The effect of pncA expression and POA efflux rate in the level of PZA-resistance

描述（由申请人提供）：背景和理由。吡嗪酰胺是最重要的抗结核药物之一，因为它缩短了结核病的治疗过程。它仍然是对抗结核分枝杆菌潜伏期最有效的药物，但其耐药机制尚不完全清楚。与异烟肼或利福平等其他抗结核一线药物相比，吡嗪酰胺的研究很少，因为：(1)它专门用于结核病治疗，即没有很高的利润；(2)它在检测微生物敏感性方面存在固有的困难，特别是pH值的微妙平衡和接种浓度范围小。然而，它在治疗结核病方面具有巨大的社会和临床意义。PZA是一种前药，需要通过结核分枝杆菌吡嗪酰胺酶转化为吡嗪酸。这种分子通过一个低效的、不具特征的外排泵输送到外部环境，在那里它被质子化。质子分子被动地返回到杆菌中并释放一个质子，导致细胞质酸化和致命的膜运输中断。当这个循环未完成时，就会产生对PZA具有抗性的菌株。大多数pza抗性菌株不能启动这一循环，因为它们具有由编码基因（pncA）突变引起的无功能吡嗪酰胺酶。与此相反，由于我们之前的NIH R03拨款，我们之前证明了pncA突变的吡嗪酰胺酶的酶活性仅解释了结核分枝杆菌pza耐药水平的30%。其他改变吡嗪酸循环的功能因子应该参与更复杂的pza耐药机制。例如，吡嗪酸流出速率的改变可能影响细胞质中质子的积累；或者即使pyrazinamidase的功能是合适的，但它也可能没有表达到适当的水平以保证PZA的完全水解。假设。结核分枝杆菌对PZA的耐药水平受吡嗪酰胺酶活性以外的其他因素调节。PZA耐药的机制可能有：(1)吡嗪酸外排泵的改变（由其编码基因的突变介导）可降低或增加杆菌外泵送吡嗪酸的速率，使其低于或高于正常值；(2)pncA基因表达水平的改变可降低PZA在杆菌胞质中转化为吡嗪酸的总体速率。在这两种情况下，无论pncA基因和活性吡嗪酰胺酶是否存在，菌株都对PZA具有抗性。方法。我们计划了解吡嗪酰胺酶动力学参数、吡嗪酸流出率、吡嗪酰胺通量（收入）率以及pncA基因表达水平对PZA抗性水平的总体和调整效应。为此，将研究pncA突变的耐pza结核分枝杆菌菌株。在重组酶中测定吡嗪酰胺酶的动力学参数。用PZA孵育后，通过测定体外和体内杆菌中吡嗪酸的含量来估计吡嗪酸的外排率。pncA基因的表达将通过逆转录和real - time PCR检测细菌中RNA的数量来获得。采用BACTEC 460TB、7H9培养MIC和Wayne活性3种不同的方法检测所有菌株对PZA的敏感性。此外，鉴于吡嗪酸外排泵在pza耐药机制中的潜在重要性，我们计划测试3个潜在的候选基因来编码POA外排泵，并通过生物信息学研究合理选择。为此，我们计划敲除耻垢分枝杆菌中的这些基因，耻垢分枝杆菌是一种天然耐pza的分枝杆菌，具有高度活跃的POA外排泵。如果我们发现敲除后POA外排率降低，该基因将被转移到pza易感结核分枝杆菌菌株H37Rv中。将分析转化菌株的PZA抗性水平和吡嗪酸外排率，以验证转移基因与POA外排的关联。

DESCRIPTION (provided by applicant): Background and justification. Pyrazinamide is one of the most important antituberculous drugs because it shortened the course of treatment for tuberculosis. It is still the most effective drug against the latent stage of Mycobacterium tuberculosis but its mechanism of resistance is not completely understood yet. In contrast to other antituberculous first line drugs like isoniazid or rifampicin, pyrazinamide has been studied for few groups because: (1) it is used exclusively for tuberculosis therapy i.e. not represent a highly profit, and (2) it has intrinsic difficulties to test the microbiological susceptibility in particular the delicate balance of pH and small range of inoculums concentration. However, it has enormous social and clinical importance in the treatment of tuberculosis. PZA is a pro drug that needs to be converted to pyrazinoic acid by the M. tuberculosis pyrazinamidase. This molecule is transported by an inefficient and uncharacterized efflux-pump to the external environment where it is protonated. The protoned molecule returns to the bacilli passively and release a protonwhich cause cytoplasm acidification and lethal disruption of the membrane transport. The generation of strains resistant to PZA occurs when this cycle is uncompleted. Most of the PZA-resistant strain cannot initiate this cycle because they have no-functional pyrazinamidases caused by mutations in its coding gene (pncA). In contrast to this fact, thanks to our previous NIH R03 grant, we previously demonstrated that the enzymatic activity of the pyrazinamidases with pncA mutations explains only 30% of PZA-resistance level in M. tuberculosis. Other functional factors altering the cycle of the pyrazinoic acid should be involved in a more complex PZA-resistance mechanism. For example an altered efflux rate of the pyrazinoic acid may affect the accumulation of protons in the cytoplasm; or even though the pyrazinamidase function is appropriate, it may not be expressed in appropriate level to assure the complete hydrolysis of the PZA. Hypothesis. The level of resistance to PZA in M. tuberculosis is modulated by other factors than the pyrazinamidase activity. Alternate mechanisms of PZA resistance could be: (1) The alteration of the pyrazinoic acid-efflux pump (mediated by mutations in its encoding gene) that could decrease or increase the rate of pumping pyrazinoic acid outside the bacilli below or above its normal value, and (2) the alteration of the level of expression of the pncA gene that could reduce the rate of overall conversion of PZA into pyrazinoic acid in the bacilli cytoplasm. In both situations, the strains would be resistant to PZA regardless the presence of a normal pncA gene and active pyrazinamidase. Methods. We plan to understand the total and adjusted effects of the pyrazinamidase kinetic parameters, the pyrazinoic acid efflux rate, and the pyrazinamide flux (income) rate, and the level of expression of the pncA gene on the level of resistance to PZA. For this purpose, PZA-resistant M. tuberculosis strains with pncA mutations will be studied. The pyrazinamidase kinetic parameters will be measured in their recombinant enzymes. The pyrazinoic acid efflux rate will be estimated by measuring the pyrazinoic acid in the external and internal bacilli contents after incubation with PZA. The expression of the pncA gene will be obtained by measuring the amount of RNA in the bacilli by reverse transcription and real time PCR. All these strains have been tested for PZA susceptibility by 3 different methods: BACTEC 460TB, 7H9 culture MIC, and Wayne activity. Additionally and given the potential importance of the pyrazinoic acid efflux pump in the PZA-resistance mechanism, we plan to test 3 potential gene candidates to encode the POA efflux pump, rationally selected by bioinformatical studies. For this we plan to knock out these genes in M. smegmatis, a naturally PZA-resistant mycobacterium with a highly active POA efflux pump. If we found a reduction in the POA efflux rate after the knock out, that gene will be transferred into the PZA-susceptible M. tuberculosis strain, H37Rv. The transformed strain will be analyzed for its PZA level of resistance as well as for the pyrazinoic acid efflux rate to verify the association of the transferred gene with the POA efflux. PUBLIC HEALTH RELEVANCE: The current project proposes to obtain several pieces of evidence to have an integral and more complete understanding of the functional factors at the molecular level, related to the mechanisms of resistance to pyrazinamide in Mycobacterium tuberculosis. We plan to measure several parameters within a same sample of M. tuberculosis PZA-resistant and PZA- susceptible clinical strains: PZAse activity (Kcat, KM, Efficiency), POA efflux rate, level of pncA expression (the PZAse coding gene), to jointly explain the level of microbiological PZA resistance of the strains. This study will let us understand the individual contribution of each factor individually and adjusted on the complex mechanism of PZA resistance. Also, in this study we will carry out studies to characterize POA efflux pump-mediated PZA resistance in M. tuberculosis by using M. smegmatis as the model organism.