Mycobacterium fortuitum is an important human pathogenic NTM, which resists stress conditions inside macrophages by exploitation of specific genes. TnphoA-based transposon mutagenesis was employed to identify membrane genes responsible for survival of M. fortuitum under such stress conditions. A library of about 450 mutants was constructed after electroporation of vector pRT291 into wild-type M. fortuitum. On the basis of blue color development and alkaline phosphatase assay, 20 mutants were shortlisted to screen for growth and survival under acidic stress at pH 6.5, 5.5, 4.5, and 3.5. Mutant MT727 showed reduced growth and survival under acidic stress. The acid susceptible mutant MT727 was subjected to other in vitro stress conditions prevalent inside macrophages including oxidative, nutrient starvation and nitrosative stress. However, the mutant showed no appreciable difference in growth behavior under oxidative, nutrient starvation and nitrosative stress conditions in comparison to the wild type. Genomic and bioinformatics analysis of MT727 led to identification of putative ribosomal maturation factor RimP of M. fortuitum to be affected by mutagenesis, showing closest homology to M. abscessus RimP. In silico functional interaction of RimP protein using STRING database showed its interaction with proteins of ribosomal assembly and maturation. Results indicate role of rimP gene in survival of M. fortuitum under acidic stress conditions which may be further explored for use as a potential drug target against M. fortuitum and other mycobacterial infections.
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Financial support in the form of research grant provided by Department of Science and Technology, Science and Engineering Research Board (DST-SERB) Project (SR/FT/LS-117/2012) to RS is gratefully acknowledged. We also acknowledge Jaypee University of Information Technology, Waknaghat, Solan (H.P.) administration for providing research facility and fellowship to Ms. Poonam. Authors are thankful to CDRI, Lucknow, for providing the bacterial strains and plasmids.
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Poonam, Yennamalli, R.M., Bisht, G.S. et al. Ribosomal maturation factor (RimP) is essential for survival of nontuberculous mycobacteria Mycobacterium fortuitum under in vitro acidic stress conditions. 3 Biotech 9, 127 (2019). https://doi.org/10.1007/s13205-019-1659-y
- Mycobacterium fortuitum
- Acidic stress
- Transposon mutagenesis
- Drug target