Pyruvate carboxylase is critical for biofilm formation in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) forms biofilms. Biofilm formation is critical in Mtb's virulence. The metabolic pathways governing mycobacterial biofilm formation remain largely unexplored. This study evaluated how different carbon sources influence Mtb biofilm formation. Fermentable substrates-glucose, glycerol, and pyruvate-significantly promoted thick, mature biofilm formation compared to non-fermentable alternatives. In this study, we analyzed the role of pyruvate carboxylase (Pca), an anaplerotic enzyme that converts pyruvate to oxaloacetate, in biofilm formation, as it regulates carbon metabolic flux into the tricarboxylic acid (TCA) cycle and gluconeogenesis. To this end, a transposon mutant of the pca gene was investigated for its ability to form biofilms. We observed that the pca transposon mutant is deficient in pellicle, submerged, and macrocolony biofilm formation. Confocal microscopy indicated that the pca mutant does not accumulate extracellular cellulose in the biofilms and has lower biomass. These defects could be rescued upon complementing the mutant with an episomal copy of the pca gene or adding glucose or pyruvate to the medium. These observations suggest that biofilm formation requires a regulated flow of carbon flux into gluconeogenesis and the TCA cycle to ensure the supply of precursors for EPS biosynthesis and sustained energy production to fuel EPS synthesis.