Mycobacterium abscessus,a fast-growing,non-tuberculous mycobacterium resistant to most antimicrobial drugs,causes a wide range of serious infections in humans,posing a significant public health challenge.The developme...Mycobacterium abscessus,a fast-growing,non-tuberculous mycobacterium resistant to most antimicrobial drugs,causes a wide range of serious infections in humans,posing a significant public health challenge.The development of effective genetic manipulation tools for M.abscessus is still in progress,limiting both research and therapeutic advancements.However,the clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein(Cas)systems have emerged as promising tools for generating highly specific double-strand breaks(DSBs)in its genome.One of the mechanisms that repair these DSBs is the error-prone nonhomologous end-joining(NHEJ)pathway,which facilitates targeted gene editing.In this study,we introduced a novel application of the CRISPR-NHEJ approach in M.abscessus.We demonstrated that NrgA from M.marinum plays a crucial role in repairing DSBs induced by the CRISPRCas system in M.abscessus.Contrary to previous findings,our study also revealed that inhibiting or overexpressing components of homologous recombination/single-strand annealing significantly reduces the efficiency of NHEJ repair in M.abscessus.This discovery challenges current perspectives and suggests that NHEJ repair in M.abscessus may involve components from both homologous recombination and single-strand annealing pathways,highlighting the complex interactions among the three DSB repair mechanisms in M.abscessus.展开更多
Treatment of Mycobacterium abscessus(Mab)infections is very challenging due to its intrinsic resistance to most available drugs.Therefore,it is crucial to discover novel anti-Mab drugs.In this study,we explored an int...Treatment of Mycobacterium abscessus(Mab)infections is very challenging due to its intrinsic resistance to most available drugs.Therefore,it is crucial to discover novel anti-Mab drugs.In this study,we explored an intrinsic resistance mechanism through which Mab resists echinomycin(ECH).ECH showed activity against Mab at a minimum inhibitory concentration(MIC)of 2μg/ml.A embC strain in which the embC gene was knocked out showed hypersensitivity to ECH(MIC:0.0078-0.0156μg/ml).The MICs of ECH-resistant strains screened with reference to AembC ranged from 0.25 to 1μg/ml.Mutations in EmbB,including D306A,D306N,R350G,V555l,and G581S,increased the Mab's resistance to ECH when overexpressed in AembC individually(MIC:0.25-0.5μg/ml).These EmbB mutants,edited using the CRISPR/Cpf1 system,showed heightened resistance to ECH(MIC:0.25-0.5μg/ml).The permeability of these Mab strains with edited genes and overexpression was reduced,as evidenced by an ethidium bromide accumulation assay,but it remained significantly higher than that of the parent Mab.In summary,our study demonstrates that ECH exerts potent anti-Mab activity and confirms that EmbB and EmbC are implicated in Mab's sensitivity to ECH.Mutation in EmbB may partially compensate foralossof EmbCfunction.展开更多
基金supported by the National Key R&D Program of China(2021YFA1300900 and 2022YFE0210600)the National Natural Science Foundation of China(21920102003)+5 种基金the Guangdong Province Grant for Belt and Road Joint Laboratory(2022B1212050004)the State Key Lab of Respiratory Diseasethe Postdoctoral Fellowship Program of CPSF(GZC20232688)the Guangzhou Institute of Respiratory Diseases,First Affiliated Hospital of Guangzhou Medical University(SKLRD-Z-202301,SKLRD-Z-202412)the Guangzhou Science and Technology(2024A04J4273)the Chinese Academy of Sciences,President's International Fellowship Initiative(2023VBC0015).
文摘Mycobacterium abscessus,a fast-growing,non-tuberculous mycobacterium resistant to most antimicrobial drugs,causes a wide range of serious infections in humans,posing a significant public health challenge.The development of effective genetic manipulation tools for M.abscessus is still in progress,limiting both research and therapeutic advancements.However,the clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein(Cas)systems have emerged as promising tools for generating highly specific double-strand breaks(DSBs)in its genome.One of the mechanisms that repair these DSBs is the error-prone nonhomologous end-joining(NHEJ)pathway,which facilitates targeted gene editing.In this study,we introduced a novel application of the CRISPR-NHEJ approach in M.abscessus.We demonstrated that NrgA from M.marinum plays a crucial role in repairing DSBs induced by the CRISPRCas system in M.abscessus.Contrary to previous findings,our study also revealed that inhibiting or overexpressing components of homologous recombination/single-strand annealing significantly reduces the efficiency of NHEJ repair in M.abscessus.This discovery challenges current perspectives and suggests that NHEJ repair in M.abscessus may involve components from both homologous recombination and single-strand annealing pathways,highlighting the complex interactions among the three DSB repair mechanisms in M.abscessus.
基金This work was supported by the National Key R&D Program of China(2021YFA1300900)the National Natural Science Foundation of China(21920102003,82022067,and 22037006)+5 种基金the Chinese Academy of Sciences Grants(154144KYSB20190005 and YJKYYQ20210026)the Key R&D Program of Sichuan Provenience(2023YFSY0047)the State Key Laboratory of Respiratory Disease,Guangzhou Institute of Respiratory Diseases,First Affiliated Hospital of Guangzhou Medical University(SKLRD-Z-202414,SKLRD-OP-202324,SKLRD-Z-202301,SKLRD-OP-202113,and SKLRD-Z-202412)Guangzhou Scienceaand Technology Plan-Youth Doctoral"Sail"Project(2024A04J4273)President's International Fellowship Initiative-CAS(2023VBC0015)the National Foreign Young Talent Program(QN2022031002L).
文摘Treatment of Mycobacterium abscessus(Mab)infections is very challenging due to its intrinsic resistance to most available drugs.Therefore,it is crucial to discover novel anti-Mab drugs.In this study,we explored an intrinsic resistance mechanism through which Mab resists echinomycin(ECH).ECH showed activity against Mab at a minimum inhibitory concentration(MIC)of 2μg/ml.A embC strain in which the embC gene was knocked out showed hypersensitivity to ECH(MIC:0.0078-0.0156μg/ml).The MICs of ECH-resistant strains screened with reference to AembC ranged from 0.25 to 1μg/ml.Mutations in EmbB,including D306A,D306N,R350G,V555l,and G581S,increased the Mab's resistance to ECH when overexpressed in AembC individually(MIC:0.25-0.5μg/ml).These EmbB mutants,edited using the CRISPR/Cpf1 system,showed heightened resistance to ECH(MIC:0.25-0.5μg/ml).The permeability of these Mab strains with edited genes and overexpression was reduced,as evidenced by an ethidium bromide accumulation assay,but it remained significantly higher than that of the parent Mab.In summary,our study demonstrates that ECH exerts potent anti-Mab activity and confirms that EmbB and EmbC are implicated in Mab's sensitivity to ECH.Mutation in EmbB may partially compensate foralossof EmbCfunction.
