MECHANISM OF INH AND RIFAMPICIN RESISTANCE IN TB

结核病（TB）是世界上最重要的感染之一， 发病率为8 × 106/年，死亡率为2.5 × 106/年。 的 美国的年发病率一直在下降， 财富和改善生活条件，30，145例和2，968例死亡 （1977年死亡率为10%）。 1991年，纽约市统计了3,673例新病例， 比1989年增加了42%，这是由于艾滋病的流行 感染、吸毒和无家可归。 纽约市占美国人口的15% 发病率为50.2/10万，是全国的5倍， 率 由于这些条件，许多新的结核病菌株已经被发现。 对一种或多种药物具有耐药性， 治疗 特定目的1：基因的分离和表征 负责INH耐药M。TB. INH或异烟肼的靶点 M. TB.是未知的，但代谢途径已被提出，包括 分枝菌酸和NAD再循环途径。 最近， 过氧化氢酶基因已被证明负责15-25%的INH 耐药结核菌株。 我们建议确定其他相关基因 INH抵抗。 1A. 克隆并鉴定所涉及的DNA片段 异烟肼耐药的M. TB.通过从这两个库中生成库， 异烟肼抗性和敏感菌株以及鉴定 导致抗性菌株转化为敏感菌株， 亦然 1B.为了列举目前 通过利用各种菌株作为供体和受体而存在。 具体目标2：确定M。tb RNA β亚基 赋予利福平抗性的聚合酶。 的机理 利福平的作用已被证明可抑制RNA的β亚基 聚合酶和RNA合成。coli.. Beta的排序 利福平耐药菌落的亚基显示了许多不同的 突变聚集在基因的中心。 我们最近分离出 来自利福平耐药临床试验的β亚基基因 分离的M. TB.正在进行基因测序 作为 扩展到这些研究，我们建议确定存在的突变 在RNA聚合酶的β亚基中形成一系列利福平 耐药和敏感临床分离株的M. TB. 2A. DNA将 从利福平敏感和耐药临床分离株中提取， 基因的中心部分将通过PCR扩增。这些PCR 将直接对产品进行测序，以识别潜在突变 或遗传改变。 2B. 为了确定碱基对 变化是负责抗性，以及使用引物定向 含有碱基的限制性片段的诱变或取代 pair转化为正常基因，转化E.大肠杆菌和选择 在含有利福平的平板上。 具体目标3：关联MDR-TB基因 利用RFLP分析和开发快速评估MDR的诊断方法- TB：3A。 为了确定删除 过氧化氢酶基因和INH抗性是一个有用的工具， 应用聚合酶链鉴定耐药临床分离株 建立了检测INH耐药的PCR方法 基于在特定目标1中发现的遗传变化。3B. 我们将 开发PCR方法，以快速识别利福平耐药结核病， 临床分离株。 3C. 为了对临床分离株进行分类， 特征在于特异性目标1和2以及多重耐药菌株， 通过对插入序列IS 6110的RFLP分析， 有抗药性

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.