Molecular diagnosis of pyrazinamide resistance and molecular understanding of the pyrazinamidase functionality in Mycobacterium tuberculosis

Pyrazinamide, together with isoniazid and rifampin are the pillars of tuberculosis therapy. Pyrazinamide has permitted the currently short-course chemotherapy to develop because of its sterilizing property. The emergence of resistant strains are threatening the control of global tuberculosis. Rapid and effective methods to determine drug susceptibilities are urgently needed to direct tuberculosis treatment. New drugs are also needed as alternative drugs especially those with sterilizing activity like pyrazinamide. Better understanding of the molecular mechanism of action and resistance to pyrazinamide will help to design effective drugs.;The microbiological determination of pyrazinamide sensitivity is slow and the results are inconsistent because the test needs to be done at a low pH. To overcome these problems molecular methods have been developed however these methods are not accessible for low-income countries.;This research is presented in three papers. In the first paper, we improved the polymerase chain reaction--single strand conformational polymorphism (PCR-SSCP) for the detection of pncA mutations directly from sputum samples. The PCR-SSCP results were compared with the Bactec microbiological test. The capacity of PCR-SSCP to detect resistant strains was 85% (17/20) (sensitivity) and the capacity to detect susceptible strains was 92% (12/13) (specificity) in approximately 1 day.;In the second paper, we selected clinical isolates from the first paper having missense pncA mutations and variable pyrazinamidase activity (Wayne test) for cloning. Kinetic parameters were measured in the expressed proteins. The mutant pyrazinamidases had diverse activity, ranging from no activity (metal-binding site directly affected) to activity similar to the wild-type pyrazinamidase. Values of kcat/K m correlated significantly with susceptibility parameters indicating that mutant pyrazinamidases may retain activity.;In the third paper, we studied the metal requirements of the recombinant-wild type and mutant pyrazinamidases selected from the second paper. Physical assays demonstrated that zinc is the physiologically relevant metal in pyrazinamidase. However, metals such as cobalt, manganese, zinc and cadmium all activated wild-type pyrazinamidase. Pyrazinamidases with mutations affecting the metal-binding site had not been stimulated by any metal while pyrazinamidases with mutation in other regions were activated as the wild-type pyrazinamidase.;The conclusions of these papers are: (1) Pyrazinamide resistance was detected by PCR-SSCP directly from smear negative and positive sputum samples. This method showed moderate sensitivity, had high specificity and was faster than the conventional assays. PCR-SSCP may be used as a rapid and specific screening method to determine pyrazinamide resistance. (2) Although, pncA mutations corresponded well to pyrazinamide resistance, our results demonstrated that some pyrazinamidases with mutations retain activity and their kinetic parameters could predict the level of resistance. (3) M. tuberculosis pyrazinamidase is a metalloenzyme, that contains zinc however in vitro it is activated additionally by cobalt, manganese and cadmium. Our findings revealed information on the molecular mechanisms of pyrazinamidase functionality and its dependence with metal when mutations are present.