<i>AceE</i> affects the optimum growth and biofilm formation of <i>Mycobacterium tuberculosis</i> via cell wall lipid remodeling

Central carbon metabolism is fundamental to the growth and virulence of Mycobacterium tuberculosis (Mtb). The aceE gene encodes the E1 subunit of the pyruvate dehydrogenase complex that irreversibly converts pyruvate to acetyl-CoA. There, we investigated the impact of aceE inactivation on the physiological metabolism of Mtb. An aceE deletion in Mtb H37Rv was constructed, and the mutant was compared to wild type (WT) by in vitro growth assays, colony morphology, biofilm formation, stress tolerance, lipidomics, RNA-seq, and BALB/c mouse infection. Mtb Δ aceE exhibited markedly slower planktonic growth, smooth colony morphology, impaired biofilm formation, and was more sensitive to acid, NaNO 2 , and some commonly used antibiotics. Lipidomics revealed global depletion of mycolic acids and major phospholipids in Mtb Δ aceE mutant. Transcriptome analysis showed downregulation of TCA cycle and fatty-acid biosynthetic genes. Two weeks after intravenous infection, lung bacillary loads were 0.6 log10 lower for Δ aceE than in WT, and spleen enlargement was absent, yet the mutant persisted, and pathology was otherwise comparable to WT. AceE is required for robust lipid anabolism, biofilm formation, and maximal growth of Mtb in vitro , especially on carbohydrate-based media, but its absence only modestly attenuates acute virulence in mice, underscoring the pathogen's metabolic plasticity during infection.IMPORTANCEThe present study demonstrated that the aceE gene, a crucial enzyme that links glycolysis and the TCA cycle, plays a vital role in regulating the normal physiological metabolism of Mycobacterium tuberculosis (Mtb). The aceE gene not only aids in the bacteria's energy metabolism but also promotes lipid synthesis, forming a thicker cell wall that helps Mtb resist various intracellular stresses, further favoring its survival within the host cells. During in vivo survival, the increased expression of the aceE gene in the virulent Mtb H37Rv strain may enhance the conversion of pyruvate into acetyl-CoA, thereby providing more precursor materials for the synthesis of lipids and amino acids.