Infection and Drug Resistance (Feb 2022)
Insertion Mutation of MSMEG_0392 Play an Important Role in Resistance of M. smegmatis to Mycobacteriophage SWU1
Abstract
Zhen Zhang,1,2 Zhulan Yang,3 Junfeng Zhen,1 Xiaohong Xiang,4 Pu Liao,1 Jianping Xie2 1Department of Clinical Laboratory, Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing, People’s Republic of China; 2Institute of Modern Biopharmaceuticals, State Key Laboratory Breeding Base of Three Gorges Eco-Environment and Bioresources, Eco-Environment Key Laboratory of the Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing, 400715, People’s Republic of China; 3Department of Clinical Laboratory, Southwest Hospital, Army Medical University, Chongqing, People’s Republic of China; 4School of Pharmacy, Chongqing Medical and Pharmaceutical College, Chongqing, People’s Republic of ChinaCorrespondence: Jianping Xie; Pu Liao, Tel/Fax +8623-68367108, Email [email protected]; [email protected]: Phage is a new choice for the treatment of multi-drug-resistant bacteria, and phage resistance is also an issue of concern. SWU1 is a mycobacteriophage, and the mechanism of its resistance remain poorly understood.Methods: The mutant strains which were stably resistant to SWU1 were screened by transposon mutation library. The stage of phage resistance was observed by transmission electron microscope (TEM). The insertion site of transposon was identified by thermal asymmetric interlaced PCR (TAIL-PCR). The possible relationship between insertion site and phage resistance was verified by gene knockout technique. The fatty acid composition of bacterial cell wall was analyzed by Gas Chromatography-Mass Spectrometer (GC-MS). Through the amplification and sequencing of target genes and gene complement techniques to find the mechanism of SWU1 resistance.Results: The transposon mutant M12 which was stably resistant to mycobacteriophage SWU1 was successfully screened. It was confirmed that resistance occurred in the adsorption stage of bacteriophage. It was verified that the insertion site of the transposon was located in the MSMEG_3705 gene, but after knocking out the gene in the wild type M. smegmatis mc2 155, the resistance of the knockout strain to SWU1 was not observed. Through the amplification and sequencing of the target gene MSMEG_0392, it was found that there was an adenine insertion mutation at position 817. After complementing MSMEG_0392 in M12, it was found that M12 returned to sensitivity to SWU1.Conclusion: We confirmed that the resistance of M12 to SWU1 was related to the functional inactivation of MSMEG_0392 and this phenomenon may be caused by the change of cell wall of M. smegmatis.Keywords: phage resistance M. smegmatis, mycobacteriophage, SWU1, insertion mutation
