GLN TO LEU MUTATION AT CODON 513 OF rpoB GENE IN RIFAMPIN-RESISTEANT Mycobacterium tuberculosis L1

• Main Author: Syahrani (NIM 205 05 015) , Puti
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/6627
• Institution: Institut Teknologi Bandung

ABSTRACT: <br /> <br /> Tuberculosis (TB) is an infectious and contagious disease caused by Mycobacterium tuberculosis. This bacillus currently infected more than one-third of the worlds population. Our country, Indonesia, is the third country with largest number of TB cases in the world. One of the factors that make TB hard to eradicate is the emergence of M. tuberculosis drug resistance. Multidrug-resistant TB (MDR-TB) is defined as resistance to at least isoniazid (INH) and rifampin (RIF), two of the first-line anti-TB drugs. This resistance phenotype resulting from missense mutations in the genes codes for drug target or one that involved in drug-target interaction. Rifampin resistance is caused by mutations in the rpoB gene, which encodes B-subunit RNA polymerase (RNAP), especially in the 81- base pair RIF resistance determining region (codon 507-533), with the highest frequency at codon 526 and 531. These mutations lead to RIF losing its ability to inhibit transcription initiation process. Out of forty-two Bandung clinical isolate of MDR M. tuberculosis strains obtained by our research group of Biochemistry Division, ITB, there are MDR isolates that have mutation at codon 315 of katG gene conferring INH resistance but have no mutation at codon rpoB526 and rpoB531 described above. Hence, it is assumed that RIF resistance phenotype is caused by mutation that occurs at other sites. The purpose of this work is to determine the genotypic basis of RIF resistance in those MDR M. tuberculosis clinical isolates, and also to find the relationship between mutation and its resistance phenotype. <br /> <br /> The work consists of several steps: multiplex allele-specific (MAS) rpoB Polymerase Chain Reaction (PCR) and agarose gel electrophoresis, nucleotide sequencing, and in silico analysis. MAS-PCR and its electrophoresis was performed to confirm mutation at codon rpoB526 and rpoB531 of clinical isolates, using two outer primers (RF and RR) and one inner primer (R526 or R531), depending on the codon position. MAS-PCR result then confirmed by sequencing of PCR fragment harboring RIF resistance determining region (done by Macrogen Inc., Seoul, Korea). The sequences obtained were in silico analyzed by aligning the isolate and M. tuberculosis H37Rv wild-type strain sequences, using SeqManTMII and MegAlignTM DNASTAR program. This alignment was aimed to identify mutation in isolate sequences. In addition, we also performed protein modeling using Open-Source version of PyMOL program to find out the mutation effect in RNAP-RIF interaction. MAS-PCR and agarose gel electrophoresis of MDR M. tuberculosis L1 isolate showed two DNA bands either on rpoB526 (0.25 kb and 0.18 kb band) or rpoB531 (0.25 kb and 0.17 kb) MAS-PCR. Sequencing electrophoregram together with alignment of L1 isolate and M. tuberculosis H37Rv sequences using SeqManTMII and MegAlignTM DNASTAR showed a substitution mutation, adenine (A) to thymine (T), at position 1295 of rpoB gene, which is a second base of codon 513 (change CAA to CTA). In silico translation analysis also showed that CAA encoding glutamine (Gln) is mutated into CTA encoding leucine (Leu). PyMOL protein modeling showed that the mutation, Gln513Leu, increase the distance between the side chain and hydroxyl group of RIF from 2.63 A to 3.71 A. Sequencing and alignment results described above showed a mutation at another site of L1 isolate rpoB, which is located at codon 513 and we suggest as the cause of RIF resistance phenotype, consistent to previous works. Shorter DNA band (0.18 kb or 0.17 kb) amplified by rpoB526 and rpoB531 MAS-PCR using inner primer (R526 or R531) and outer primer RR could be explained based on sequencing result which reveal that those codons are wild-type alleles. Gln513 is known as a residue involved in RIF binding on RNAP B subunit by forming hydrogen bond between its polar side chain and hydroxyl group of RIF. This mutation has changed it to Leu, which has non polar side chain and causing the distance between the side chain and hydroxyl group of RIF become longer, as shown by PyMOL protein modeling. Those changes are strongly believed causing no hydrogen bond formed, that will reduce the binding affinity of RIF to RNAP. If codon 513 mutation is the only mutation in rpoB gene of MDR M. tuberculosis L1 isolate, then Gln513Leu alteration that gave rise to RIF failure in inhibiting RNAP in transcription process was strongly suggested as a factor for RIF resistance phenotype of the isolate. This resistance mechanism information may be useful as a consideration of novel drug design to overcome MDR-TB problems.