MECHANISM OF INH AND RIFAMPICIN RESISTANCE IN TB

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Tuberculosis (TB) is one of the most important infections worldwide with an incidence of 8X106/year and a death rate of 2.5 x 106/year. The annual incidence rate has been decreasing in the US due to increasing wealth and improving living conditions with 30,145 cases and 2,968 deaths (a 10% mortality rate) in 1977. In 1991, NYC counted 3,673 new cases, a 42% increase over 1989, which has been fueled by the epidemic of HIV infection, drug abuse and homelessness. NYC accounts for 15% of all US cases and its incidence rate, 50.2 per 105, is five times the national rate. Due to these conditions, many new strains of TB have been identified that are resistant to one or more drugs that are used in treatment. SPECIFIC AIM 1: Isolation and Characterization Of the Gene(s) Responsible for INH Resistant M. tb. The target of INH or isoniazid in M. tb. is unknown, but metabolic pathways have been suggested including the mycolic acid and NAD recycling pathways. Recently, deletions in the catalase gene has been shown to be responsible for 15-25% of INH resistant strains of TB. We propose to identify the other genes involved in INH resistance. 1A. To clone and identify the DNA segments involved in isoniazid resistance in M. tb. by generating libraries from both isoniazid resistant and sensitive strains and identifying clones that result in transformation of a resistant strain to sensitive and vice versa. 1B. To enumerate the mechanisms of resistance that presently exist by utilizing various strains both as donor and recipients. SPECIFIC AIM 2: Identify Mechanisms in the M. tb Beta Subunit of RNA Polymerase that Confers Resistance to Rifampicin. The mechanism of action of rifampicin has been shown to inhibit the Beta subunit of RNA polymerase and RNA synthesis in E. coli.. Sequencing of the Beta subunit from rifampicin resistant colonies revealed many different mutations clustered in the center of the gene. We have recently isolated the gene for the Beta subunit from a rifampicin resistant clinical isolated of M. tb. and are in the process of sequencing the gene. As an extension to those studies, we propose to determine the mutations present in the Beta subunit of RNA polymerase form a series of rifampicin resistant and sensitive clinical isolates of M. tb. 2A. DNA will be extracted from rifampicin sensitive and resistant clinical isolated and the central portion of the gene will be amplified by PCR. These PCR products will be directly sequenced to identify the potential mutation(s) or genetic alteration(s). 2B. To definitively determine if base pair changes are responsible for resistance, well use primer directed mutagenesis or substitution of restriction fragments containing the base pair change into the normal gene, transformation of E. coli and selection on plates containing rifampicin. Specific Aim 3: Correlate MDR-TB Genes With RFLP Analysis and Develop Diagnostics For Rapid Assessment of MDR- TB: 3A. To determine whether the correlation between deletion of the catalase gene and INH resistance is a useful tool for rapid identification of resistant clinical isolates using polymerase chain reaction (PCR) and to develop PCR methods for detection of INH resistance based on the genetic changes found in specific aim 1.. 3B. We will develop PCR methods to rapidly identify rifampicin resistant TB from clinical isolates. 3C. To classify the clinical isolates we are characterizing in specific aims 1 and 2 and multi-drug resistant strains, by RFLP analysis for the insertion sequence, IS6110, and to correlate with drug resistance patterns.