Abstract
BackgroundThe integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant (aceE-mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis (M. smegmatis). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis.ResultsWe observed that the colony morphology of aceE-mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis. Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE-mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE-mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE-mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceE gene restored the composition of MA.ConclusionsOver all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.
Highlights
The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria
Studies have found that the genes involved in gluconeogenic pathway and glyoxylate cycle are up-regulated in Mycobacterium tuberculosis (Mtb) isolated from macrophages, mouse lung tissues and tissue samples of patients, suggesting that the compensatory metabolism of acetyl-coA is necessary for intracellular growth and persistence in vivo [10, 11]
The aceE-Mut exhibited unusual colony morphology By screening the M. smegmatis transposon library, a transposon mutant showed obvious differences in colony morphology on agar plate when compared to the parental WT M. smegmatis strain (Fig. 1a)
Summary
The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. Pyruvate dehydrogenase (PDH) is an enzyme complex that catalyzes the conversion of pyruvate into acetyl-coA in vivo. The complex mainly consists of three enzymes, which are respectively called E1, E2 and E3 components of PDH according to the order in which they participate in the reactions. Through a series of chemical reactions of pyruvate decarboxylation, the glycolytic pathway (the final product is pyruvate) and the tricarboxylic acid cycle (the initial reactant is acetyl-coA) can be effectively connected [7]. Studies have found that the genes involved in gluconeogenic pathway and glyoxylate cycle are up-regulated in Mtb isolated from macrophages, mouse lung tissues and tissue samples of patients, suggesting that the compensatory metabolism of acetyl-coA is necessary for intracellular growth and persistence in vivo [10, 11]
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