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.
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.
