Towards measuring growth rates of pathogens during infections by D2O-labeling lipidomics
RATIONALE:Microbial growth rate is an important physiological parameter that is challenging to measurein situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times ofS. aureusin cystic fibrosis (CF) infections can be determined by D2O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to
... ately quantify trace incorporation of deuterium into phospholipids.METHODS:Lipid extracts of D2O-treatedE. colicultures were measured on LC-ESI-MS instruments equipped with TOF and Orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio GC-MS. We then develop an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we apply D2O-labeling lipidomics to clinical samples from CF patients with chronic lung infections.RESULTS:Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific forS. aureusin CF sputum, indicates an average generation time of the pathogen on the order of one cell doubling per day.CONCLUSIONS:This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2O.