Fighting the oxidative assault:
The Trypanosoma cruzi journey
to infection
Dra. Lucia Piacenza
Dpto. De Bioquimica, Facultad de Medicina y
Center for Free Radical and Biomedical Research
Montevideo, 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
2 O2 + NADPH  2 O2
•–
+ NADP+ + H+
Rapid activation of NADP(H) oxidase during phagocytosis
8
min
NBT
Villalta et al, 1984
DAPI
NBT2+ + O2•─
2NBT+
NBT+ + O2
F+ + NBT 2+
Inducible Nitric Oxide Synthase
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
Reactive Oxygen
Species
Reactive Nitrogen
Species
O2
RNH2
+ 1e-
NADPH
oxidase
i NOS
Superoxide O ● _
2
(radical anion)
+ 1e-
Hydrogen
Peroxide
●
SOD
-
ONOO
H2O2
Hidroxil
Radical
NO
Nitric
Oxide
peroxinitrite
NO2¯
Nitrite
- 1e- Peroxidase/H2O2
(MPO)
+ 1e-
ONOOH
- 5e-
- 1e-
CO2
H+
L-Arginine
ONOOCO2¯
●
OH¯+ OH●
NO2
+ 1e-
- 1e●
2 H2O
Nitrogen
dioxide
NO2 + OH●
●
NO2 + CO3●¯
NO3¯
Nitrate
Peroxynitrite toxicity
Szabo, Ischiropoulos and Radi, Nature Reviews (2007)
Intraphagosomal Oxidants
Resting Macrophage
2 hrs infection
Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)
Intraphagosomal Oxidants- “Our hypothesis”
Activated macrophage
2 hrs infection
Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)
Antioxidant Defense in T. cruzi
Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)
In vitro peroxynitrite-oxidereductase activity
of T. cruzi peroxiredoxins
NADPH
TRox
(10 3 M-1s-1)
(10 3 M-1s-1)
T(SH)2
TXNox
TcCPXRed
ONOOH
10 6 M-1s-1
NADP +
TRred
TS2
TXNred
TcCPXOx
NO2-
TR = trypanothione reductase
T(SH)2= trypanothione
TXN = tryparedoxin
TcCPX= citosolic tryparedoxin peroxidase
Trujillo, M et al., J. Biol. Chem. 279, 34175 (2004)
In vivo peroxynitrite-oxidereductase activity
of T. cruzi TcMpx and tccpx peroxiredoxins
[3H]-Thymidine Incorporation (%)
100
TcCPX
IC50 480 M
Control
TcAPX
TcMPX
TcCPX
80
TcMPX
IC50 400 M
60
40
IC50 250 M
20
0
0
100
200
300 400 500
ONOO- (µM)
600
700
Piacenza et al., Biochem J 2008, 410: 359-368.
TcCPX T. cruzi overexpressers are
resistant to peroxynitrite cytotoxicity
Control
Control + peroxynitrite
TcCPX + peroxynitrite
Peroxynitrite-mediated protein oxidation
Protection by peroxiredoxins
Immuno-spin trapping
t1/2= months-years
Mason et al; 2002-2004
Intraphagosomal peroxynitrite
Peroxynitrite-derived intraphagosomal-T. cruzi protein radical detection
Immune spin-trap of
DMPO-protein nitrone
adducts
Alvarez et al., J. Biol. Chem. 286, 6627 (2011)
Intraphagosomal peroxynitrite
Peroxynitrite-derived intraphagosomal-T. cruzi protein radical detection
ONOOH+
O•
OH
•OH
OH
•NO
+ •NO2
R
R
Tyrosine
Tyrosyl radical
NO 2
2
R
3-NO2-tyrosine
Alvarez et al., J. Biol. Chem. 286, 6627 (2011)
T. cruzi CPX overexpressers
O
H2N
NH2
•OH/•
2 HN
O
NH
NO2
H
C OCH3
O
Dihydrorhodamine
CO3
●
-
C OCH3
O
Rhodamine
Intracellular amastigote oxidant detection
Alvarez et al., J. Biol. Chem. 286, 6627 (2011)
T. cruzi CPX overexpressers
Wild type
Tc CPX
Infección (%)
120
100
80
60
*
40
20
0
O2●
-
ONOO-
O2●
-
ONOO-
Macrophage infection
Alvarez et al., J. Biol. Chem. 286, 6627 (2011)
T. cruzi CPX overexpressers
Increase infectivity of TcCPX overexpressers
Trypomastigotes / 50 fields
In vivo
Days post-infection
Balb-c infections
Alvarez et al., J. Biol. Chem. 286, 6627 (2011)
Does enzymes of T. cruzi antioxidant
network are involved in infectivity?
T. cruzi strains and virulence
Biochemical and molecular diversity of T. cruzi populations
Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)
T. cruzi strain analysis
Virulent vs attenuated
TCC
CL-WT
Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)
Antioxidant enzyme contents





TcCPX
TcMPX
TcAPX
TcTS
TcTR
Contenido relativo de enzima
Antioxidant enzyme evaluated:
TcCPX
TcMPX
TcTS
TcCPX
TcMPX
TcTS
3 days epimastigotes
Metacyclic trypomastigotes
Specific antibodies used
Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)
Relative enzyme content
(metacyclic trypomastigote)
Antioxidant enzymes: virulence factors
Trypomastigotes/100 fields
Trypomastigotes/100 fields
Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)
First line of defenses: Superoxide Dismutases
2 O2• – + 2H+
O2 + H2O2
Prokaryotes
Cu/Zn
Most Eukaryotes
Mn
Fe
Trypanosomatids
T. cruzi contains 4 isoforms : SOD-A/C mitochondrial
SODB-1 Cytosol
SODB-2 Glycosomes
Fe-Superoxide Dismutases
Studies with recombinant enzymes
100
Cytosolic
% Activity
80
60
TbFeSODA
TcFeSODB
40
20
Mitochondrial
0
0
250
500
750
1000
Cytosolic
TbFeSODA
NO2-Tyr detection
Mitochondrial
TcFeSODB
Inactivation due to Tyrosine nitration
specific residues
1250
-
[ONOO ] (M)
Different enzyme sensitivities against
peroxynitrite-dependent inactivation
Different cellular functions?
Mitochondrial FeSOD-A and cell signaling
SIN-1: gives intracellular and equimolar fluxes of NO and O2Parasite viability
Intracellular probe oxidation
140
300
200
100
0
0
5
SIN-1 (mM)
10
[3H]Thymidine incorporation (%)
Rhodamine 123
400
120
100
80
60
40
CL-Brener
20
TcSOD-A
0
0
2
4
SIN-1 (mM)
 SOD-A inhibits peroxynitrite formation efficiently eliminating O2•- radicals
6
Role of FeSODs in cellular signaling and survival
Programmed cell death in T. cruzi
Death stimuli: Fresh human serum (FHS):
Control
FHS
CTL HIS FHS
Ctl
FHS
TUNEL staining
DNA fragmentation
Complement activation
Phosphatidyl serine exposure
Ca2+ influx and mitochondrial
dysfunction
Piacenza et al., Proc. Natl. Acad. Sci. U.S.A. 98, 7301 (2001)
Irigoín, F et al., Biochem. J (418)-595, 2009
Mitochondrial FeSOD-A and cell signaling
Mitochondrial Fe-SODA overexpression
Control
bp IHS
FHS
TcSODA
HIS
FHS
Piacenza et al., Biochem. J. 403, 323 (2007)
Mitochondrial FeSOD-A and cell signaling
Intramitochondrial O2- detection: MitoSOX oxidation
Mito-Hidroetidio
HE
(Mitosox)
CL-Brener + DMNQ (superoxide generator)
Smith and Murphy, 2003
Mitochondrial FeSOD-A and cell signaling
Parasite mitochondrial-O2- production triggers programmed cell death
Piacenza et al., Biochem. J. 403, 323 (2007)
Apoptotic parasites in the vertebrate host
Chronic infection
In vitro cardiomiocytes infection
In vivo hearth infection(14 dpi)
Peritoneal macrophages
Death stimuli unknown
Souza EM et al., 2003 Cell and Tissue Research.
Mitochondrial respiration
Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)
Mitochondrial FeSOD-A and cell signaling
Intramitochondrial NO-dependent MitoSOX oxidation:
80
TcSODA
CL-Brener
10
0
10
1
2
10
MitoSOX
TcSOD-A
10
3
10
4
Ctl
DMNQ (1 mM)
SPM-NO (3 mM)
10
0
10
1
2
10
MitoSOX
10
3
10
4
Oxidized Mito-SOX
Control
60
40
CL-Brener
20
SOD-A
0
0
5
NOC-18 (mM)
10
 SOD-A protects NO-derived mitochondrial parasite dysfunction
Cardiomyocyte-induced T. cruzi oxidative stress
T. cruzi cardiomyocyte infection and detection of
mitochondrial O2•- production by MitoSOX oxidation
in intracellular amastigotes
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 cytotoxin
against internalized T. cruzi
 Wild type strains of high virulence have increase
expression of antioxidant enzymes
 Levels of mitochondrial FeSOD could modulate
parasite 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 player
In the control and progression of the disease
 Evaluate cardiomyocyte-derived NO as a cell
mediator for the signaling of amastigote apoptosis in
vivo
Biochemistry Department
Gonzalo Peluffo
María Noel Alvarez
Alejandra Martínez
Madia Trujilo
Martín Hugo
Dolores Piñeyro
Carlos Robello
Rafael Radi
International Colaboration
Shane Wilkinson, UK
Martin Taylor, UK
John Kelly, UK
Paola Zago, Argentina
Miguel Basombrio,
Argentina
Howard Hughes Medical Institute, USA
National Institute of Health (NIH), USA
PEDECIBA (Uruguay)
CONICYT (Uruguay)
PDT (Uruguay)
Different mitochondrial membrane potential in infective T. cruzi stages
obtained from infected cardiomiocytes
Activación Clásica
Activación Clásica
Activación Alterna
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
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