PIRAZINAMIDASA MYCOBACTERIUM TUBERCULOSIS

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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 - PowerPoint PPT Presentation

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  • PIRAZINAMIDASA MYCOBACTERIUM TUBERCULOSIS

  • TuberculosisTuberculosis is an airborne communicable disease caused by Mycobacterium tuberculosis

    80% of tuberculosis is pulmonar

    Infectious disease causing highest mortality worldwide

  • Tuberculosis: Global epidemiologyInfected = 2 billionCases = 20 millionNew cases = 8 million per yearDeaths = 2 million per yearSource: Organization, W.H., Global Tuberculosis Control Surveillance, Planning, Financing. 2007.

  • Areas with high prevalence

    Association HIV/AIDS TB

    Increase in the number of multi-drug resistant tuberculosis (MDR-TB)Factors threatening TB controlOccurs after inappropriate treatmentIncreasing number of compromised drugs (XDR-TB strains)Isoniazid and rifampin resistant strains

  • In Peru, TB control is improving but MDR-TB is increasingPulmonary TBMulti-drug resistant TB

  • First line TB TreatmentDrugs EffectIsoniazid (H)Rapid growingEthambutol (E)Rapid growingRifampin (R)Semi-dormantPyrazinamide (Z)Semi-dormant/acid pH

    Sterilizing activityReduction of TB treatment from 9 months to 6 months

  • 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

  • NNC NHO2Passive diffusionDefectiveeffluxPassive diffusionpyrazinamidase conversionPOA- + H+ HPOANAD metabolism?Acid pH typical of an inflamated tissue [POA-] HPOAAcidification of cytoplasmDisruption of membrane energy and functionPZAH+PZA Mode of actionExtracellular bacilliZhang, et al., 2004Major mechanism of resistance

  • PZA and pyrazinamidasepncA gene (561bp)Pyrazinamidase (181aa)

    Hoja1

    D12A

    D12GK48TG78C

    G24DY34DD49NH51RF94LT135P

    1123243546

    G78C

    Y71Y

    P62L

    C14GT76P

    D12AL86P

    D12GK48TP54LL116PH137P

    L4SQ10RG24DY34DD49NH51RF94LG105DW119LT135P

    1123243546

    1123243546

    Hoja2

    Hoja3

  • PZA Major mechanism of resistanceMutations in pncA geneAmino acid substitutions in pyrazinamidaseLoss of enzimatic acitivityHighly diverseAlong the entire geneRare silent mutations

    Hoja1

    D12A

    D12GK48TG78C

    G24DY34DD49NH51RF94LT135P

    1123243546

    G78C

    Y71Y

    P62L

    C14GT76P

    D12AL86P

    D12GK48TP54LL116PH137P

    L4SQ10RG24DY34DD49NH51RF94LG105DW119LT135P

    1123243546

    1123243546

    Hoja2

    Hoja3

  • PZA resistance and pncA mutationsMutations (72 98%)PZaseno activityPZaseinactivePZaseactivePZaseactiveMutations likely affect PZase structureMutations do not likely affect PZase structureAlternate mechanism Mutations in regulatory regions of PZase expressionAlternate mechanism Levels of activity rather than yes/noNo mutations (2 28%)pncA gene in PZA resistant strains

  • pncA mutants characterization in PZA-resistant strainsMutations: 22 missense (74%) 3 nonsense (11%)5 insertions (5%)4 deletions (4%) 1 50 100 150 183DNA regionsAmino acid numberMutationspncA geneNovel mutationesClustered

  • pET28a:: His6-PncAE.coli BL21(DE3)pLys Broth LB + Kanamycin + IPTGpncA cloning and Pzase expressionCells

  • Purification by affinity chromatografy Soluble portionColumn His-TrapCells rupture Freezen and sonicationPZase elution with 60mM Imidazole Tubes 10 11 12 13 14 M 15 16 12% SDS-PAGEProtein concentration and dialysis - 10Kb AMICONPurity of the fractionsAffinity chromatography

  • Estimation of wild-type PZase kinetic parametersSlope = Km/VmaxIntercept = 1/Vmax

    ActivityKmkcatEfficmM[POA].uM -1 [PZase].min-1mMmin-1mM . Min-10.412.34622265

    Grfico10

    8.5

    5.6666666667

    2.8333333333

    1.5454545455

    0.9444444444

    0.7083333333

    0.6666666667

    0.693877551

    0.7906976744

    1/PZA (1/mM)

    1/Velocity (min/mM)

    Lineweaver-Burk plot

    y = 1.2131x + 0.5176R2 = 0.9962

    Hoja1

    FechaProteinaCodigoaa_changeResultadosExperimentoPZA mMOD1OD2MeanValueRestaPOA mMPorcentaje_consumoConc PZasa mix (uM)Conc PZAse Rx (uM)Tiempo minVelocidad mMPOA/min1/PZA1/Veloc1/PZA_valor1/Veloc_valorvalidopendienteinterceptor2kmvmaxkcatmin_1kcat/kmactiv (du)activ (4mM PZA)comentarios

    11 September 2007JonH37RVwild type110.00.040.040.0400.0000.1270.1010.0000.000.0012.862.601.001.100.394043.71130610563675.11177.363.37

    11 September 2007JonH37RVwild type10.30.0450.0450.0450.0050.07290.1270.1010.0714.0014.004.0014.00

    11 September 2007JonH37RVwild type10.50.0490.0480.04850.00850.12240.1270.1010.1212.008.242.008.24

    11 September 2007JonH37RVwild type11.00.0530.0520.05250.01250.18180.1270.1010.1791.005.601.005.60

    11 September 2007JonH37RVwild type11.50.0550.0550.0550.0150.21140.1270.1010.2140.674.670.674.67

    11 September 2007JonH37RVwild type12.00.0560.0560.0560.0160.23110.1270.1010.2290.504.380.504.38

    11 September 2007JonH37RVwild type12.50.060.060.060.020.29110.1270.1010.2860.403.500.403.50

    11 September 2007JonH37RVwild type13.00.0610.0610.0610.0210.30100.1270.1010.3000.333.330.333.33

    11 September 2007JonH37RVwild type13.50.060.060.060.020.2980.1270.1010.2860.293.500.293.50

    11 September 2007JonH37RVwild type14.00.0630.0620.06250.02250.3280.1270.1010.3210.253.110.253.11

    12 September 2007JonH37RVwild type120.00.0410.040.040500.000.2160.1610.0000.00031.845.640.985.640.181093.9707769774193.889.360.40valores bajos

    12 September 2007JonH37RVwild type20.30.0410.040.040500.0000.2160.1610.0004.000.0028.917.140.904.050.14864.5433482035213.5110.300.40

    12 September 2007JonH37RVwild type20.50.0420.0410.04150.0010.0130.2160.1610.0142.0070.002.0070.00

    12 September 2007JonH37RVwild type21.00.0430.0420.04250.0020.0330.2160.1610.0291.0035.001.0035.00

    12 September 2007JonH37RVwild type21.50.0440.0420.0430.00250.0420.2160.1610.0360.6728.000.6728.00

    12 September 2007JonH37RVwild type22.00.0430.0420.04250.0020.0310.2160.1610.0290.5035.00

    12 September 2007JonH37RVwild type22.50.0470.0410.0440.00350.0520.2160.1610.0500.4020.00

    12 September 2007JonH37RVwild type23.00.0440.0440.0440.00350.0520.2160.1610.0500.3320.000.3320.00

    12 September 2007JonH37RVwild type23.50.0470.0460.04650.0060.0920.2160.1610.0860.2911.670.2911.67

    12 September 2007JonH37RVwild type24.00.0450.0450.0450.00450.0620.2160.1610.0640.2515.56

    12 September 2007JonH37RVwild type130.00.0420.0690.04200.000.3130.2310.0000.0016.883.570.981.930.281193.2355476802619.1629.880.73

    12 September 2007JonH37RVwild type30.30.0430.0430.0430.0010.0160.3130.2310.0144.0070.004.0070.00

    12 September 2007JonH37RVwild type30.50.0460.0460.0460.0040.06110.3130.2310.0572.0017.502.0017.50

    12 September 2007JonH37RVwild type31.00.0490.0480.04850.00650.0990.3130.2310.0931.0010.771.0010.77

    12 September 2007JonH37RVwild type31.50.0520.0530.05250.01050.15100.3130.2310.1500.676.670.676.67

    12 September 2007JonH37RVwild type32.00.0510.0510.0510.0090.1360.3130.2310.1290.507.780.507.78

    12 September 2007JonH37RVwild type32.50.0540.0540.0540.0120.1770.3130.2310.1710.405.830.405.83

    12 September 2007JonH37RVwild type33.00.0540.0540.0540.0120.1760.3130.2310.1710.335.830.335.83

    12 September 2007JonH37RVwild type33.50.0540.0540.0540.0120.1750.3130.2310.1710.295.830.295.83

    12 September 2007JonH37RVwild type34.00.0540.0540.0540.0120.1740.3130.2310.1710.255.830.255.83

    13 September 2007JonH37RVwild type140.00.0390.040.039500.000.6030.4510.00013.233.670.910.880.27602.4976540248684.5733.040.46

    13 September 2007JonH37RVwild type40.30.0440.0440.0440.00450.06260.6030.4510.0644.0015.564.0015.563.303.470.910.950.29638.1432583754670.0532.34

    13 September 2007JonH37RVwild type40.50.0450.0450.0450.00550.08160.6030.4510.0792.0012.732.0012.73

    13 September 2007JonH37RVwild type41.00.0490.050.04950.010.14140.6030.4510.1431.007.001.007.00

    13 September 2007JonH37RVwild type41.50.0580.0520.0520.01250.18120.6030.4510.1790.675.600.674.52

    13 September 2007JonH37RVwild type42.00.0540.0530.05350.0140.20100.6030.4510.2000.505.000.505.00

    13 September 2007JonH37RVwild type42.50.0540.0530.05350.0140.2080.6030.4510.2000.405.000.405.00

    13 September 2007JonH37RVwild type43.00.0660.0550.0550.01550.2270.6030.4510.2210.334.520.333.33

    13 September 2007JonH37RVwild type43.50.0520.0520.0520.01250.1850.6030.4510.1790.295.60

    13 September 2007JonH37RVwild type44.00.0540.0540.0540.01450.2150.6030.4510.2070.254.830.254.83

    13 September 2007GinaH37RVwild type150.00.050.0480.04900.001.3951.0510.00017.484.780.961.570.21199.8942809122127.706.160.16

    13 September 2007GinaH37RVwild type50.30.0590.0470.047-0.002-0.03-111.3951.051-0.0294.00-35.00

    13 September 2007GinaH37RVwild type50.50.0550.050.05250.00350.05101.3951.0510.0502.0020.002.0020.00

    13 September 2007GinaH37RVwild type51.00.0540.0560.0550.0060.0991.3951.0510.0861.0011.671.0011.67

    13 September 2007GinaH37RVwild type51.50.0520.0570.0570.0080.1181.3951.0510.1140.678.750.678.75

    13 September 2007GinaH37RVwild type52.00.0510.0560.0560.0070.1051.3951.0510.1000.5010.000.5010.00

    13 September 2007GinaH37RVwild type52.50.0550.060.05750.00850.1251.3951.0510.1210.408.240.408.24

    13 September 2007GinaH37RVwild type53.00.0750.0550.0550.0060.0931.3951.0510.0860.3311.67

    13 September 2007GinaH37RVwild type53.50.0570.0570.0570.0080.1131.3951.051