Abstract
Tuberculosis is still on the top of infectious diseases list on both mobility and mortality, especially due to drug-resistance of Mycobacterium tuberculosis (M.tb). Ethionamide (ETH) is one of effective second line anti-TB drugs, a synthetic compound similar to isoniazid (INH) structurally, with existing severe problem of ETH resistance. ETH is a prodrug, which is activated by Etha inside M.tb, and etha is transcriptionally repressed by Ethr. We found that c-di-GMP could bind Ethr, enhanced the binding of Ethr to the promoter of etha, and then repressed the transcription of etha, thus caused resistance of M.tb to ETH. Through docking analysis and in vitro validation, we identified that c-di-GMP binds 3 amino acids of Ethr, i.e., Q125, R181 and E190, while the first 2 were the major binding sites. Homology analysis showed that Ethr was highly conservative among mycobacteria. Further docking analysis showed that c-di-GMP preferentially bound proteins of TetR family at the junction hole of symmetric dimer or tetramer proteins. Our results suggest a possible drug-resistance mechanism of ETH through the regulation of Ethr by c-di-GMP.
Highlights
Tuberculosis (TB) with its causative pathogen Mycobacterium tuberculosis (M.tb) has afflicted humans for millennia and remains a huge threat on human life and health
Using UV cross-linking and Bio-Layer interference (BLI), we found that EthrQ125A, EthrR181A and EthrE125A had a significantly decreased binding to c-di-GMP, especially EthrQ125A and EthrR181A (Fig. 4b and c, respectively)
The results showed that the three c-di-GMP binding sites Q125, R181, and E190 with red star mark are highly conservative among these bacteria (Fig. 5a)
Summary
Hai-Nan Zhang[1], Zhao-Wei Xu1, He-Wei Jiang[1], Fan-Lin Wu1, Xiang He1, Yin Liu[1], Shu-Juan Guo1,Yang Li1, Li-Jun Bi4,5,6,7, Jiao-Yu Deng[8], Xian-En Zhang4 & Sheng-Ce Tao 1,2,3. Competition experiment showed that the binding of bio-c-di-GMP and Ethr could be significantly inhibited upon addition of unlabeled c-di-GMP To rule out this possibility that the GST tag may interference with the binding, a 6× His tagged Ethr was constructed and tested, similar results as that of GST-Ethr was obtained (Supplementary Fig. S1a). These results indicate c-di-GMP regulates etha mRNA level through enhancing the binding of Ethr to etha promoter. These results suggest that c-di-GMP increases M.sm resistance to ETH depending on Ethr. Plasmids or Strains E.coli DH5a BL21 M. smegmatis mc2155 M.sm/WT M.sm-pMV261 M.sm-pMV261-ethr M.sm-pMV261-dgc M.sm-ethr::hyg M.sm-ethr::hyg-pMV261
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