Title: Apresentao do PowerPoint
1New Chemo- and Immunotherapeutic Approaches in
Chagas Disease
Rosa A. Maldonado Medina Federal University of
São Paulo (UNIFESP)
2Chagas Disease (American trypanosomiasis)
- Chagas disease affects 16-18 million people in
Latin America, from Mexico to Argentina. - The ethiological agent is the protozoan
parasite - Trypanosoma cruzi
- Current treatment (benznidazole, Rochagan) is
partially effective (60 cure). - Recently, drug-resistant strains have been
found.
.
3Chemotherapeutic Target Properties
a) The molecule must perform essential functions
in the parasite.
Basic Metabolism
Life or death
b) It can be selectively inhibited
Present in T. cruzi, but not in mammals
4Oleate (D-12) desaturase (ODTc ) A New
Chemotherapeutic Target in Chagas Disease
5X CoA or ACP (acyl carrier protein)
or phospholipid.
- OD is absent in humans, then it can be
selectively inhibited. - Linoleic acid is a major component of GPI
anchor of T. cruzi trypomastigote mucins and
phospholipids. So, the product of OD seems to
perform essential functions for the parasite.
OD fulfills all the requirements as a potential
chemotherapeutic target.
6Cloning of the T. cruzi Oleate Desaturase (ODTc)
7Chromosomal localization
Y CL
CL-Brener
Mb
Mb
Mb
1.03
0.95
0.85
0.85
0.45
Strain
Chrom. Mb Dm28c Sylvio CL Y
CL Band X10/7 Brener IX 1.03 VII 0.95 VI 0.
85 I 0.45
Phylogenetic groups I (sylvatic cycle) Dm28c,
Sylvio X10/7 II (domestic cycle) Y, CL Hybrid
CL Brener
8Alignment of Trypanosomatid ODs vs. Plant OD
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
Putative transmembrane motifs
His-rich motifs possibly participating in
catalysis
PSORT II - k-nearest neighbor (k-NN) algorithm
44.4 endoplasmic reticulum
9Homologous expression of ODTc fused to GFP
5-Xba I
gDNA
Xba I -5
PCR
GFP
ODTc
1 x 106 epimastigotes/ 200 mL
Eco RV
Xba I
Xba I
Neor
Electroporation
pTEX
50 mg plasmid
AMPr
Selection
LIT G418 (100 mg/mL) 1 blood
Transfected parasites
10Co-localization of ODtc in the Endoplasmic
Reticulum
OD-GFP
ER-Tracker Blue-White DPX
Phase
Merge
11Molecular characterization of OD in other
trypanosomatids
12Biochemical studies of the native OD activity in
different trypanosomatids
13Metabolic labeling and lipid analysis
1010 parasites 5 mCi 14C-oleic acid 16 h, at
28 or 37ºC
Washed
Freeze-dried
Lipid extraction (CM, 21 CMW, 120.8)
Total lipids
HPTLC Autoradiography
14Fatty acid hydrogenation of Trypanosomatid
phospholipids
Phospholipids (PC, PE, PI)
Alkaline hydrolysis
Methylation
/-Hydrogenation
RP-HPTLC Autoradiography
C183
C183
C182
C182
C181
C181
C180
C180
Hydrogenation -
- - -
- -
Tc epi Tc tryp Cf
La Hm Ls
15CONCLUSIONS
- ODTc gene has 2 copies in tandem array and is
transcribed in all T. cruzi stages. - The enzyme is localized in the endoplasmic
reticulum. - The enzymes ?-12 and ?-15 desaturases are active
in trypanosomatids. - OD seems to be promising chemotherapeutic target
in Chagas disease, leishmaniasis and African
trypanosomiasis, as well as in fungal infections.
16The Spider Antimicrobial Peptide Gomesin
as a New
Adjuvant in the Immunotherapy against T. cruzi
17Antimicrobial peptides (AMP)
- Involved in innate immunity found from bacteria
to mammals - Cationic molecules composed of 12 to 50 amino
acids - Frequently rich in cysteine residues
- Positively charged in physiological pH, and with
amphypathic structure in ?-helix, ?-sheet or both - Primary action mechanism is the permeabilization
of the microbial membrane
18Gomesin
- Isolated from hemocytes of the Brazilian
tarantula spider Acanthoscurria gomesiana (Silva
Jr. et al, JBC, 2000) - Mass of 2270 Da, 18 amino acids
(ZCRRLCYKQRCVTYCRGRa), two disulfide bonds
(Cys6-11 e Cys2-15) - Antibacterial (Gram-negative and Gram-positive)
and antifungal activity
19Gomesin kills T. cruzi epimastigotes in vitro
DL50 6.5 mM
Survival ()
Concentration (mM)
20In vivo activity of gomesin in the experimental
infection of mice BALB/c by T. cruzi
1.5107
1.0107
Parasitemia (No. of parasites/mL)
0.5107
0
0
1
2
3
4
5
6
7
8
9
10
Time (days after infection)
21In vivo activity of gomesin in the experimental
infection of mice BALB/c by T. cruzi
22How gomesin was protecting the animals if it was
not by directly killing the parasites?
- One possibility was that gomesin like defensins
(Biragyn et al., Science, 2002) was acting on the
mouse innate immune system, inducing
pro-inflammatory cytokines, through the
activation of Toll-like receptors. - TLRs are primary sensors of the innate immunity.
They recognize Pathogen-Associated Molecular
Patterns (PAMPs)(e.g. LPS, GPIs, etc), initiating
signaling pathways that stimulate the host
defense against microorganisms.
23METHODOLOGY
- Transfected CHO/CD14 cells CHO/CD14/TLR2
- CHO/CD14/TLR4
- CHO/CD14/mMD2
- CHO/CD14/mTLR4
-
- Gomesin stock solution was cleaned-up of
endotoxins using an affinity column of polimyxin
B (depletion of LPS) - The stimulation of the TLR was measured by the
expression of CD25 on the surface of the cells
by FACS
mutants not able to signal via TLR4
24Activation of TLR4 by Gomesin
Gomesin activates TLR4 at low nanomolar to
picomolar range
25Gomesin activates Toll-like receptor 4
26CONCLUSIONS
- Gomesin stimulates TLR4, so it is a new PAMP.
- Gomesin could be used as an adjuvant of the
innate immune response against Chagas infection
and, probably, other infectious diseases.
27ACKNOWLEDGMENTS
- University of São Paulo (USP)
- ICB
- Lab. Parasite Glycobiology
- Igor C. Almeida
- Renata K. Kuniyoshi
- Lab. of Biochem. and Immunol. of Arthropodes
- Sirlei Daffre
- Marcello R. Burgierman
- IQ
- Maria Terêsa Miranda
- Iolanda Cuccovia
- Federal University of São Paulo (UNIFESP)
- Antônio Miranda
- Marcos Fazio
University of Dundee Alan Fairlamb Financial
support FAPESP/ CNPq WHO/TDR The Wellcome
Trust-UK