Dual stable isotopes enhance lipidomic studies in bacterial model organism Enterococcus faecalis.

Dual stable isotope probes of deuterium oxide and 13C fatty acid were demonstrated to probe the lipid biosynthesis cycle of a Gram-positive bacterium Enterococcus faecalis. As external nutrients and carbon sources often interact with metabolic processes, the use of dual-labeled isotope pools allowed for the simultaneous investigation of both exogenous nutrient incorporation or modification and de novo biosynthesis. Deuterium was utilized to trace de novo fatty acid biosynthesis through solvent-mediated proton transfer during elongation of the carbon chain while 13C-fatty acids were utilized to trace exogenous nutrient metabolism and modification through lipid synthesis. Ultra-high-performance liquid chromatography high-resolution mass spectrometry identified 30 lipid species which incorporated deuterium and/or 13C fatty acid into the membrane. Additionally, MS2 fragments of isolated lipids identified acyl tail position confirming enzymatic activity of PlsY in the incorporation of the 13C fatty acid into membrane lipids.

Expanding lipidomics coverage: effective ultra performance liquid chromatography-high resolution mass spectrometer methods for detection and quantitation of cardiolipin, phosphatidylglycerol, and lysyl-phosphatidylglycerol.

INTRODUCTION: Lipidomics can reveal global alterations in a broad class of molecules whose functions are innately linked to physiology. Monitoring changes in the phospholipid composition of biological membranes in response to stressors can aid the development of targeted therapies. However, exact quantitation of cardiolipins is not a straightforward task due to low ionization efficiencies and poor chromatographic separation of these compounds. OBJECTIVE: The aim of this study was to develop a quantitative method for the detection of cardiolipins and other phospholipids using both a targeted and untargeted analyses with a Q-Exactive. METHODS: HILIC chromatography and high-resolution mass spectrometry with parallel reaction monitoring was used to measure changes in lipid concentration. Internal standards and fragmentation techniques allowed for the reliable quantitation of lipid species including: lysyl-phosphatidylglycerol, phosphatidylglycerol, and cardiolipin. RESULTS: The untargeted analysis was capable to detecting 6 different phospholipid classes as well as free fatty acids. The targeted analysis quantified up to 23 cardiolipins, 10 phosphatidylglycerols and 10 lysyl-phosphatidylglycerols with detection limits as low as 50 nM. Biological validation with Enterococcus faecalis demonstrates sensitivity in monitoring the incorporation of exogenously supplied free fats into membrane phospholipids. When supplemented with oleic acid, the amount of free oleic acid in the membrane was 100 times greater and the concentration of polyunsaturated cardiolipin increased to over 3.5 µM compared to controls. CONCLUSIONS: This lipidomics method is capable of targeted quantitation for challenging biologically relevant cardiolipins as well as broad, untargeted lipid profiling.

A MALDI Mass Spectrometry Imaging Sample Preparation Method for Venous Thrombosis with Initial Lipid Characterization of Lab-Made and Murine Clots.

Venous thromboembolism (VTE) and its complications affect over 900,000 people in the U.S. annually, with a third of cases resulting in fatality. Despite such a high incidence rate, venous thrombosis research has not led to significant changes in clinical treatments, with standard anti-coagulant therapy (heparin followed by a vitamin K antagonist) being used since the 1950s. Mechanical thrombectomy is an alternative strategy for treating venous thrombosis; however, clinical guidelines for patient selection have not been well-established or accepted. The effectiveness of both treatments is impacted by the heterogeneity of the thrombus, including the mechanical properties of its cellular components and its molecular makeup. A full understanding of the complex interplay between disease initiation and progression, biochemical molecular changes, tissue function, and mechanical properties calls for a multiplex and multiscale approach. In this work, we establish a protocol for using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to characterize spatial heterogeneity of biomolecules in lab-made blood clots and ex vivo murine thrombi. In this work, we compared (1) tissue preservation and cryosectioning methods, (2) various matrixes, 9-aminoacridine hydrochloride monohydrate (9AA), 2,5-dihydroxybenzoic acid (DHB), and alpha-cyano-4-hydroxycinnamic acid matrix (CHCA), (3) plasma-rich versus red-blood-cell rich lab-made blood clots, and (4) lab-made blood clots versus ex vivo murine thrombi. This project is the first step in our work to combine mass spectrometry imaging with biomechanical testing of blood clots to improve our understanding of VTE.

Removal of peptidoglycan and inhibition of active cellular processes leads to daptomycin tolerance in Enterococcus faecalis.

Daptomycin is a cyclic lipopeptide antibiotic used in the clinic for treatment of severe enterococcal infections. Recent reports indicate that daptomycin targets active cellular processes, specifically, peptidoglycan biosynthesis. Within, we examined the efficacy of daptomycin against Enterococcus faecalis under a range of environmental growth conditions including inhibitors that target active cellular processes. Daptomycin was far less effective against cells in late stationary phase compared to cells in exponential phase, and this was independent of cellular ATP levels. Further, the addition of either the de novo protein synthesis inhibitor chloramphenicol or the fatty acid biosynthesis inhibitor cerulenin induced survival against daptomycin far better than controls. Alterations in metabolites associated with peptidoglycan synthesis correlated with protection against daptomycin. This was further supported as removal of peptidoglycan induced physiological daptomycin tolerance, a synergistic relation between daptomycin and fosfomycin, an inhibitor of the fist committed step peptidoglycan synthesis, was observed, as well as an additive effect when daptomycin was combined with ampicillin, which targets crosslinking of peptidoglycan strands. Removal of the peptidoglycan of Enterococcus faecium, Staphylococcus aureus, and Bacillus subtilis also resulted in significant protection against daptomycin in comparison to whole cells with intact cell walls. Based on these observations, we conclude that bacterial growth phase and metabolic activity, as well as the presence/absence of peptidoglycan are major contributors to the efficacy of daptomycin.

Enterococcus faecalis Readily Adapts Membrane Phospholipid Composition to Environmental and Genetic Perturbation.

The bacterial lipid membrane, consisting both of fatty acid (acyl) tails and polar head groups, responds to changing conditions through alteration of either the acyl tails and/or head groups. This plasticity is critical for cell survival as it allows maintenance of both the protective nature of the membrane as well as functioning membrane protein complexes. Bacteria that live in fatty-acid rich environments, such as those found in the human host, can exploit host fatty acids to synthesize their own membranes, in turn, altering their physiology. Enterococcus faecalis is such an organism: it is a commensal of the mammalian intestine where it is exposed to fatty-acid rich bile, as well as a major cause of hospital infections during which it is exposed to fatty acid containing-serum. Within, we employed an untargeted approach to detect the most common phospholipid species of E. faecalis OG1RF via ultra-high performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). We examined not only how the composition responds upon exposure to host fatty acids but also how deletion of genes predicted to synthesize major polar head groups impact lipid composition. Regardless of genetic background and differing basal lipid composition, all strains were able to alter their lipid composition upon exposure to individual host fatty acids. Specific gene deletion strains, however, had altered survival to membrane damaging agents. Combined, the enterococcal lipidome is highly resilient in response to both genetic and environmental perturbation, likely contributing to stress survival.