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

Abstract

BackgroundThe presence of four mammalian cell entry (mce) operons in Mycobacterium tuberculosis suggests the essentiality of the functions of the genes in these operons. The differential expression of the four mce operons in different phases of in vitro growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.Here we investigate the extent of polymorphism in eight genes in the mce1 and mce4 operons of M. tuberculosis 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.ResultsWe discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of mce1 and mce4 operons revealed that yrbE1A [Rv0167] was most polymorphic in mce1 operon while yrbE4A [Rv3501c] and lprN [Rv3495c] had the highest number of SNPs in the mce4 operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to M. tuberculosis 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.ConclusionsOur results show that SNPs in the coding sequences of mce1 and mce4 operons in clinical isolates can be significantly high. Moreover, mce4 operon is significantly more polymorphic than mce1 operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in mce1 operon and synonymous substitutions are more in mce4 operon. In silico modeling predict that nonsynonymous SNP at mce1A [Rv0169], a virulence gene could play a pivotal role in causing functional changes in M. tuberculosis that may reflect upon the biology of the bacteria.

Analysis of expression profile of mce operon genes (mce1, mce2, mce3 operon) in different Mycobacterium tuberculosis isolates at different growth phases

“…The significant difference was observed in the expression profile of different mce operon in all the three M. tuberculosis isolates (MDR1, MDR2, sensitive isolates) at both growth phases, indicating complex regulation of mce operon genes expression. Pasrich et al 18 investigated the extent of polymorphism in eight genes in the mce1 and 4 operon in 112 clinical isolates of M. tuberculosis varying in their drug susceptible profile (more polymorphic mutations in mce genes in the drug sensitive isolate as compared to resistant isolates). Since drug resistance provides an extra edge to resistant isolates, the more expression (as in present investigation) or polymorphic mutation 18 of mce operon genes in susceptible isolates may also relate to virulence mechanisms after adaptability to the environment.…”

“…Moreover, M. tuberculosis grows under various stress conditions within cells, and these conditions may provide a hypermutable background, making resistance more likely. The genetic variations in mce operon genes ( mce1 and 4) have been studied in clinical isolates of M. tuberculosis and high level of polymorphism of operon genes has been reported in drug sensitive isolates as compared to resistant 18 . As the levels of expression of the mce operon genes in different M. tuberculosis isolates during different growth ( in vitro ) stages might be necessary for evaluating their putative functions, virulence mechanism, etc ., therefore, we explored the mce operon genes ( mce1 A, mce1 D, mce2 A, mce2 D, mce3 A, mce3 C) expression analysis in different M. tuberculosis isolates at early exponential and stationary growth phases ( in vitro ) by quantitative real-time PCR.…”

Single nucleotide polymorphisms in Mycobacterium tuberculosis and the need for a curated database

“…Other tools include ANNOVAR 73 and SVA (http://www.svaproject.org). In MTBC, such an approach has recently been used to look at variation in mammalian cell entry ( mce ) operons 74 . Ultimately however, polymorphisms predicted to effect protein function will have to be experimentally confirmed using other tools 75 .…”

TheMycobacterium tuberculosisrecombinant LprN protein ofmce4operon induces Th-1 type response deleterious to protection in mice

“…Moreover, LprN protein facilitates prolonged survival of M. tuberculosis in the host by maintaining inflammatory response and tissue damage during later phase of the disease. Our earlier report of high frequency of polymorphism observed in the lprN gene (Pasricha et al ., ) in conjunction with the report presented here strongly indicates that LprN is an immunogenic protein encoded by lprN gene of mce4 operon that is prone to genetic variation. There is a strong suggestion that the sequence polymorphisms should translate into modification and antigenic variation in the LprN protein and contribute to immune evasion and persistence of mycobacterial infection in the host.…”

“…Using bioinformatic tools, we studied genetic variations and sequence polymorphisms in the genes of the mce1 and mce4 operons. Of the 16 genes of the two operons in > 100 clinical isolates of M. tuberculosis , we observed that the yrbE1A gene of mce1 operon and yrbE4A and lprN genes of mce4 operon were most polymorphic (Pasricha et al ., ).…”