GrcC1 mediates low-level resistance to multiple drugs in <i>M. marinum</i> , <i>M. abscessus,</i> and <i>M. smegmatis</i>

The escalating threat of mycobacterial infectious diseases, particularly those caused by nontuberculous mycobacteria (NTM), poses a serious challenge to public health. Linezolid (LZD), an oxazolidinone antimicrobial, exhibits potent activity against <i>Mycobacterium tuberculosis</i> and NTM. Generally, mutations in the <i>rrl</i> and <i>rplC</i> genes are widely associated with resistance to LZD. However, in this study, we screened <i>Mycobacterium marinum</i> strains lacking such mutations, indicating the presence of an alternative resistance mechanism. Notably, through whole-genome sequencing, we identified a novel mutation C395T in the <i>MMAR_0911</i> (<i>grcC1</i>) gene that has never been linked to drug resistance. This mutation leads to an A132V substitution in the encoded protein, a polyprenyl diphosphate synthase potentially involved in the synthesis of cell wall components and menaquinones. We found that the overexpression of <i>grcC1</i> caused resistance to multiple drugs including LZD, clarithromycin (CLR), vancomycin (VAN), clofazimine (CFZ), rifampicin (RIF), cefoxitin (CEF), levofloxacin (LEV), and moxifloxacin (MXF) and reduced cell wall permeability, while the silence and knockout of <i>grcC1</i> showed increased cell wall permeability and susceptibility to these drugs. Using CRISPR/Cpf1-assisted gene editing, we confirmed that the A132V mutation conferred low-level resistance to the aforementioned drugs in <i>Mycobacterium abscessus</i> and <i>Mycobacterium smegmatis</i>. Furthermore, thin-layer chromatography analysis indicated reduced glycolipid polarity in the <i>grcC1</i> mutant strains, suggesting an impact on the cell envelope integrity. Our findings suggest that GrcC1 contributes to low-level drug resistance in mycobacteria by potentially reducing cell wall permeability, highlighting its potential as a novel target for antimicrobial agents and as a diagnostic marker.IMPORTANCEOur study uncovers a novel drug resistance mechanism in mycobacteria, focusing on the previously uncharacterized <i>grcC1</i> gene. We identified a new mutation, A132V, in GrcC1, which is involved in cell wall component synthesis and menaquinone production. This mutation contributes to low-level resistance not only to linezolid but also to a broad range of drugs, including clarithromycin, vancomycin, and rifampicin. Through advanced techniques like CRISPR interference and gene editing, we demonstrated that GrcC1 plays a critical role in drug susceptibility and cell wall permeability across multiple <i>Mycobacterium</i> species. These findings represent the first connection between GrcC1 and drug resistance, offering new insights into combating infections caused by nontuberculous mycobacteria (NTM). Our work highlights the potential of GrcC1 as a target for novel therapeutic approaches and as a diagnostic marker for drug-resistant NTM infections.