ISOLATION AND CLONING OF Mycobacterium transcriptional RESPONSE REGULATOR-A (MTRA) GENE FROM Mycobacterium tuberculosis

• Main Author: MASANG BAN BOLLY (NIM : 20510002); Pembimbing I : Dessy Natalia, Ph.D ; Pembimbing II : D, HENDRIKUS
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/15898
• Institution: Institut Teknologi Bandung

Tuberculosis (TB) remains a global health problem caused by Mycobacterium tuberculosis infection. Incidence, morbidity and mortality of TB infection in the world and in Indonesia are still high. There are 9 million of new cases with <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> almost 2 million mortality of TB around the world. Human Immunodefiency Virus infection, drug resistance, late diagnosis, and the natural characteristics of bacteria increase the number of M. tuberculosis cases. The infection occurs when some bacil are inhaled through aerosol and survive in the host alveolar macrophages. Activated alveolar macrophage will release a variety of oxidative <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> compounds, ROS (Reactive Oxygen Species) and nitrosative compounds (RNI, Reactive Nitric Intermediate), which have bactericidal effects against intracellular M. tuberculosis. In order to survive in the host macrophages, Mtb is able to <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> reprogram its gene expression. Moreover, M. tuberculosis has a proteasome activity, which is resistable to bactericidal effects of ROS and RNI. MtrA is a protein that plays an important role in the pathogenesis of tuberculosis in humans. MtrA has major roles in transcription activation, proliferation regulation, cell multiplication, inhibiting phagosome-lysosome fusion and as a substrate of M. tuberculosis proteasome. Understanding the role of MtrA in the protein degradation system of M. tuberculosis proteasome will provide clear understanding of tuberculosis pathogenesis in human. This study was aimed to isolate and clone the gene encoding MtrA from M. tuberculosis strains H37Rv and clinical isolates, Y, which was obtained from liquour cerebrospinalis of patients with meningitis tuberculosis,; and two isolates, R26 and R27, which were <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> MDR. Genes encoding MtrA of all isolates were amplified successfully by the Polymerase Chain Reaction (PCR) method using a forward primer HMTAR-F and a reverse primer HMTAR-R. Both primers were designed based on the nucleotide <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> sequence of M. tuberculosis mtrA (Genebank Accession number U01971.1). Gene fragments of mtrA (0,687 kb) from H37Rv strain and Y clinical isolate had been cloned into the vector pGEM-T and digested by XhoI and XbaI. However, we <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> could not obtain the fragment of mtrA gene from the vector due to DNA methylation of GATC sequences on XbaI restriction site. The sequencing results of the mtrA gene from H37Rv and Y clinical isolate showed 687 bp encoding 228 amino acids. Furthermore, the mtrA gene sequence from R26 and R27 isolate showed 680 bp and 682 bp, respectively. Sequence homology analysis from all samples showed 100% homology with that of mtrA in the Genebank database. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> There was a silent mutation C261T, which was not change Lys-85 from the Y clinical isolate and there was no mutation on the mtrA gene from the R26 and R27 clinical isolates. These results implied that the MtrA protein is still active in regulating genes expression of MDR M. tuberculosis and meningococal tuberculosis.