The Use Of Rap-PCR In Studying Mycobacterium tuberculosis Intracellular Gene During Macrophage Infection

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes, which were specifically expressed, or upregulated during intracellular infection ofJ774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine macrophage cell line was infected with M. tuberculosis (H37Rv strain) at 10:1 multiplicity of infection (MOI). RNA wasdifferentially extracted from M. tuberculosis infecting J774 macrophage cell line. The control in this case was RNA from extracellular broth grown bacteria. Approximately 50 ng of RNA from intracellular derived bacteria and extracellular derived bacteria (control) were subjected to random arbitrarily primed PCR (RAP-PCR) using 50 primer combinations. Eleven differential RAP-PCR products were observed. All RAP-PCR products were cloned into pCR®2.1 and sequenced in order to determine the identity of the products. Four of the eleven products were derived from macrophage genes and another 4 products were derived from the M. tuberculosis rRNA genes (three 23S and one 16S rRNA). The 3 remaining RAP-PCR products were found to be mycobacterial genes other than ribosomal genes. The three products were genes encoding enzyme involving in a shikimate pathway, a putative carboxyphosphonoenolpyruvate phosphonomutase and a serine protease with homology to HtrA. Of the 3 mycobacterial genes other than ribosomal genes detected, none were specifically expressed during intracellular infection but competitive RT-PCR showed that aroF gene was upregulated two-fold in intracellular derived bacilli

Strain-Specific Differences in the Genetic Control of Two Closely Related Mycobacteria

The host response to mycobacterial infection depends on host and pathogen genetic factors. Recent studies in human populations suggest a strain specific genetic control of tuberculosis. To test for mycobacterial-strain specific genetic control of susceptibility to infection under highly controlled experimental conditions, we performed a comparative genetic analysis using the A/J- and C57BL/6J-derived recombinant congenic (RC) mouse panel infected with the Russia and Pasteur strains of Mycobacterium bovis Bacille Calmette Guérin (BCG). Bacillary counts in the lung and spleen at weeks 1 and 6 post infection were used as a measure of susceptibility. By performing genome-wide linkage analyses of loci that impact on tissue-specific bacillary burden, we were able to show the importance of correcting for strain background effects in the RC panel. When linkage analysis was adjusted on strain background, we detected a single locus on chromosome 11 that impacted on pulmonary counts of BCG Russia but not Pasteur. The same locus also controlled the splenic counts of BCG Russia but not Pasteur. By contrast, a locus on chromosome 1 which was indistinguishable from Nramp1 impacted on splenic bacillary counts of both BCG Russia and Pasteur. Additionally, dependent upon BCG strain, tissue and time post infection, we detected 9 distinct loci associated with bacillary counts. Hence, the ensemble of genetic loci impacting on BCG infection revealed a highly dynamic picture of genetic control that reflected both the course of infection and the infecting strain. This high degree of adaptation of host genetics to strain-specific pathogenesis is expected to provide a suitable framework for the selection of specific host-mycobacteria combinations during co-evolution of mycobacteria with humans

Optimization Of Rna Extraction In Mycobacterium Tuberculosis For Studying Intracellular Gene Expression

Mycobacterium tuberculosis is the leading cause of death due to infectious disease after Human immunodeficiency virus. There has been an upsurge in the incidence of tuberculosis since 1980s. In order to reverse this trend, there is need to understand the biology of the organism. This can be brought about by studying gene expression at transcriptional level. The success of this hinges on RNA of good quality. In this paper, five methods (hot phenol, sonication with guanidinium thiocyanate (GTC) solution, beadbeating method with Trizol, FastPrep machine with Divolab as detergent and GTC solution, and FastPrep machine with Trizol) of extracting RNA from bacteria were compared to find which of the method would be suitable for mycobacteria. The study found that physical method of lysing bacteria was necessary for extraction of RNA from mycobacteria. FastPrep machine gave the highest yield and also provided the speed necessary for optimum RNA extraction. FastPrep and Trizol as reagent for extraction of RNA was applied to macrophage infected with M. tuberculosis (H37Rv) after removing the macrophage RNA. We were able to demonstrate the expression of dnaK gene in both intracellular and broth grown bacilli. The expression of dnaK gene was found to be downregulated in macrophage compared to broth. African Journal of Clinical and Experimental Microbiology Vol. 10 (2) 2009: pp. 64-7