Download PZase - UPCH

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
Cases
New cases
Deaths
=
=
=
=
2 billion
20 million
8 million per year
2 million per year
Source: Organization, W.H., Global Tuberculosis Control Surveillance, Planning, Financing. 2007.
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)
In Peru, TB control is improving but
MDR-TB is increasing
60,000
50,000
Pulmonary
TB
40,000
30,000
20,000
10,000
0
80
81
82
84
85
87
91
92
93
94
95
96
97
98
99
´00
'01
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
17.8
18
15.4
16
14
13.4
12
Multi-drug resistant
TB
10
8
RP
MDR P
6
4
2.4
3
2
0
1993
1996
1999
First line TB Treatment
Drugs
Isoniazid (H)
Ethambutol (E)
Rifampin (R)
Pyrazinamide (Z)
Effect
Rapid growing
Rapid growing
Semi-dormant
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
PZA Mode of action
Extracellular
bacilli
O
N
C
NH
PZA
N
2
HPOA
POA- + H+
Passive
diffusion
pyrazinamidase
conversion
Major
mechanism
of
resistance
Passive
diffusion
[POA-]
Acid pH
typical of an
inflamated
tissue
Defective
efflux
H+
NAD
Acidification
metabolism?
of cytoplasm
HPOA
Disruption of
membrane
energy and
function
Zhang, et al., 2004
PZA and pyrazinamidase
pncA gene (561bp)
Pyrazinamidase
(181aa)
β1
α1
β2
β3
α2
β4
α3
β5
α4
β6
PZA Major mechanism of resistance
Highly diverse
Mutations in pncA gene
Along the entire gene
Rare silent mutations
Amino acid substitutions in
pyrazinamidase
β1
α1
β2
β3
α2
β4
Loss of enzimatic acitivity
α3
β5
α4
β6
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
Alternate
mechanism
Mutations in regulatory
regions of PZase
expression
Mutations do not
likely affect PZase
structure
Levels of activity
rather than
yes/no
PZase
inactive
Mutations likely
affect PZase
structure
Alternate
mechanism
pncA mutants characterization in
PZA-resistant strains
Mutations
Novel
mutationes
DNA regions
Amino acid
number
1
50
100
150
pncA gene
Mutations:
–
–
–
–
22 missense (74%)
3 nonsense (11%)
5 insertions (5%)
4 deletions (4%)
Clustered
183
pncA cloning and Pzase expression
pET28a:: His6-PncA
E.coli BL21(DE3)pLys
Broth LB +
Kanamycin +
IPTG
Cells
Purification by affinity chromatografy
Cells rupture
Freezen and
sonication
Purity of the fractions
Tubes
10
11
12 13
14
M
15 16
Soluble portion
Affinity
chromatography
Column His-Trap
PZase elution with
60mM Imidazole
12% SDS-PAGE
Protein concentration and
dialysis - 10Kb AMICON
Estimation of wild-type PZase
kinetic parameters
Lineweaver-Burk plot
1/Velocity (min/mM)
Velocity (mM/min)
Velocity vs. [Substrate]
2.0
1.5
1.0
0.5
0.0
0
5
10
15
20
y = 1.2131x + 0.5176
R2 = 0.9962
10
Slope = Km/Vmax
8
6
4
Intercept = 1/Vmax
2
0
0
1
2
3
4
1/PZA (1/mM)
PZA (mM)
Activity
Km
kcat
Effic
mM[POA].uM -1
[PZase].min-1
mM
min-1
mM . Min-1
0.41
2.34
622
265
5
6
7
Kinetic parameters of PZase
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
MBS = Metal binding site
AS = Active site
Low
efficiency
Some
efficiency
High
efficiency
100 200 300 400
0
0
200 400 600 800
PZA MIC (ug/ml)
0
1000
PZA + PZase
R = -0.92
P = <0.00001
0
R = -0.60
P = 0.038
0
R = -0.63
P = 0.0274
Efficiency (min-1)
100 200 300 400
Efficiency (min-1)
100 200 300 400
Pzase efficiency correlate with
susceptibility parameters
20
40
60
80
%Growth in BACTEC
100
[PZA-PZase]
Km
Weak
Wayne
Negative
POA + PZase
kcat
Efficiency
Positive
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
Control
PZase
6 h at 25˚C
EDTA dialysis (Ultrafiltration)
PZase
Activity
0.7
Control PZase
0.6
Chelated PZase
OD
0.5
0.4
0.3
10mM EDTA
0.2
20mM EDTA
40mM EDTA
0.1
0
1
2
3
4
Pzase activation
Chelated
Pzase
FeSO4.7H2O
+
30 min at 37˚C
Metal ions
+
3 min at 37˚C
PZase
Activity
PZA
Stop reaction
20% ferrous ammonium sulphate
0.1 M glycine–HCl buffer, pH 3.4
Absorbance at 450nm
CuCl2.2H2O
Cd(NO3)2.4H2O
MgSO4.7H2O
CoSO4.7H2O
MnSO4.H2O
CaCl2
Zn (NO3) 2 .6H2O
4000
Metal re-activation of metal-depleted
H37Rv PZase
2000
Re-activation
Zn
Mn
Mg
Fe
Cu
Co
Cd
Ca
No metal
0
Co > Mn> Zn > Cd
id
% Recovered activity = (metal-depleted PZase activity with
metal /metal-depleted PZase activity with no metal) x 100.
id
No synergism
Zn+Cd
Mn+Zn
Mn+Cd
Co+Zn
Co+Mn
Co+Cd
Cd
Zn
Mn
Co
No metal
0
2000
4000
Re-activation with combined metal
of metal-depleted H37Rv PZase
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
Effect of metals in the PZase activity of
metal-depleted mutant enzymes
3
2
1
F94L
K48T
id
id
H51R
Zn
non-ch
No metal
Ca
Cd
Co
Cu
Fe
Mg
Mn
0
.01 .02 .03 .04
id
PZase activity
Zn
non-ch
No metal
Ca
Cd
Co
Cu
Fe
Mg
Mn
0
Zn
non-ch
No metal
Ca
Cd
Co
Cu
Fe
Mg
Mn
0
.25
PZase activity
4
.5
(mM [POA] · µM-1 PZase · min-1)
Mutation
affecting the
metal-binding
site
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
Analysis of zinc in H37RV PZase by
Atomic Absorption Spectroscopy
Adicion estandar
0,25
Absorbancia
0,2
y = 0,03726x + 0,00423
R2 = 0,95627
0,15
0,1
0,05
0
-1
1
3
ug de Zn agregado
0.1 Zn ions per Pzase
molecule
5
7