Lipidomic analysis by LC-MS/MS of wildtype, mutant and VBNC strains of Francisella tularensis LVS.

Francisella tularensis is one of the many bacteria that can enter a viable but nonculturable state (VBNC). The VBNC state acts as an adaptive strategy that allows for long-term survival of bacteria in unfavorable environments. A defining characteristic of VBNC bacteria is a loss of culturability, making VBNC bacteria difficult to detect in the environment using standard plating assays. Failure to identify VBNC bacteria in the food industry or water distribution centers poses a serious risk to human health. Our preliminary data suggests that lipid metabolism plays a role in VBNC adaptation in F. tularensis. To investigate further, genetic deletion mutants have been constructed from two predicted lipid metabolism genes, FTL_1569 (phosphoglycolate phosphatase) and FTL_1570 (phospholipase D family protein). Lipid extracts were collected from wildtype, mutant and VBNC strains of F. tularensis to determine any lipid species associated with the VBNC state. A single-phase lipid extraction method was used to obtain lipids from F. tularensis cultures grown for 24 hours (culturable) and 336 hours (VBNC). Liquid chromatography with tandem mass spectrometry (LC-MS-MS) and the ThermoFisher Compound Discoverer software were utilized for lipid identification, and comparative analyses. Differences in the lipid profile of wildtype, mutant and VBNC strains of F. tularensis have been identified.  (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)