Functional characterization of GplA (MSMEG-0394) reveals a novel determinant of glycopeptidolipids homeostasis in Mycobacterium smegmatis

Introduction Glycopeptidolipids (GPLs) are major constituents of the cell envelope of non-tuberculous mycobacteria (NTM) and play essential roles in colony morphology, surface motility, biofilm formation, antimicrobial susceptibility, and host–pathogen interactions. Although the GPL biosynthetic locus has been extensively characterized, the function of several conserved genes within this cluster remains unresolved. Here, we functionally characterized GplA (MSMEG-0394), a conserved small protein encoded within the GPL biosynthetic locus of Mycobacterium smegmatis . Methods The role of GplA was investigated by combining genetic and phenotypic analyses, lipid profiling by thin-layer chromatography and electrospray ionization mass spectrometry, transcriptional analyses, complementation studies, and structural modelling. Results Deletion of gplA resulted in extensive alterations of cell-envelope-associated phenotypes, including rough colony morphology, impaired sliding motility, delayed biofilm formation, increased cell aggregation, reduced growth, and enhanced susceptibility to rifampicin. Biochemical analyses demonstrated a marked remodeling of the GPL profile in the mutant strain, supporting a role for GplA in maintaining GPL homeostasis. Transcriptional analyses further showed that gplA is co-transcribed with MSMEG_0393 and MSMEG_0395 and that deletion of gplA results in upregulation of this operon, suggesting that GplA contributes, directly or indirectly, to the control of GPL-associated gene expression. Complementation substantially restored the wild-type phenotype, confirming that the observed alterations were specifically associated with loss of gplA . Finally, structural modelling predicts that GplA is a small soluble protein lacking recognizable catalytic features, consistent with a non-enzymatic role and supporting the hypothesis that it may act through protein-protein interactions. Discussion Collectively, our findings identify GplA as a determinant of glycopeptidolipid homeostasis and suggest that this conserved small protein contributes to the coordination of cell-envelope composition, surface-associated physiology, and operon expression in M. smegmatis . These findings expand the current understanding of the regulatory network underlying GPL homeostasis and provide a framework for future studies aimed at defining the molecular mechanism of GplA function in clinically relevant NTM.