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PIRAZINAMIDASA MYCOBACTERIUM TUBERCULOSIS

PIRAZINAMIDASA MYCOBACTERIUM TUBERCULOSIS. Tuberculosis. Tuberculosis is an airborne communicable disease caused by Mycobacterium tuberculosis 80% of tuberculosis is pulmonar Infectious disease causing highest mortality worldwide. Tuberculosis: Global epidemiology. Infected = 2 billion

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PIRAZINAMIDASA MYCOBACTERIUM TUBERCULOSIS

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  1. PIRAZINAMIDASA MYCOBACTERIUM TUBERCULOSIS

  2. Tuberculosis • Tuberculosis is an airborne communicable disease caused by Mycobacterium tuberculosis • 80% of tuberculosis is pulmonar • Infectious disease causing highest mortality worldwide

  3. Tuberculosis: Global epidemiology • Infected = 2 billion • Cases = 20 million • New cases = 8 million per year • Deaths = 2 million per year Source: Organization, W.H., Global Tuberculosis Control Surveillance, Planning, Financing. 2007.

  4. Factors threatening TB control • Areas with high prevalence • Association HIV/AIDS – TB • Increase in the number of • multi-drug resistant • tuberculosis (MDR-TB) Isoniazid and rifampin resistant strains • Occurs after inappropriate treatment • Increasing number of compromised drugs (XDR-TB strains)

  5. In Peru, TB control is improving but MDR-TB is increasing Pulmonary TB Multi-drug resistant TB

  6. First line TB Treatment DrugsEffect Isoniazid (H) Rapid growing Ethambutol (E) Rapid growing Rifampin (R) Semi-dormant Pyrazinamide (Z) Semi-dormant/acid pH Sterilizing activity Reduction of TB treatment from 9 months to 6 months

  7. Pyrazinamide (PZA) • Only active against M. tuberculosis complex • Action against semi-dormant tubercle bacilli in acidic environments • Reduction of treatment time of (6 months) • Pro-drug converted by pyrazinamidase (PZase) in the active molecule • 30% of MDR-TB cases are resistant to (PZA) reported by INS based on the Wayne test

  8. PZA Mode of action Extracellular bacilli O N C NH Acid pH typical of an inflamated tissue 2 PZA POA- + H+HPOA N Passive diffusion Defectiveefflux Passive diffusion pyrazinamidase conversion [POA-]HPOA H+ Major mechanism of resistance NAD metabolism? Disruption of membrane energy and function Acidification of cytoplasm Zhang, et al., 2004

  9. PZA and pyrazinamidase pncA gene (561bp) Pyrazinamidase (181aa)

  10. PZA Major mechanism of resistance Highly diverse Along the entire gene Rare silent mutations Mutations in pncA gene Amino acid substitutions in pyrazinamidase Loss of enzimatic acitivity

  11. PZA resistance and pncA mutations pncA gene in PZA resistant strains No mutations (2 – 28%) Mutations (72 – 98%) PZase active PZase no activity PZase active PZase inactive Alternate mechanism Mutations in regulatory regions of PZase expression Mutations do not likely affect PZase structure Mutations likely affect PZase structure Levels of activity rather than yes/no Alternate mechanism

  12. pncA mutants characterization in PZA-resistant strains Mutations Novel mutationes DNA regions Amino acid number 1 50 100 150 183 pncA gene Mutations: • 22 missense (74%) • 3 nonsense (11%) • 5 insertions (5%) • 4 deletions (4%) Clustered

  13. pncA cloning and Pzase expression pET28a:: His6-PncA E.coli BL21(DE3)pLys Broth LB + Kanamycin + IPTG Cells

  14. Purification by affinity chromatografy Cells rupture Purity of the fractions Freezen and sonication Tubes 10 11 12 13 14 M 15 16 Soluble portion Affinity chromatography 12% SDS-PAGE Column His-Trap PZase elution with 60mM Imidazole Protein concentration and dialysis - 10Kb AMICON

  15. Estimation of wild-type PZase kinetic parameters Slope = Km/Vmax Intercept = 1/Vmax

  16. Kinetic parameters of PZase Low efficiency Some efficiency High efficiency MBS = Metal binding site AS = Active site

  17. Pzase efficiency correlate with susceptibility parameters R = -0.60 P = 0.038 R = -0.92 P = <0.00001 R = -0.63 P = 0.0274 PZA + PZase [PZA-PZase] POA + PZase Km kcat Efficiency

  18. Theoretical model of M. tuberculosisH37RV PZase • Nicotinamidase • 37% sequence identity with Pzase Pyrococcus horikoshii 999 • 6 beta sheets • 4 alpha helixs • Active site: • D8, A134, C138 • Metal-binding site: H51, H71, D49

  19. 80mM EDTA Pzase Control PZase Pzase chelation 6 h at 25˚C EDTA dialysis (Ultrafiltration) PZase Activity Chelated PZase

  20. Chelated Pzase Pzase activation + 30 min at 37˚C Metal ions + PZA 3 min at 37˚C PZase Activity Stop reaction 20% ferrous ammonium sulphate 0.1 M glycine–HCl buffer, pH 3.4 Absorbance at 450nm

  21. Metal re-activation of metal-depleted H37Rv PZase Re-activation Co > Mn> Zn > Cd % Recovered activity = (metal-depleted PZase activity with metal /metal-depleted PZase activity with no metal) x 100.

  22. Re-activation with combined metal of metal-depleted H37Rv PZase No synergism

  23. Effect of metals in the PZase activity of metal-depleted mutant enzymes (mM [POA] · µM-1 PZase · min-1) Mutations affecting the metal-binding site

  24. Effect of metals in the PZase activity of metal-depleted mutant enzymes (mM [POA] · µM-1 PZase · min-1) F94L K48T Mutation affecting the metal-binding site H51R

  25. X-ray fluorescent spectroscopy of recombinant H37Rv PZase Purified and concentrated E. coli extract without plasmid TRIS Purification buffer PZase in TRIS 0.3 Zn ions per PZase molecule

  26. Analysis of zinc in H37RV PZase by Atomic Absorption Spectroscopy 0.1 Zn ions per Pzase molecule

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