MECHANISM OF INH AND RIFAMPICIN RESISTANCE IN TB

结核病是世界范围内最重要的传染病之一。 发病率为8×106/年，死亡率为2.5×106/年。这个 美国的年发病率一直在下降，原因是 财富和改善生活条件30145例和2968例死亡 (10%的死亡率)。1991年，纽约市统计了3673例新病例， 比1989年增加了42%，这是由艾滋病毒流行推动的 感染、吸毒和无家可归。纽约市占美国总人口的15% 病例及其发病率为50.2/105，是全国 费率。由于这些情况，许多新的结核病菌株已经 已确定对一种或多种药物产生抗药性 治疗。特异性目标1：基因的分离和鉴定(S) 对耐异烟肼结核分枝杆菌负责。异烟肼或异烟肼的靶点 结核分枝杆菌。是未知的，但已经提出的代谢途径包括 霉菌酸和NAD循环途径。最近，删除了 过氧化氢酶基因已被证明与15%-25%的异烟肼有关 具有抗药性的结核菌株。我们建议确定其他涉及的基因 在异烟肼抵抗中。1A.克隆和鉴定涉及的DNA片段 结核分枝杆菌对异烟肼的耐药性。通过从两个库中生成库 异烟肼耐药株和敏感株的鉴定 导致抗性菌株转变为敏感和有害的 反过来说。1B.列举目前抗药性的机制 通过利用各种菌株作为捐赠者和接受者而存在。 特定目标2：确定结核分枝杆菌核糖核酸Beta亚基的机制 对利福平产生抗药性的聚合酶。它的作用机制 利福平的作用已被证明抑制RNA的Beta亚基 在大肠杆菌中聚合酶和RNA的合成。测试版的测序 利福平耐药菌落的亚基表现出许多不同 突变集中在基因的中心。我们最近分离出了 利福平耐药临床病例的β亚基基因 分离出结核分枝杆菌。正在对基因进行测序。作为一种 作为这些研究的延伸，我们建议确定存在的突变 在RNA聚合酶的Beta亚基中形成一系列利福平 结核分枝杆菌耐药和敏感的临床分离株。2a.DNA将会是 从对利福平敏感和耐药的临床分离株和 该基因的中心部分将通过聚合酶链式反应进行扩增。这些聚合酶链反应 将对产品进行直接测序以确定潜在突变(S) 或基因改变(S)。2B。最终确定碱基对是否 变化是造成抗性的原因，用好引子定向 含有碱基的限制性片段的突变或替换 配对转化为正常基因、大肠杆菌的转化及选择 放在含有利福平的盘子里。具体目标3：耐多药结核病相关基因 利用RFLP分析和开发快速评估MDR的诊断学- 结核病：3A。以确定删除 过氧化氢酶基因和异烟肼抗性是快速检测的有用工具 聚合酶链法鉴定临床分离的耐药菌株 并建立检测异烟肼耐药性的聚合酶链式反应方法 根据在特定目标1中发现的基因变化..3B。我们会 建立快速鉴定耐利福平结核病的聚合酶链式反应方法 临床分离株。3C。为了对临床分离株进行分类，我们是 具有特异性目标1和2以及多重耐药株的特征， 通过对插入序列IS6110进行RFLP分析，并将其与 有耐药模式。

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.