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