PIRAZINAMIDASA MYCOBACTERIUM TUBERCULOSIS
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Transcript of 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• Cases = 20 million• New cases = 8 million per year• Deaths = 2 million per year
Source: Organization, W.H., Source: Organization, W.H., Global Tuberculosis Control Surveillance, Planning, FinancingGlobal Tuberculosis Control Surveillance, Planning, Financing. 2007.. 2007.
• Areas with high prevalence
• Association HIV/AIDS – TB
• Increase in the number of multi-drug resistant tuberculosis (MDR-TB)
Factors threatening TB control
•Occurs after inappropriate treatment•Increasing number of compromised drugs (XDR-TB strains)
Isoniazid and rifampin
resistant strains
0
10,000
20,000
30,000
40,000
50,000
60,000
Tto. 7,000 5,000 6,000 8,000 13,000 13,836 38,000 52,500 53,000 51,372 48,074 47,498 47,062 46,223 41,730 39,790 38,269
Dx 16,011 21,925 21,579 22,792 24,438 24,702 38,000 52,500 53,000 51,372 48,074 47,498 47,062 46,223 41,730 39,790 38,269
80 81 82 84 85 87 91 92 93 94 95 96 97 98 99 ´00 '01
In Peru, TB control is improving but MDR-TB is increasing
1993 1996 1999
13.4
15.4
2.4
17.8
3
0
2
4
6
8
10
12
14
16
18
RP
MDR P
Pulmonary TB
Multi-drug resistant TB
First line TB Treatment
Drugs Effect
Isoniazid (H) Rapid growingEthambutol (E) Rapid growingRifampin (R) Semi-dormantPyrazinamide (Z) Semi-dormant/acid pH
Sterilizing activity
Reduction 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
N
N
C NH
O
2
Passive diffusion
Defectiveefflux
Passive diffusionpyrazinamidase
conversion
POA- + H+ HPOA
NAD metabolism?
Acid pH typical of an inflamated
tissue
[POA-] HPOA
Acidification of cytoplasm
Disruption of membrane energy and function
PZA
H+
PZA Mode of action
Extracellular bacilli
Zhang, et al., 2004
Major mechanism
of resistance
PZA and pyrazinamidase
α4 β6α2 β4 α3 β5β1 α1 β2 β3
pncA gene (561bp)
Pyrazinamidase (181aa)
PZA Major mechanism of resistance
α4 β6α2 β4 α3 β5β1 α1 β2 β3
Mutations in pncA gene
Amino acid substitutions in pyrazinamidase
Loss of enzimatic acitivity
Highly diverse
Along the entire gene
Rare silent mutations
PZA resistance and pncA mutations
Mutations (72 – 98%)
PZaseno activity
PZaseinactive
PZaseactive
PZaseactive
Mutations likely affect PZase
structure
Mutations do not likely affect
PZase structure
Alternate mechanism
Mutations in regulatory regions of
PZase expression
Alternate mechanism
Levels of activity rather than
yes/no
No mutations (2 – 28%)
pncA gene in PZA resistant strains
pncA mutants characterization in PZA-resistant strains
Mutations: – 22 missense (74%) – 3 nonsense (11%)– 5 insertions (5%)– 4 deletions (4%)
1 50 100 150 183
DNA regions
Amino acid number
Mutations
pncA gene
Novel mutationes
Clustered
pET28a:: His6-PncA
E.coli BL21(DE3)pLys
Broth LB + Kanamycin + IPTG
pncA cloning and Pzase expression
Cells
Purification by affinity chromatografy
Soluble portion
Column His-Trap
Cells rupture Freezen
and sonication
PZase elution with 60mM Imidazole
Tubes 10 11 12 13 14 M 15 16
12% SDS-PAGE
Protein concentration and dialysis - 10Kb AMICON
Purity of the fractions
Affinity chromatography
Estimation of wild-type PZase kinetic parameters
Activity Km kcat Effic
mM[POA].uM -1
[PZase].min-1
mM min-1 mM . Min-1
0.41 2.34 622 265
Lineweaver-Burk ploty = 1.2131x + 0.5176
R2 = 0.9962
0
2
4
6
8
10
0 1 2 3 4 5 6 7
1/PZA (1/mM)
1/V
elo
city
(m
in/m
M)
Velocity vs. [Substrate]
0.0
0.5
1.0
1.5
2.0
0 5 10 15 20
PZA (mM)
Vel
oci
ty (
mM
/min
)
Slope = Km/Vmax
Intercept = 1/Vmax
Km Kcat
Efficiency Mutated region
mM min-1 mM . Min-1
H51R Und 0.00 0.00 MBS
D49N 0.6 0.19 0.3 MBS
T135P 0.9 9.3 12.4 Close AS
G78C 1.4 110 80 Loop
D24D 1.1 133 128 Loop
D12A 2.4 478 161 Close AS
F94L 0.8 161 208 Close AS
Y34D 2.3 514 219 Loop
D12G 3.6 821 226 Close AS
Wild type 2.1 574 280 --
K48T 1.5 536 377 Close MBS
Kinetic parameters of PZase
Low efficiency
Some efficiency
High efficiency
MBS = Metal binding siteAS = Active site
010
020
030
040
0
Effi
cien
cy (
min
-1)
0 200 400 600 800 1000
PZA MIC (ug/ml)0
100
200
300
400
Effi
cien
cy (
min
-1)
0 20 40 60 80 100
%Growth in BACTEC
010
020
030
040
0
Effi
cien
cy (
min
-1)
Positive Weak Negative
Wayne
R = -0.63P = 0.0274
R = -0.60P = 0.038
R = -0.92P = <0.00001
Pzase efficiency correlate with susceptibility parameters
PZA + PZase [PZA-PZase] POA + PZase
Km
Efficiency
kcat
Theoretical model of M. tuberculosis H37RV 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
Pzase chelation
80mM EDTA Pzase
6 h at 25˚C
EDTA dialysis (Ultrafiltration)
Control PZase
40mM EDTA20mM EDTA
10mM EDTA
Control PZase
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 2 3 4
OD
PZaseActivity
Chelated PZase
Pzase activation
Chelated Pzase
Metal ions
PZaseActivity
30 min at 37˚C
FeSO4.7H2O
CuCl2.2H2O
Cd(NO3)2.4H2O
MgSO4.7H2O
CoSO4.7H2O
MnSO4.H2O
CaCl2
Zn (NO3) 2 .6H2O
PZA3 min at 37˚C
Stop reaction20% ferrous ammonium sulphate 0.1 M glycine–HCl buffer, pH 3.4
Absorbance at 450nm
+
+
02000
4000
% R
eco
vere
d a
ctiv
ity
No m
eta
l
Ca
Cd
Co
Cu
Fe
Mg
Mn
Zn
id
Metal re-activation of metal-depleted H37Rv PZase
% Recovered activity = (metal-depleted PZase activity with metal /metal-depleted PZase activity with no metal) x 100.
Co > Mn> Zn > Cd
Re-activation
020
0040
00
% R
ecov
ered
act
ivity
No
met
al Co
Mn
Zn
Cd
Co+
Cd
Co+
Mn
Co+
Zn
Mn+
Cd
Mn+
Zn
Zn+
Cd
id
Re-activation with combined metal of metal-depleted H37Rv PZase
No synergism
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
0.2
5.5
PZ
ase
activ
ity
non-
ch
No
met
al Ca
Cd
Co
Cu
Fe
Mg
Mn
Zn
id
01
23
4
PZ
ase
activ
ity
non-
ch
No
met
al Ca
Cd
Co
Cu
Fe
Mg
Mn
Zn
id
0.0
1.0
2.0
3.0
4
PZ
ase
activ
ity
non-
ch
No
met
al Ca
Cd
Co
Cu
Fe
Mg
Mn
Zn
id
Effect of metals in the PZase activity of metal-depleted mutant enzymes
(mM [POA] · µM-1 PZase · min-1)
F94L K48T
H51R
Mutation affecting the metal-binding site
X-ray fluorescent spectroscopy of recombinant H37Rv PZase
PZase in TRIS
TRIS
Purification buffer
Purified and concentrated E. coli extract without plasmid
0.3 Zn ions per PZase molecule
Adicion estandar
y = 0,03726x + 0,00423
R2 = 0,95627
0
0,05
0,1
0,15
0,2
0,25
-1 1 3 5 7
ug de Zn agregado
Ab
sorb
anci
a
0.1 Zn ions per Pzase molecule
Analysis of zinc in H37RV PZase by Atomic Absorption Spectroscopy