Milena Della Gala, Rosangela Marasco, Ida De Chiara, Martina Montebuglio, Luigi Russo, Severina Pacifico, Lidia Muscariello
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.
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.