Research On The Self-resistance Mechanism Of Azinomycin B In STREPTOMYCES SAHACHIROI

The genotoxic antibiotic azinomycin B with an active epoxide moiety and an unusual aziridine ring,is a hybrid polyketide/nonribosomal peptide antibiotic isolated from Streptomyces sahachiroi.It can tandemly nucleophilically attack the N7 position of purine bases in the 5'-d?Pu NPy?-3'sequence located on the two strands to form DNA interstrand crosslinks?ICLs?.This antibiotic exhibits prominent in vitro cytotoxicity and broad-spectrum in vivo antitumor activity,suggesting its potential for development into pharmaceutical drug.As the producer of the strong DNA cross-linking reagent azinomycin B,S.sahachiroi should have multiple self-resistance protection mechanisms to prevent from the attack of antibiotic on the chromosome of the host.The research on the self-resistance mechanism of genotoxic antibiotics producing bacteria not only provides a research direction for genetically engineered antibiotic production,but also sets up a new idea for the solution of drug resistance and the repair of DNA damage.The aziE,aziF,azi N,orf8 and orf10 genes in the azinomycin B biosynthetic gene cluster became the candidates of antibiotic resistance determinants in our study and the main results were showed as follows:1.The azi N gene is a functional unknown gene adjacent to the right edge of the azinomycin B biosynthetic gene cluster.Deletion of azi N significantly reduced the yield of azinomycin B,suggesting that Azi N is closely related to the drug biosynthesis.Base on the orf1 gene deletion strain,the azi N gene deletion mutant strain was constructed and showed increased sensitivity to azinomycin B than the original strain.Heterologous expression of Azi N in an azinomycin B-sensitive strain susceptible strain conferred the host resistance to azinomycin B,indicating the resistance function of Azi N against azinomycin B.Bioinformatics analysis showed that Azi N belonged to the Glyoxalase₂ superfamily and shared homology with the mitomycin C binding protein Mrd,which suggested that Azi N might be an antibiotic binding protein.Equilibrium fluorescence titration and microscale thermophoresis?MST?assays confirmed that Azi N could specifically recognize and bind azinomycin B.The drug-binding proteins usually sequestrate or inactivate drugs to exert resistance.However,bioassay and crosslinking activity detection found that the activity of azinomycin B was not affected by the binding of Azi N.EMSA experiments showed that Azi N could specifically bind to the azinomycin B induced cross-linking sites by the strong affinity with the drug.A series of DNA cross-linking repair experiments in vitro were then conducted and confirmed that Azi N could catalyze the hydrolytic cleavage of phosphodiester bonds at the cross-linking site and cause DNA strand breakage.For example,at the cross-linking site of 5'-GCC-3',Azi N first cleaves the phosphodiester bond at 3'-end of the G-G cross-linking site on forward strand,and then continues to cleave the phosphodiester bond at 5'-end the G-G cross-linking site on reverse strand,eventually leading to DNA double-strand breakage.Azi N is a new nuclease-like enzyme,which can induce the breakage of DNA strands through specific recognition and cleavage of the azinomycin B induced cross-linked DNA,and then different DNA repair pathways such as nucleoside excision?NER?,homologous recombination or non-homologous end-joining can be induced,which provides strong protection for the host to produce a large number of azinomycin B in the later stage.2.In the heterologous host,the expression of Orf8 and Orf10 did not change the azinomycin B sensitivity of host,suggesting that Orf8 and Orf10 were not implicated in the drug resistance mechanism.3.aziE is responsible for encoding the main facilitator superfamily?MSF?transporter which are usually involved in the efflux of small molecules such as drugs.After deletion of the azi E gene,concentration of extracellular azinomycin B decreased significantly,indicating that Azi E may be involved in the efflux of azinomycin B.In addition,heterologous expression experiment showed that the expression of Azi E conferred resistance to the host against azinomycin B,which further confirmed that azi E as a drug transporter could significantly reduce the concentration of intracellular drugs by timely transporting intracellular antibiotics to extracellular,thus protecting antibiotic-producing bacterium.4.Azi F shows homology to acetyl-CoA carboxylase ? subunit.All attempts to disrupt the azi F gene in the azinomycin B producer S.sahachiroi failed.The azi F deletion mutant strain was achieved by using ?azi B in which the azinomycin B biosynthetic pathway was abolished.Obviously,azi F is an essential resistance factor for azinomycin B biosynthesis.Deletion of the azi F gene significantly reduced the resistance to azinomycin B and heterologous expression of Azi F could significantly enhance the resistance of the drug sensitive strain to azinomycin B,which confirmed that azi F is another resistance determinant found in the azinomycin B producing strain.In this study,the self-resistance mechanisms of S.sahachiroi were investigated.Besides the reported antibiotic binding protein Azi R and transglucosylase Alk Z?orf1encoding product?,the proteins encoded by azi E,azi F and azi N also can protect the producing strain from the toxicity of azinomycin B.A new DNA repair mechanism induced by nuclease-like enzyme Azi N was found by systematically analyzing the self-resistance mechanism of this genotoxic antibiotic-producing strain.