Fighting the oxidative assault: The Trypanosoma cruzi journey to infection
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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
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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
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Central rol of macrophages in the control of T. cruzi infections
Phagocytes: First line of defense against invading T. cruzi
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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+
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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
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● 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●¯
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Szabo, Ischiropoulos and Radi, Nature Reviews (2007)
Peroxynitrite toxicity
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Intraphagosomal Oxidants
Resting Macrophage
2 hrs infection
Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)
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Activated macrophage
2 hrs infection
Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)
Intraphagosomal Oxidants- “Our hypothesis”
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Antioxidant Defense in T. cruzi
Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)
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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)
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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.
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Control Control + peroxynitrite TcCPX + peroxynitrite
TcCPX T. cruzi overexpressers are
resistant to peroxynitrite cytotoxicity
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Peroxynitrite-mediated protein oxidationProtection by peroxiredoxins
Immuno-spin trapping
Mason et al; 2002-2004
t1/2= months-years
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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
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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+
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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
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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)
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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
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Does enzymes of T. cruzi antioxidant
network are involved in infectivity?
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T. cruzi strains and virulence
Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)
Biochemical and molecular diversity of T. cruzi populations
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TCC CL-WT
T. cruzi strain analysis
Piacenza et al., Int. J. Parasitol. 39, 1455 (2009)
Virulent vs attenuated
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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
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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)
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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
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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
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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
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Role of FeSODs in cellular signaling and survival
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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
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Mitochondrial FeSOD-A and cell signaling
Control TcSODA
IHS FHS HIS FHSbp
Mitochondrial Fe-SODA overexpression
Piacenza et al., Biochem. J. 403, 323 (2007)
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CL-Brener + DMNQ (superoxide generator)
Intramitochondrial O2- detection: MitoSOX oxidation
Mitochondrial FeSOD-A and cell signaling
Smith and Murphy, 2003
HEMito-Hidroetidio
(Mitosox)
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Mitochondrial FeSOD-A and cell signaling
Parasite mitochondrial-O2- production triggers programmed cell death
Piacenza et al., Biochem. J. 403, 323 (2007)
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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
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Mitochondrial respiration
Piacenza et al., Curr. Opin. Microbiol. 12, 415 (2009)
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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
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T. cruzi cardiomyocyte infection and detection of mitochondrial O2
•- production by MitoSOX oxidation in intracellular amastigotes
Cardiomyocyte-induced T. cruzi oxidative stress
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Cardiomyocyte-induced T. cruzi oxidative stress
Control Activated
Cardiomyocyte-derived NO as a cellular mediator in the control of parasite proliferation
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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
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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
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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
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Different mitochondrial membrane potential in infective T. cruzi stagesobtained from infected cardiomiocytes
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Activación Clásica
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Activación Alterna
Activación Clásica
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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