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

pncA表达和POA流出率对PZA抗性水平的影响

• 批准号: 7942080
• 负责人: Mirko Zimic
• 金额: $ 5.28万
• 依托单位: UNIVERSIDAD PERUANA CAYETANO HEREDIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7942080
• 关键词: Affect; Animal Model; Bacillus (bacterium); Candidate Disease Gene; Clinical; Code; Complex; Cytoplasm; Drug Design; Environment; Enzymes; Equilibrium; Future; Gene Expression; Gene Transfer; Generations; Genes; Genus Mycobacterium; Goals; Grant; Hydrolysis; Income; Individual; Kinetics; Knock-out; Knowledge; Measures; Mediating; Methods; Molecular; Mutation; Mycobacterium tuberculosis; Normal Range; Pharmaceutical Preparations; Predisposition; Prodrugs; Proteins; Protons; Pump; Pyrazinamide; Pyrazinamide resistance; RNA; Recombinants; Resistance; Reverse Transcription; Rifampin; Sampling; Staging; Testing; Time; Transmembrane Transport; Transportation; Tuberculosis; United States National Institutes of Health; efflux pump; isoniazid; public health relevance; pyrazinamide deamidase; pyrazinoic acid; resistance mechanism; resistant strain; social; tuberculosis treatment

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.

描述（由申请人提供）：背景和依据。吡嗪酰胺因其能缩短结核病的疗程而成为最重要的抗结核药物之一。它仍然是治疗潜伏期结核分枝杆菌最有效的药物，但其耐药机制尚未完全了解。与其他抗结核一线药物（如异烟肼或利福平）相比，吡嗪酰胺仅在少数几组中进行了研究，因为：（1）其仅用于结核病治疗，即不代表高利润，（2）其具有固有的困难来测试微生物敏感性，特别是pH值的微妙平衡和接种物浓度的小范围。然而，它在结核病的治疗中具有巨大的社会和临床重要性。PZA是一种前体药物，需要通过M.结核吡嗪酰胺酶该分子通过低效且未表征的流出泵输送到外部环境，在该外部环境中其被质子化。质子化的分子被动地回到杆菌中并释放质子，这导致细胞质酸化和膜运输的致命破坏。当这个周期未完成时，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，因为它们具有由其编码基因（pncA）突变引起的无功能吡嗪酰胺酶。与此相反，由于我们先前的NIH R 03资助，我们先前证明了具有pncA突变的吡嗪酰胺酶的酶活性仅解释了M中30%的PZA抗性水平。结核改变吡嗪酸循环的其他功能因子应该参与更复杂的PZA抗性机制。例如，吡嗪酸的流出速率改变可能影响质子在细胞质中的积累;或者即使吡嗪酰胺酶功能是适当的，它可能不能以适当的水平表达以确保PZA的完全水解。假说. M.结核病受吡嗪酰胺酶活性以外的其它因素调节。PZA耐药性的替代机制可能是：（1）吡嗪酸外排泵的改变（由其编码基因中的突变介导），其可以降低或增加将吡嗪酸泵出杆菌的速率低于或高于其正常值，和（2）pncA基因表达水平的改变可降低杆菌中PZA向吡嗪酸的总转化率细胞质在这两种情况下，无论是否存在正常的pncA基因和活性吡嗪酰胺酶，菌株都将对PZA具有抗性。方法.我们计划了解吡嗪酰胺酶动力学参数、吡嗪酸流出率和吡嗪酰胺通量（收入）率以及pncA基因表达水平对PZA抗性水平的总体和调整影响。为此，抗PZA M.将研究具有pncA突变的结核菌株。将在其重组酶中测量吡嗪酰胺酶动力学参数。通过测量与PZA孵育后外部和内部杆菌内容物中的吡嗪酸来估计吡嗪酸外排率。pncA基因的表达将通过逆转录和真实的时间PCR测量杆菌中RNA的量来获得。所有这些菌株已通过3种不同的方法测试PZA敏感性：BACTEC 460 TB、7 H9培养物MIC和韦恩活性。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，通过生物信息学研究合理选择。为此，我们计划敲除M中的这些基因。耻垢病是一种天然的抗PZA分枝杆菌，具有高活性的POA外排泵。如果我们发现敲除后POA外排速率降低，则该基因将被转移到PZA敏感的M中。结核菌株H37 Rv。将分析转化菌株的PZA抗性水平以及吡嗪酸外排速率，以验证转移基因与POA外排的关联。 公共卫生关系：目前的项目建议获得一些证据，以便在分子水平上对与结核分枝杆菌对吡嗪酰胺耐药机制相关的功能因子有一个完整和更完整的了解。我们计划在M的同一样本内测量几个参数。结核病PZA耐药和PZA敏感临床菌株：PZAse活性（Kcat、KM、Efficiency）、POA外排率、pncA表达水平（PZAse编码基因），共同解释菌株的微生物PZA耐药水平。这项研究将使我们了解每个因素的单独贡献，并对PZA耐药的复杂机制进行调整。此外，在这项研究中，我们将进行研究，以表征POA外排泵介导的PZA耐药性在M。结核分枝杆菌M.作为模式生物。

项目成果

Evaluation of a lens-free imager to facilitate tuberculosis diagnostics in MODS.

• DOI: 10.1016/j.tube.2015.12.001
• 发表时间: 2016-03
• 期刊: Tuberculosis
• 影响因子: 3.2
• 作者: L. Solís;J. Coronel;D. Rueda;R. Gilman;P. Sheen;M. Zimic

A multiple genome analysis of Mycobacterium tuberculosis reveals specific novel genes and mutations associated with pyrazinamide resistance.

• DOI: 10.1186/s12864-017-4146-z
• 发表时间: 2017-10-11
• 期刊: BMC genomics
• 影响因子: 4.4
• 作者: Sheen P;Requena D;Gushiken E;Gilman RH;Antiparra R;Lucero B;Lizárraga P;Cieza B;Roncal E;Grandjean L;Pain A;McNerney R;Clark TG;Moore D;Zimic M
• 通讯作者: Zimic M

Detecting Mutations in the Mycobacterium tuberculosis Pyrazinamidase Gene pncA to Improve Infection Control and Decrease Drug Resistance Rates in Human Immunodeficiency Virus Coinfection.

• DOI: 10.4269/ajtmh.15-0711
• 发表时间: 2016-12-07
• 期刊: The American journal of tropical medicine and hygiene
• 作者: Dudley MZ;Sheen P;Gilman RH;Ticona E;Friedland JS;Kirwan DE;Caviedes L;Rodriguez R;Cabrera LZ;Coronel J;Grandjean L;Moore DAJ;Evans CA;Huaroto L;Chávez-Pérez V;Zimic M
• 通讯作者: Zimic M

Nicotinamidase/pyrazinamidase of Mycobacterium tuberculosis forms homo-dimers stabilized by disulfide bonds.

结核分枝杆菌的烟酰胺酶/吡嗪酰胺酶形成由二硫键稳定的同型二聚体。

• DOI: 10.1016/j.tube.2014.08.008
• 发表时间: 2014
• 期刊: Tuberculosis (Edinburgh, Scotland)
• 作者: Rueda,Daniel;Sheen,Patricia;Gilman,RobertH;Bueno,Carlos;Santos,Marco;Pando-Robles,Victoria;Batista,CesarV;Zimic,Mirko
• 通讯作者: Zimic,Mirko