Characterization of Mycobacterium abscessus with mutation at the putative cell wall hydrolase gene MAB_1974

• Main Authors: Jia-Huei Chen, 陳家輝
• Other Authors: Shiau-Ting Hu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/13287150991715580834

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 99 === Abstract Mycobacterium abscessus is an important emerging nontuberculous mycobacteria (NTM). It grows fast and ubiquitous in our environment. It causes a wide spectrum of disease in immunodeficient patients, such as chronic lung disease, post-traumatic wound and soft tissue infections. Previous studies indicated that M. abscessus could switch morphotype between rough and smooth form spontaneously. The morphoptype switch affects its virulence, growth rate, sliding motility, and biofilm formation. The content and composition of glycopeptidolipid (GPL) of M. abscessus might be the major cause for the morphoptype switch. The clinical strain M. abscessus cs1c used in this study also reveals the same morphologic switch. To investigate the mechanism, the rough strain of M. abscessus cs1c was mutated by transposon mutagenesis method. Twenty mutants with smooth morphology were obtained. The mutant of my study was a strain with transposon inserted into the MAB_1974. To investigate the function of MAB_1974 in M. abscessus, we first compared some properties of M. abscessus wild type and MAB_1974::Tn mutant. The results indicated that MAB_1974::Tn strain displayed weaker lysozyme resistance and biofilm formation ability than the wild type strain. Using thin layer chromatography and MALDI-TOF MS to analyze the glycopeptidolipid of both strains, only MALDI-TOF MS revealed an obvious difference in the content and composition of GPL. The MAB_1974 protein was predicted to contain an Nlpc/p60 domain that might have bacterial cell wall hydrolyzing activity. The morphotype change to smooth form of MAB_1974::Tn strain may be due to the structural change of cell wall caused by the cell wall peptidases. To investigate whether MAB_1974 protein possesses the bacterial cell wall peptidases activity, we expressed MAB_1974 in E. coli and analyze its activity by zymogram experiment. The results showed no activity. The lack of appropriate protein modulation or folding on MAB_1974 may be one of the causes leading to losing activity. Using M. smegmatis to express MAB_1974 protein may be needed to solve this problem in the future.