Determining the Contribution of Exogenous Lipids to Antimicrobial Resistance in Staphylococcus aureus Using Comprehensive Lipidomics and Online Paterno-Buchi Reactions

Staphylococcus aureus adapts to the host environment by utilizing exogenous fatty acids (eFA) to reduce energy consumption from de novo fatty acid biosynthesis, bypass the innate immune response, and withstand drug activity. Host-derived unsaturated FAs (UFA) can be incorporated into the bacterial lipids, with or without further elongation via the type II fatty acid synthesis pathway (FASII); however, several questions concerning lipidomic changes remain and identifying the double bond location of incorporated eFAs continues to be a challenge. Here, we use hydrophilic interaction liquid chromatography (HILIC) ion mobility-mass spectrometry (IM-MS) to elucidate factors affecting the utilization of eFAs by S. aureus and obtain detailed structural information on altered lipids via the Paterno-Buchi (PB) reaction with 2-acetylpyridine (2-ACP). The PB reaction is coupled online with HILIC-tandem mass spectrometry by installing a flow microreactor post HILIC separation, but before ESI, to preserve lipid retention time, while the PB reagent, 2-ACP, is teed-in via a syringe pump (HILIC-PB-MS/MS). Bacterial strains were grown in various eFA environments, and total lipids were extracted and analyzed in both positive and negative ionization modes. Our work shows a) the substrate specificity of S. aureus secreted lipases, b) human serum albumin can serve as a buffer of eFAs for S. aureus, c) the FASII inhibitor AFN-1252 leads to an increase of UFAs in the membrane with or without eFAs, and d) the carbon-carbon double bond location of host-derived UFAs that are further metabolized by S. aureus. Overall, this work provides a detailed understanding of the metabolism of UFAs in S. aureus.