Identification of Genes Involved in IN VIVO Virulence of Mycobacterium Fortuitum as Potential Drug Target

Abstract

Mycobacterium fortuitum is an important human pathogenic NTM, which resists stress conditions inside macrophages by exploitation of virulence specific genes. TnphoA-based transposon mutagenesis was employed to identify membrane protein-encoding genes responsible for virulence of M. fortuitum, as potential drug target. A library of about 5000 mutants was constructed after electroporation of plasmid pRT291 into wild-type M. fortuitum. On the basis of blue color development, and alkaline phosphatase assay, six mutants were shortlisted for in vivo virulence studies. Three mutants were obtained with virulence attenuation, of which mutant MT726 showed highest attenuation followed by mutant MT725 and MT727. Genomic and bioinformatics analysis of attenuated mutants led to identification of four novel M. fortuitum ORFs namely, short-chain dehydrogenase (Mfsdr) in mutant MT726, anthranilate synthase component I (MftrpE) and anthranilate synthase component II (MftrpG) in mutant MT725, and ribosomal maturation factor (MfrimP) in mutant MT727. Growth kinetics analyses of attenuated mutants suggested role of Mfsdr for survival under acidic stress, hypoxic stress, and nutrient starvation conditions; role of anthranilate synthase encoding gene MftrpE and MftrpG under acidic and hypoxic stress; and role of MfrimP under acidic, hypoxic and detergent stress conditions. Structural and functional characterization of most potent ORF Mfsdr was done using in silico approaches. MfSdr was predicted to be localized on membrane, using TMpred database. In silico functional interaction of short-chain dehydrogenase (SDR) protein, using STRING database predicted its interaction with proteins involved in mycolic acid synthesis pathway, indicating probable role of MfSdr in mycolic acid synthesis. Secondary structure of MfSdr generated using Robetta server showed presence of Rossmann fold which is a characteristic of short chain dehydrogenase/reductase protein family. Virtual screening of MfSdr structure as drug target, using molecular docking too