Fighting the oxidative assault: The Trypanosoma cruzi journey to infection Dra. Lucia Piacenza Dpto....

Fighting the oxidative assault:

The Trypanosoma cruzi journey

to infection

Dra. Lucia PiacenzaDpto. De Bioquimica, Facultad de Medicina yCenter for Free Radical and Biomedical ResearchMontevideo, Uruguay

Life cycle of Trypanosoma cruzi

Triparedoxina peroxidasa

Ascorbato peroxidasa

Triparedoxina

Tripanotiona sintetasa

Fe-Superóxido Dismutasa

Adapted from “The Trypanosoma cruzi Proteome” Atwood III et al; Science Vol 309; 2005

Central rol of macrophages in the control of T. cruzi infections

Phagocytes: First line of defense against invading T. cruzi

Macrophage-T cruzi interactions

Villalta et al, 1984

8 min

Rapid activation of NADP(H) oxidase during phagocytosis

2 O2 + NADPH 2 O2

• – + NADP+ + H+

NBT

DAPINBT2+ + O2

•─ NBT+ + O2

2NBT+ F+ + NBT 2+

Activators: IFN-γ, TNF-α, IL-1β, IL-6,

pathogen-derived products

(viral, bacterial and protozoa)

Inhibitors: IL-4, IL-10, IL-13, TGF-β

Apoptotic cell removal

Inducible Nitric Oxide Synthase

● NO Nitric Oxide

NO2¯ Nitrite

Nitrogendioxide

● NO2

NO3¯

Nitrate

RNH2 L-Arginine

- 1e-

- 1e-

- 1e-

i NOS

Reactive NitrogenSpecies

Peroxidase/H2O2 (MPO)

- 5e-

Superoxide(radical anion)

+

HydrogenPeroxide

OH● HidroxilRadical

H2O2

2 H2O

O2

Reactive OxygenSpecies

NADPHoxidase

OH¯

SOD

+ 1e-

+ 1e-

+ 1e-

+ 1e-

O2●

_

peroxinitrite

●NO2 + CO3●¯

ONOO

ONOOH

●NO2 + OH●

H+ CO2

ONOOCO2¯

-

●NO2 + CO3●¯

Szabo, Ischiropoulos and Radi, Nature Reviews (2007)

Peroxynitrite toxicity

Intraphagosomal Oxidants

Resting Macrophage

2 hrs infection

Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)

Activated macrophage

2 hrs infection

Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)

Intraphagosomal Oxidants- “Our hypothesis”

Antioxidant Defense in T. cruzi

Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)

NADPH

NADP +

TRox

TRred

T(SH)2

TS2

TXNox

TXNred

TcCPXRed

TcCPXOx

ONOOH

NO2-

In vitro peroxynitrite-oxidereductase activity

of T. cruzi peroxiredoxins

TR = trypanothione reductase

T(SH)2= trypanothione

TXN = tryparedoxin

TcCPX= citosolic tryparedoxin peroxidase

10 6 M-1s-1

(10 3 M-1s-1) (10 3 M-1s-1)

Trujillo, M et al., J. Biol. Chem. 279, 34175 (2004)

0

20

40

60

80

100

0 100 200 300 400 500 600 700

ONOO- (µM)

ControlTcAPX

TcCPXTcMPX

[3 H]-

Th

ymid

ine

Inco

rpo

rati

on

(%

) TcCPXIC50 480

M

TcMPXIC50 400

M

IC50 250 M

In vivo peroxynitrite-oxidereductase activity

of T. cruzi TcMpx and tccpx peroxiredoxins

Piacenza et al., Biochem J 2008, 410: 359-368.

Control Control + peroxynitrite TcCPX + peroxynitrite

TcCPX T. cruzi overexpressers are

resistant to peroxynitrite cytotoxicity

Peroxynitrite-mediated protein oxidationProtection by peroxiredoxins

Immuno-spin trapping

Mason et al; 2002-2004

t1/2= months-years

Intraphagosomal peroxynitrite

Peroxynitrite-derived intraphagosomal-T. cruzi protein radical detection

Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

Immune spin-trap ofDMPO-protein nitroneadducts

Intraphagosomal peroxynitrite

Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

Peroxynitrite-derived intraphagosomal-T. cruzi protein radical detection

OH

R

Tyrosine

R

O•

Tyrosyl radical

•NO2

R

OHNO2

3-NO2-tyrosine

ONOO-

•OH + •NO2

H+

O NH2H2N

C

O

OCH3

H

H2 N

C

O

OCH3

O NH•OH/• NO2

CO3●-

Dihydrorhodamine Rhodamine

T. cruzi CPX overexpressers

Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

Intracellular amastigote oxidant detection

T. cruzi CPX overexpressers

0

20

40

60

80

100

120

Infe

cció

n

(%)

Tc CPXWild type

O2●- ONOO-

O2●-

*

ONOO-

Macrophage infection

Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

T. cruzi CPX overexpressers

Balb-c infections

In vivo

Days post-infection

Try

po

ma

stig

ote

s /

50

fie

lds

Alvarez et al., J. Biol. Chem. 286, 6627 (2011)

Increase infectivity of TcCPX overexpressers

Does enzymes of T. cruzi antioxidant

network are involved in infectivity?

T. cruzi strains and virulence

Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)

Biochemical and molecular diversity of T. cruzi populations

TCC CL-WT

T. cruzi strain analysis

Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)

Virulent vs attenuated

Antioxidant enzyme contents

Specific antibodies used

TcCPX TcMPX TcTS TcCPX TcMPX TcTS

Con

teni

do r

ela

tivo

de e

nzim

a

3 days epimastigotes

Metacyclic trypomastigotes

Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)

Antioxidant enzyme evaluated:

TcCPXTcMPXTcAPXTcTSTcTR

Trypomastigotes/100 fields Trypomastigotes/100 fields

Re

lativ

e e

nzy

me

co

nte

nt

(me

tacy

clic

try

po

ma

stig

ote

) Antioxidant enzymes: virulence factors

Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)

First line of defenses: Superoxide Dismutases

2 O2• – + 2H+ O2 + H2O2

Cu/Zn Mn Fe

Most Eukaryotes

Prokaryotes

Trypanosomatids

T. cruzi contains 4 isoforms : SOD-A/C mitochondrial SODB-1 Cytosol SODB-2 Glycosomes

Fe-Superoxide Dismutases

Different cellular functions?

TcFeSODB TbFeSODA

NO

2-T

yr d

ete

ctio

n

Inactivation due to Tyrosine nitrationspecific residues

0 250 500 750 1000 1250

0

20

40

60

80

100

% A

ctiv

ity

[ONOO-] (M)

TbFeSODATcFeSODB

Different enzyme sensitivities againstperoxynitrite-dependent inactivation

Cytosolic

Mitochondrial

Studies with recombinant enzymesMitochondrial Cytosolic

Mitochondrial FeSOD-A and cell signaling

SIN-1: gives intracellular and equimolar fluxes of NO and O2-

SOD-A inhibits peroxynitrite formation efficiently eliminating O2•- radicals

Intracellular probe oxidationParasite viability

Role of FeSODs in cellular signaling and survival

Programmed cell death in T. cruzi

TUNEL staining

Death stimuli: Fresh human serum (FHS):

Control FHS CTL HIS FHS

DNA fragmentation

Phosphatidyl serine exposure

Ctl

FHS

Piacenza et al., Proc. Natl. Acad. Sci. U.S.A. 98, 7301 (2001)Irigoín, F et al., Biochem. J (418)-595, 2009

Complement activation

Ca2+ influx and mitochondrial

dysfunction

Mitochondrial FeSOD-A and cell signaling

Control TcSODA

IHS FHS HIS FHSbp

Mitochondrial Fe-SODA overexpression

Piacenza et al., Biochem. J. 403, 323 (2007)

CL-Brener + DMNQ (superoxide generator)

Intramitochondrial O2- detection: MitoSOX oxidation

Mitochondrial FeSOD-A and cell signaling

Smith and Murphy, 2003

HEMito-Hidroetidio

(Mitosox)

Mitochondrial FeSOD-A and cell signaling

Parasite mitochondrial-O2- production triggers programmed cell death

Piacenza et al., Biochem. J. 403, 323 (2007)

Peritoneal macrophages

Souza EM et al., 2003 Cell and Tissue Research.

In vivo hearth infection(14 dpi)In vitro cardiomiocytes infection

Apoptotic parasites in the vertebrate host

Chronic infection

Death stimuli unknown

Mitochondrial respiration

Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)

100 101 102 103 104MitoSOX

CtlDMNQ (1 mM)

SPM-NO (3 mM)

100 101 102 103 104MitoSOX

CL-Brener TcSOD-A

Mitochondrial FeSOD-A and cell signaling

Intramitochondrial NO-dependent MitoSOX oxidation:

SOD-A protects NO-derived mitochondrial parasite dysfunction

Control TcSODA

T. cruzi cardiomyocyte infection and detection of mitochondrial O2

•- production by MitoSOX oxidation in intracellular amastigotes

Cardiomyocyte-induced T. cruzi oxidative stress

Cardiomyocyte-induced T. cruzi oxidative stress

Control Activated

Cardiomyocyte-derived NO as a cellular mediator in the control of parasite proliferation

Conclusions, hypothesis and perspectives

Intraphagosomal peroxynitrite is a potent cytotoxinagainst internalized T. cruzi

Wild type strains of high virulence have increaseexpression of antioxidant enzymes

Levels of mitochondrial FeSOD could modulateparasite fate during chronic infection

Conclusions, hypothesis and perspectives

Evaluation of the behavior in chronic models of infection of stable overepressers of TcSODA, TcSODB,

TcCPX and TcMPX

In chronic infection we postulate NO as a key playerIn the control and progression of the disease

Evaluate cardiomyocyte-derived NO as a cell mediator for the signaling of amastigote apoptosis in vivo

Howard Hughes Medical Institute, USANational Institute of Health (NIH), USA

PEDECIBA (Uruguay)CONICYT (Uruguay)

PDT (Uruguay)

Biochemistry Department

Gonzalo Peluffo María Noel AlvarezAlejandra Martínez Madia TrujiloMartín HugoDolores PiñeyroCarlos RobelloRafael Radi

International Colaboration

Shane Wilkinson, UKMartin Taylor, UK

John Kelly, UK

Paola Zago, ArgentinaMiguel Basombrio,

Argentina

Different mitochondrial membrane potential in infective T. cruzi stagesobtained from infected cardiomiocytes

Activación Clásica

Activación Alterna

Activación Clásica

Eficiencia en el establecimineto de la infección

1- Capacidad de las células inmunes de establecer de forma temprana

una respuesta proinflamatoria con inducción de la iNOS en macrófagos

2- Niveles de enzimas antioxidantes en los tripomastigotas metacíclicos

3- Presencia de parásitos apoptóticos en el inóculo infectivo