we characterized the full repertoire of mprF mutations within 10,000 DAP-R clones. We identified 24 nonsynonymous mutations in mprF, of which 6 were novel mutations. Introduction of individual mprF mutation into a daptomycin-susceptible S. aureus strain led to DAP-R, resistance to CAMPs, and enhanced biosynthesis of L-PG. We confirmed that these mprF mutations were gain-of-function by expressing these mprF alleles in Escherichia coli and assessing the de novo biosynthesis of L-PG. To directly measure the impact of L-PG on daptomycin actions, we utilized the established bacterial membrane model coupled with high resolution neutron reflectometry.1 Increase of L-PG impaired daptomycin penetration and membrane solubilization despite of daptomycin association with the membranes. Replacement of L-PG with DOTAP reproduced this inhibition of daptomycin actions, indicating that increase of cationic charge was the mechanism behind mprF-mediated DAP-R. Interestingly, mutations in both mprF and cardiolipin synthase 2 (cls2) made MRSA hyper resistant to daptomycin, indicating that the L-PG mediated DAP-R mechanism is independent of a previously characterized mechanism by the enhanced cardiolipin biosynthesis.1 To disrupt this DAP-R mediated by MprF, we generated a loss-of-function mprF-D731A mutant, which disrupted the active site of L-PG synthesis as a proof of principle. This D731A mutation rendered the MRSA strain L-PG deficient and hypersusceptible to daptomycin, confirming the importance of L-PG in S. aureus. Together, our results illustrate that S. aureus is capable of utilizing diverse metabolic strategies to circumvent attacks from antibiotics and immune systems, providing important insights into membrane-targeting therapeutic strategies against this significant pathogen.

  1. Jiang JH, Bhuiyan S, Shen HH, et al., Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation. PNAS 2019. 116(9):3722-27. Doi: 10.1073/pnas.1812066116.