Molecular studies and comparative genomic analysis related to the virulence genes of Mycobacterium tuberculosis strain H37Ra versus H37Rv

Mycobacterium tuberculosis (Mtb) is a leading cause of death in humans around the world. The Mtb 20S proteasome, an ATP-dependent protease, is a major cytosolic protein degradation system. The proteasome helps protect Mtb from the killing effect of nitric oxide, and the inhibition of proteasome activity diminishes growth and recovery from nitrosative stress. In continuing studies of this system, we analyzed the strain Mtb H37Ra through complete sequence analysis of the proteasomal accessory and substrate genes (mpa, panB, fabD, the paf operon), the housekeeping cytochrome c oxidase genes (ctaD, ctaC, ctaE), and the ITS regions. We hypothesized that the sequences of strains Mtb H37Ra and H37Rv are very similar and that the H37Ra could be a useful and a less hazardous model for proteasome-specific inhibitor drug assays. PCR conditions were optimized for all of the sequences under study. The addition of DMSO (10%) gave better PCR results for the paf operon. Further Real-time PCR analyses were carried out to test the amplification efficiency of mRNA transcribed from the mpa, panB, fabD, the paf operon, ctaD, ctaC, ctaE, and the ITS regions. These studies should help develop an understanding of the relation between efficiency % (doubling of the amount of DNA after each RT-PCR cycle), GC % and length of the template. Secondary structure analysis of paf suggested hairpin loop structures that could interfere with the polymerase. Upon sequencing and in silico analysis, a 100% sequence identity was observed for our genes of interest between the Mtb H37Ra and H37Rv strains. There was no one factor that controlled the amplification efficiency of the templates used in the study, and all of these, and perhaps other factors (unknown inhibitors) influence the amplification efficiency. We also studied the effect of varying concentrations (0.3 mM, 3.0 mM and 30 mM) of nitric oxide (NaNO2) to induce stress on prcA, prcB, mpa, panB, fabD, the paf operon, and ctaE RNA expression. The mRNA expression level for prcA and prcB were not significantly different up to 3.0 mM NaNO2. The paf operon showed down-regulation at 3.0 mM NaNO2, and the signal was very much less at 30 mM NaNO2. The mpa gene may be down-regulated slightly at 30 mM NaNO2. Significant changes were observed in the fabD and panB RNA levels at the 30 mM concentrations. The proteasome activity at higher nitric oxide stress levels could more likely be supported by Mpa, PrcA, PrcB, rather than by the Paf operon. Thus, Mpa, PrcA, and PrcB proteins look to be good targets for antimycobacterial therapy, as they are not down-regulated under high nitrosative stress conditions. These studies are useful to the understanding of the proteasomal function in Mtb.