Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach

Abstract

Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1 kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy.

Keywords: Antisense; Mycobacterium tuberculosis; Polycistronic operon; mce.