Mechanisms of MSMEG_3312-mediated collective antibiotic tolerance to erythromycin in mycobacteria revealed by quantitative proteomic analysis

Abstract:

[Objective] The effects of antibiotics on bacteria are complex, and bacterial response to antibiotics is just beginning to be understood using systems biology. We previously showed that a hemerythrin-like protein, MSMEG_3312, is involved in erythromycin susceptibility. In this study, we explore the mechanisms of collective antibiotic tolerance to erythromycin in mycobacteria through the hemerythrin-like protein MSMEG_3312.[Methods] We analyzed MSMEG_3312 secondary structure using spectrophotometric and circular dichroism (CD) methods. Tandem mass tag(TMT)-labeled quantitative proteomics was used to compare protein level changes between the wild type strain mc² 155 and the knockout strain Δmsmeg_3312, following bioinformatics analysis. Differentially expressed proteins were also verified by qPCR. To confirm our analyses' conclusions that transporters are involved in MSMEG_3312-related erythromycin susceptibility, we also measured the concentration of mycobacterial erythromycin in vivo in the wild type strain mc² 155 and Δmsmeg_3312 using an erythromycin ELISA kit.[Results] Initially, we confirmed that MSMEG_3312 is a redox-related hemerythrin-like protein using spectrophotometric and CD analysis. Quantitative proteomic analysis revealed that Δmsmeg_3312 has eight up-regulated proteins, including three transporters, and 14 down-regulated proteins, compared with the wild type strain mc² 155, while growing in 7H9 medium. In contrast, 448 proteins were identified as being differentially expressed between mc² 155 and Δmsmeg_3312, when treated with erythromycin, of which 11 were identified as up-regulated transporter proteins, and 26 were associated with amino acid synthetic pathways. The intracellular erythromycin concentration in Δmsmeg_3312 was also lower than in mc² 155.[Conclusion] We show that MSMEG_3312 mediates erythromycin resistance due to collective antibiotic tolerance arising from antibiotic titration and high-density populations.