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

Staphylococcus aureus reduces daptomycin susceptibility and evades host innate immune response by altering membrane phospholipid composition (#32)

Jhih-Hang Jiang 1 , Md Saruar Bhuiyan 1 , David Cameron 1 , Thusitha Rupasinghe 2 , Pamela Hall 3 , Xenia Kostoulias 1 , Hsin-Hui Shen 1 , Malcolm J. McConville 4 , Michael J. Hickey 3 , Graham Lieschke 5 , Anton Y. Peleg 1 6
  1. Department of Microbiology, Monash University, Clayton, VIC, Australia
  2. Metabolomics Australia, Bio21 Institute of Molecular Science and Biotechnology, Parkville, VIC, Australia
  3. Centre for Inflammatory Diseases, Department of Medicine, Monash University, Monash Medical Centre, Clayton, VIC, Australia
  4. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, Australia
  5. Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia
  6. Department of Infectious Diseases, The Alfred Hospital, Melbourne, VIC, Australia

Staphylococcus aureus is a significant human bacterial pathogen. Due to the emergence of multidrug resistance, the treatment of staphylococcal infection now relies on last-line antibiotics, including daptomycin. Daptomycin is bactericidal by interacting with the bacterial cell membrane in the presence of calcium. Recent studies have revealed that clinically-derived daptomycin-resistant isolates are persistent in animal models compared to their daptomycin-susceptible progenitors, raising the question about the correlation between daptomycin resistance and immune evasion. Whole-genome analysis showed that daptomycin-resistant isolates contain mutations in genes related to phospholipid biogenesis, including cardiolipin (CL) synthase 2 (cls2), which is responsible for production of one CL molecule from two phosphatidylglycerol (PG) molecules. To investigate the importance of cls2 mutations in daptomycin resistance and host immune response, single nucleotide polymorphisms were generated in a daptomycin-susceptible clinical strain to mimic mutations observed after daptomycin exposure and resistance emergence in patients (Cls2A23V, Cls2T33N and Cls2L52F). All mutants had greater CL and less PG due to increased Cls2 activity. Two of these point mutants (Cls2T33N and Cls2L52F) were independently associated with reduced susceptibility to daptomycin. All mutants also evaded host immune responses, with significantly less neutrophils migrating to the site of infection in an in vivo zebrafish model compared to wild-type. Injection of purified liposomes confirmed that these altered immune responses were due to changes in membrane phospholipid composition. Mutation repair (Cls2T33N back to Cls2N33T) returned daptomycin susceptibility, phospholipid composition and neutrophil migration to the same level as wild-type. Finally, using isolated PG and CL, we showed that PG was a novel bacterial-driven chemoattractant in a mammalian neutrophil emigration model. Modification of membrane phospholipid content is a stealth mechanism that S. aureus employs to simultaneously evade antibiotic and host immune responses.