Multi-Dimensional Analysis Of Drug-Resistant Mycobacterium Tuberculosis

Tuberculosis remains one of the main threats to mankind causing 8 million newcases and 2 million deaths a year. The problem is becoming more critical with the emergence and spread of multidrug resistant(MDR) strains of M.tuberculosis, defined as resistant to at least isoniazid(INH) and rifampin(RIF). China is not only one of the 22 high-burden countries (HBCs) that collectively account for approximately 80% of the world's tuberculosis cases, but also the hotspot area of very high prevalence of MDR TB identified by WHO(4,5). The increasing prevalence of tuberculosis makes this disease a major focus for drug research and for the development of novel diagnostic tools. Mutations in the 81-bp rifampin resistance determining region(RRDR) and mutation V176F locating in the beginning of the ropB gene were analyzed by DNA sequencing of 86 Mycobacterium tuberculosis clinical isolates(72 resistant and 14 sensitive) from different parts of China. Sixty-five mutations of 22 distinct kinds, 21 point mutations and one insertion , were found in 65 of 72 resistant isolates. The most frequent mutations were in codon 531(41%), 526(40%), and 516(4%). Mutations were not found in seven(10%) of the resistant isolates. Six new alleles within the RRDR, along with five novel mutations outside the RRDR, are reported. None of isolates contained the V176 mutation. The information obtained from this study will assist not only in design of a suitable diagnostic method for rapid detection of MDR TB but also in the identification of any hot-spot regions in the country for proper implementation of TB control strategy. Based on information about ropB gene mentioned above. A moderate densityarray method that rapidly identified Mycobacterium tuberculosis rifampin resistant strains was developed. The method is based on detection of point mutations and other rearrangements in the rpoB gene region determining rifampin resistance. Rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Fifty-three rifampin-resistant M.tuberculosis and fifteen rifampin-sensitive M.tuberculosis were tested, results were concordant with those based on culture drug susceptibility testing and sequencing. Rifampin resistant clinical isolates were detected in as little as 1.5 hour after PCR amplication with visual results. It is demonstrated that oligonucleotide microchip is efficient, specialized technique to implement and could be used as a rapid method for detecting rifampin resistance to complement standard culture-based method.A secreted protein from culture supernatants of INH-resistant M.tuberculosis were purified by HPLC, and identified by MALDI-MS. The protein was trehalose-phosphate phosphatase involved in the biosynthesis of trehalose. Because trehalose is neither synthesized by, nor utilized in, mammalian cells, but is probably an essential molecule for the structural integrity of the mycobacterial cell wall, trehalose-phosphate phosphatase represent excellent potential target sites for chemotherapy against Mycobacterium tuberculosis.We used a proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry to compare the cellular protein composition of two INH resistant strains of Mycobacterium tuberculosis with two INH sensitive strains of Mycobacterium tuberculosis, in order to identify unique proteins of these strains. Emphasis was given to the identification of INH resistant strains specific proteins, because we consider these proteins to represent putative candidates for development of drug and diagnosis of tuberculosis.26 spots were exclusively detected in the INH resistant M.tuberculosis. Six spots of those were identified by MALDI-MS. The results in this study provide a basis for further develop novel anti-mycobacterial agents and diagnostic approach of drug-resistant tuberculosis. The differentially expressed genes in macrophages in response to infection with INH-resistant M.tuberculosis and INH-...