Functions And Regulatory Networks Of Transcriptional Factor MtrA In Actinomycetes

Actinomycetes is pervasively existed gram-positive bacteria including industrial actinomycetes for antibiotics production(such as Saccharopolyspora erythraea),which is widely used in the fields of industry,agriculture and medical treatment.The pathogenic actinomycetes,represented by Mycobacterium tuberculosis,is the emphasis of global pathogenicity,multi-drug resistance and asymptomatic latent infection of tuberculosis.Bacterial drug resistance is a well-known serious threat of human health.In addition to the common chromosomal mutation mechanism,the two-component regulatory systems should be concerned and further studied as well.Here,we systematically investigated the function and regulatory network of the transcriptional factor MtrA in the two-component regulatory system MtrA-MtrB of actinomycetes,and expanded its regulatory role in drug resistance.The enzyme characteristics of Eis(a drug resistance related protein)was also analyzed,the MtrA mediated regulatory network of drug resistance was expected to be established,which provided an enrichment of pathogen biology and the foundation for new therapeutic discovery efforts.The main research contents are as follows:(1)Transcriptional factor MtrA regulated drug resistance and erythromycin synthesis in Saccharopolyspora erythraea.We revealed that the MtrA in Saccharopolyspora erythraea E3 strain(a high erythromycin-producing strain)had a two amino acid(H197 and V198)deletion(MtrAdel)in the DNA recognition helices of the C-terminal domain compared to the wild type strain NRRL2338.An MtrA target gene mepA(encoding a membrane protein related to metalloendopeptidases)was then identified influencing cell peptidoglycan.MtrAdel lost the DNA-binding activity towards mepA,and resulted in the decreased of drug resistance.In addition,MtrAdel increased erythromycin biosynthesis,deregulate osmoprotection,and improved material transport.These findings demonstrated that MtrA plays an important pleiotropic regulation role in series of physiological processes especially drug resistance.(2)Transcriptional factor MtrA mediated drug resistance in Mycobacterium smegmatis.We found that MtrA enhanced drug resistance of Mycobacterium smegmatis by directly inhibiting whiB2(a member of WhiB protein family)transcription.The supposed drug resistance related genes panD and katG were then identified by CHIP-Seq.We also found that the resuscitation-promoting factor E(rpfE)as another regulatory target of MtrA.MtrA regulated latent/activation of mycobacterium by directly activating rpfE transcription,and affected the bacterial response to drugs.These results expanded the drug resistance regulatory network in mycobacterium mediated by MtrA and provided evidence for the development of new highly effective anti-tuberculosis drugs.(3)Enzyme characteristics of Eis acetyl transferase which is the downstream target protein of mycobacterium MtrA-WhiB.Eis(enhanced intracellular survival)protein is a regulatory target of the WhiB protein family,which enables bacterial resistance by acetylating aminoglycoside antibiotics.We found that Eis proteins have an unprecedented ability in acetylating arylalkylamines,suggesting a novel type of aryl alkyl amine N-acetyltransferase AANAT(EC 2.3.1.87).In vitro acetylation experiments and bioinformatics analysis confirmed that the arylamine N-acetyltransferase activity of Eis was conserved in actinomycetes.Due to the fundamental role of arylalkylamines such as histamine in immune reactions,the AANAT activity of Eis protein is conducive to further broadening our understanding of the interaction between gram-positive pathogens and hosts.In-depth analysis of the molecular mechanism of drug resistance genes could facilitate pathological research and the development of therapeutic drugs.