Functional Analysis Of Mycobacterium Hemerythrin-like Protein MSMEG₆212

Tuberculosis （TB）, caused by Mycobacterium tuberculosis （M.tb）, is an infectious disease with high mortality. The mortality rate was significantly declined when induction of chemotherapy. Recently, the prevailing of drug-resistant M. tb strains, causing multiple drug-and extensively drug-resistant TB, becomes new worldwide public health challenges. The research on the mechanism of drug resistance can provide scientific theoretical basis for the prevention and treatment of TB.Hemerythrin-like proteins are non-heme, di-iron and O2 binding proteins that are ubiquitous from bacteria to mammals and have oxygen-related functions such as oxygen reverse, delivery and transport. The characteristic motif of hemerythrin-like proteins is H-HxxxE-HxxxH-HxxxxD, designed as HHE domain. Bioinformatic analysis indicated that M. smegmatis, a model organism for M. tb, possesses three hemerythrin-like proteins, MSMEG₂415, MSMEG₃312 and MSMEG₆212. The previous studies in our lab showed that MSMEG₂415 is involved in H2O2 response and MSMEG₃312 is related to macrolide antibiotics resistance.In this study, we characterized the biological functions of a mycobacterial hemerythrin-like protein MSMEG₆212. To define the potential roles of MSMEG₆212, its knockout strain （A6212） was constructed by using the mycobacteriophage-based specialized transduction. We measured the minimum inhibitory concentration （MIC） of different class antibiotic drugs and H2O2 against msmeg₆212 knockout strain Δ6212 and wild type strain mc2155.Surprisingly, among all examine conditions, our results showed that Δ6212 strain, like Δ3312 strain showed resistance to macrolides antibiotics, comparing with the wild type strains mc2155.Consistence with above results, overexpression of msmeg₆212 increases susceptibility to erythromycin. Moreover, in contrast MSMEG₂415 and MSMEG₃312, MSMEG₆212 has not involved into H2O2 response. Furthermore, the unchanged level resistance of erythromycin in the double-knockout mutant strain Δ3312-6212 suggested that no superposition effect was shown in double-knockout mutant Δ3312-6212. To determine the order of MSMEG 3312 and MSMEG₆212, we analyzed the mRNA expression level of msmeg₆212 in Δ3312 and the mRNA expression level of msmeg₃312 in Δ6212. The knockout of MSMEG₃312 has affected the mRNA level of msmeg₆212, but not in versea. According to our result, we reasoned that MSMEG₃312 is located at the upstream of a potential pathway connecting MSMEG₃312 and MSMEG₆212.