The effects of Lsr2 on biofilm formation, resistance to oxidative stress and tetracycline-class antibiotics in Mycobacterium abscessus.

• Main Authors: You-Jhen Lu, 盧宥蓁
• Other Authors: Shiau-Ting Hu
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/v9jzq8

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 103 === Lsr2, a global transcription regulator highly conserved in mycobacteria, is involved in biofilm formation, oxidative stress tolerance, antibiotic resistance, and virulence of M. smegmatis and M. tuberculosis, while its role in M. abscessus has not been studied. M. abscessus is a rapid-growing and most chemotherapy-resistant bacterium, causing localized soft tissue infections, pulmonary disease and disseminated infections. To investigate whether Lsr2 plays the same role in M. abscessus as in other mycobacteria, the Δlsr2 mutant strain was generated by recombineering (recombination-mediated genetic engineering) method. It was discovered that lsr2 is required for the formation of pellicle, a kind of biofilm covered on the liquid surface. It was known that some mycolic acid-biosynthesis associated genes were associated with biofilm formation in M. smegmatis. To investigate whether Lsr2 affects the expression of these genes, qRT-PCR assay was performed to quantify the RNA level of lsr2 and these genes in wild type strain during pellicle formation. The results revealed that the expression of these tested genes was increased, which was correlated to the result that all the mycolic acid-biosynthesis associated genes were down regulated in Δlsr2 mutant. Taken together of these results, it was postulated that Lsr2 might affect the expression of mycolic acid-biosynthesis associated genes, in turn regulating the biofilm formation. Since the lsr2 positively affected the expression of the biofilm-associated genes that are involved in the biosynthesis (kasA, kasB, groEL1, inhA, and mmaA4) and the transport (mmpL11) of mycolic acid, the lipid profiles of the wild type, Δlsr2 mutant, and complement strain were analyzed by thin layer chromatography (TLC) and MALDI-TOF. No difference of lipid profile was observed among these tested bacteria by TLC analysis, but MALDI-TOF analysis revealed that the Δlsr2 mutant had different peaks at 1002 ~ 1131 (Region 1) m/z and 1200 ~1273 (Region 2) m/z. This result indicated that the lsr2 indeed affected the production of lipid composition in M. abscessus. In addition, it was found that deletion of lsr2 reduced the H2O2 tolerance and tetracycline-class antibiotic resistance of M. abscessus. Since the Δlsr2 mutant was more sensitive to all tetracycline-class antibiotics, it was postulated that these antibiotics might be accumulated in Δlsr2 mutant. The tetracycline fluorescence assay confirmed this hypothesis. To test whether this accumulation was due to the function of efflux pump inhibited in the absence of lsr2, the efflux pump inhibitor CCCP was added with tetracycline for MIC test. The result showed that CCCP indeed reduced the MIC of tetracycline for these strains in the same degree, indicating that Lsr2 did affect the efflux pump that was inhibited by CCCP. Besides, the CCCP non-related gene mab_1409c, a putative drug antiporter precursor was also investigated for its association with lsr2. The result demonstrated that the expression of mab_1409c was decreased in Δlsr2 mutant, indicating that lsr2 maintain the gene expression of this putative efflux pump. To the best of our knowledge, this is the first report indicating that lsr2 contributed to tetracycline resistance and the involved mechanism in mycobacteria. In conclusion, this study demonstrates that lrs2 is an important modulator that mediates many biological functions in M. abscessus.