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bioRxiv · 10.64898/2025.12.18.695065

Linking MSMEG_1353 to lipid metabolism and envelope integrity in Mycolicibacterium smegmatis

Xie, Z.·Lioe, T. S.·Sahu, A.·Cui, J.·Ruiz Carrillo, D.·Kadowaki, T.·Tefsen, B.
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Abstract

Mycobacterium tuberculosis (Mtb) poses a significant global health burden. Rv0647c, an essential Mtb cell wall protein, is a potential drug target. We studied its Mycolicibacterium smegmatis (MSMEG) homologue, MSMEG_1353, using a CRISPRi conditional knockdown. MSMEG_1353 depletion increased cell width and volume, delayed log-phase initiation, slowed aggregation, reduced biofilm formation, and heightened susceptibility to antibiotics and sodium dodecyl sulfate (SDS). AlphaFold, sequence alignment, and UNIPROT analyses suggest MSMEG_1353 functions as a protein kinase, with conserved residues in intermediate high-confidence regions potentially forming an ATP-binding site. Investigating its role in cell envelope biosynthesis, mass spectrometry revealed elevated levels of mycolic acid biosynthesis proteins upon MSMEG_1353 knockdown. RT-qPCR confirmed upregulation of the fabD-acpM-kasA-KasB-accD6 operon, encoding key mycolic acid synthesis enzymes. Notably, a strong negative correlation with MSMEG_0911, the predominant isocitrate lyase, important in the glyoxylate cycle, was observed via both MS and RT-qPCR. Collectively, MSMEG_1353 deficiency compromises cell wall integrity, likely due to altered lipid composition resulting from dysregulated mycolic acid biosynthesis and lipid metabolism. These findings support the development of models explaining MSMEG_1353s involvement in these pathways.

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Xie, Z., Lioe, T. S., Sahu, A., Cui, J., Ruiz Carrillo, D., Kadowaki, T., Tefsen, B.. 2025-12-18. Linking MSMEG_1353 to lipid metabolism and envelope integrity in Mycolicibacterium smegmatis. https://doi.org/10.64898/2025.12.18.695065

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