Thin layer chromatography for identification of lipid accumulation within Mycolicibacterium smegmatis

• Malcolm Blenkhorn, Biological Sciences, University of Delaware

• Karl Schmitz, Department of Biological Sciences, University of Delaware

Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis, remains a global health threat due to the rise of multidrug-resistant strains (MDR-TB) resistant to existing first- and second-line antibiotics. This resistance arises through genetic mutations, drug efflux pumps, and stress response systems, driving an urgent need for novel antibiotics. The essential ATP-dependent ClpC1P1P2 protease complex represents a promising therapeutic target due to its critical role in mycobacterial protein quality control under stress conditions. Preliminary microscopy indicated that knocking down ClpC1 in the model organism Mycolicibacterium smegmatis triggers an intracellular accumulation of lipids, likely triacylglycerols, in the form of lipid droplets. To validate this phenotype using thin layer chromatography (TLC), an experimental control was required.  Lipid accumulation and the accompanying droplet formation is a phenotype confirmed to have been seen in M. smegmatis under nitrogen deprivation. Existing nitrogen deprivation and lipid extraction protocols for M. smegmatis were adapted, both to validate the functionality of TLC and to establish a positive control for future ClpC1 experiments. When tested with TLC, lipid levels appeared higher in cells that had been deprived of nitrogen, compared to cells that had grown at normal nitrogen levels. With TLC validated as a viable method for observing lipid accumulation, the next stage is to perform lipid extraction and TLC on strains of M. smegmatis with ClpC1 knocked down to see if similar results are obtained.