[Quantitative proteomic analysis of mkl gene function in Mycobacterium marinum using iTRAQ].

Objective: To identify differentially expressed proteins in Mycobacterium marinum wild-type (WT) and mkl::Tn mutant strains, and provide new clues for exploring the functions of mkl gene. Methods: Cellular proteins were extracted from cultures of M. marinum WT and mkl::Tn strains, and labelled with isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex. Differentially expressed proteins were identified with LC-MS/MS and subjected to biological information analysis. Results: A total of 566 differentially expressed proteins were revealed, among which 232 proteins were up-regulated (ratio≥1.4) and 334 proteins were down-regulated (ratio≤0.7). These proteins are mainly associated with lipid metabolism, cell wall and cell processes, intermediary metabolism and respiration, and hypothetical proteins. The most down-regulated protein DesA3, is a fatty acid desaturase and involved in the synthesis of oleic acid. Further experiments showed that the growth of mkl::Tn strain was attenuated on 7H10-ADC agar plate without oleic acid, suggesting that mkl may play a role in the biosynthesis of oleic acid. Conclusion: Differentially expressed proteins were identified in M. marinum mkl::Tn compared to WT, and these results shed light on the mechanisms of mkl gene in mycobacterial pathogenesis.

CpsA, a LytR-CpsA-Psr Family Protein in Mycobacterium marinum, Is Required for Cell Wall Integrity and Virulence.

LytR-CpsA-Psr family proteins play an important role in bacterial cell wall integrity. Although the pathogenic relevance of LytR-CpsA-Psr family proteins has been studied in a few bacterial pathogens, their function in mycobacteria remains uncharacterized. In this work, a transposon insertion mutant (cpsA::Tn) of Mycobacterium marinum was studied. We found that inactivation of CpsA altered bacterial colony morphology, sliding motility, cell surface hydrophobicity, and cell wall permeability. Besides, the cpsA mutant exhibited a decreased arabinogalactan content, indicating that CpsA plays a role in cell wall assembly. Moreover, the mutant shows impaired growth within macrophage cell lines and is severely attenuated in zebrafish larvae and adult zebrafish. Taken together, our results indicated that CpsA, a previously uncharacterized protein, is important for mycobacterial cell wall integrity and is required for mycobacterial virulence.