Construction of lipU anti-sense knock out mutant of Mycobacterium fortuitum and its in-silico analysis to determine its potential role in pathogenesi

Abstract

Mycobacterium fortuitum is a non-tuberculous mycobacterium (NTM) causing various clinical symptoms such as cutaneous infections, respiratory infections, joint infections and disseminated infections in immuno-compromised patients. Since the mechanism of pathogenesis of M. fortuitum is not known, be that as it may, it has been reported that it taints macrophages in an indistinguishable way from that of Mycobacterium tuberculosis. Out of all the genes identified for the virulence of M. tuberculosis infection, lipU also called as cystathionine beta synthase, plays a major role coding for lipid hydrolyses necessary for the survival and virulence of M. tuberculosis and other mycobacteria. This lipase is very much similar to that of esterases of mycobacterium which implicates modification of the mycobacterial cell wall as an adaptive response to acid damage, also degrades host lipids during infection, making fatty acids available as building blocks for lipid biosynthesis. This gene helps in compensating with un-favorable acidic conditions in macrophages, where mycobacterium resides. With an aim to study the pathogenesis of M. fortuitum, multiple primer sets were designed from conserved regions of lipF of M. tuberculosis to amplify full length gene sequence of its homologue in M. fortuitum on the basis of the homology between the two genomic sequences (M. fortuitum and M. tuberculosis). Full length fragments were confirmed by sequencing. Longest sequence showing highest similarity with M. tuberculosis was submitted to GenBank database (Accession number - MH197269) as lipU of M. fortuitum ATCC 6841 homologue found using in-silico analysis. The study also followed in-silico anaylsis for sequence based homological studies and protein based homological studies using bio-informatics tools. To further confirm the role of lipU in virulence of M. fortuitum, an anti sense mutant was constructed by cloning the gene in pMV261 vector followed by electroporation into the wild type M. fortuitum. This construct can be further used to establish the role of this gene in pathogenesis of M. fortuitum which may act as a potential drug target against the infection caused by the bacteria.