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

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

Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains

<p>Abstract</p> <p>Background</p> <p>The presence of four mammalian cell entry (<it>mce</it>) operons in <it>Mycobacterium tuberculosis </it>suggests the essentiality of the functions of the genes in these operons. The differential expression of the four <it>mce </it>operons in different phases of <it>in vitro </it>growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.</p> <p>Here we investigate the extent of polymorphism in eight genes in the <it>mce1 </it>and <it>mce4 </it>operons of <it>M. tuberculosis </it>from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform.</p> <p>Results</p> <p>We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of <it>mce1 </it>and <it>mce4 </it>operons revealed that <it>yrbE1A </it>[<it>Rv0167</it>] was most polymorphic in <it>mce1 </it>operon while <it>yrbE4A </it>[<it>Rv3501c</it>] and <it>lprN </it>[<it>Rv3495c</it>] had the highest number of SNPs in the <it>mce4 </it>operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to <it>M. tuberculosis </it>using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation.</p> <p>Conclusions</p> <p>Our results show that SNPs in the coding sequences of <it>mce1 </it>and <it>mce4 </it>operons in clinical isolates can be significantly high. Moreover, <it>mce4 </it>operon is significantly more polymorphic than <it>mce1 </it>operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in <it>mce1 </it>operon and synonymous substitutions are more in <it>mce4 </it>operon. <it>In silico </it>modeling predict that nonsynonymous SNP at <it>mce1A </it>[<it>Rv0169</it>], a virulence gene could play a pivotal role in causing functional changes in <it>M. tuberculosis </it>that may reflect upon the biology of the bacteria.</p