Post on 16-Jan-2016
New Chemo- and Immunotherapeutic
Approaches in Chagas’ Disease
Rosa A. Maldonado MedinaFederal University of São Paulo
(UNIFESP)
.
• 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.
Chagas’ Disease(American trypanosomiasis)
Chemotherapeutic Target Properties
a) The molecule must perform essential functions in the parasite.
Life or death
b) It can be selectively inhibited:
Present in T. cruzi, but not in mammals
Carbohydrates Lipids Proteins
Basic Metabolism
Oleate (-12) desaturase (ODTc ):
A New Chemotherapeutic Target
in Chagas’ Disease
• 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.
9-
12- desaturase (OD)
(C18:0)---X* (C18:1)---X
NAD(P)H
(C18:2)---X
NAD(P)
9- desaturase
12-
Stearoyl (C18:0)--- Oleoyl (C18:1)---X
NAD(P)H
Linoleoyl (C18:2)---X
NAD(P)
15- desaturaseNAD(P)H
(C18:3)---X
NAD(P) -
NAD(P)H
Linolenoyl(C18: )---X
NAD(P)
9-
12- desaturase (OD)
(C18:0)---X* (C18:1)---X
NAD(P)H
(C18:2)---X
NAD(P)
9- desaturase
12-
Stearoyl (C18:0)--- Oleoyl (C18:1)---X
NAD(P)H
Linoleoyl (C18:2)---X
NAD(P)
15- desaturaseNAD(P)H
(C18:3)---X
NAD(P) -
NAD(P)H
Linolenoyl(C18: )---X
NAD(P)X* = CoA or ACP (acyl carrier protein) or phospholipid.
Cloning of the T. cruzi Oleate Desaturase (ODTc)
P1, P2= degenerated primersgDNA
P1
P2PCR
High similarity with 12-desaturase from plants
(1003 bp)
Size selection library
Sac I Genomic clone Sac I 3 kb
Mannosyl Transferase
5´-ODTc 3´- missing
Sph
I
Sac I
Kpn I
Hind
II
3.0
Kb
3´-RACE PCR
Sac I Sac I Sac I
Mannosyl Transferase
ODTcI ODTc II
3 Kb 2.7 Kb
~8 Kb
1.2 Kb
Sac I Sac I Sac I
3 Kb 2.7 Kb
Sph I Sph I
1.2 Kb
Chromosomal localization
Chrom. Mb Dm28c Sylvio CL Y CL
Band X10/7 Brener
IX 1.03
VII 0.95
VI 0.85
I 0.45
Strain
Phylogenetic groups: I (sylvatic cycle): Dm28c, Sylvio X10/7II (domestic cycle): Y, CLHybrid: CL Brener
CL-Brener
0.95
0.85
0.45
Mb
Y CL
1.03
0.85
Mb
0.45
Dm28c S.X10/7
Mb
Alignment of Trypanosomatid ODs vs. Plant OD
T.cruziL.major
T.bruceiB.officinalis
T.cruziL.major
T.bruceiB.officinalis
T.cruziL.major
T.bruceiB.officinalis
T.cruziL.major
T.bruceiB.officinalis
T.cruziL.major
T.bruceiB.officinalis
T.cruziL.major
T.bruceiB.officinalis
T.cruziL.major
T.bruceiB.officinalis
Putative transmembrane motifs
His-rich motifs: possibly participating in catalysis
PSORT II - k-nearest neighbor (k-NN) algorithm: 44.4 %: endoplasmic reticulum
1 54 .......... ...MSRVDNL TVAPGSPDVM KAVLKSGRNK .ENNVIVPS. .SALTIREIQ EKIPVKYFER .......... .......... .........M KSVLKAGLKK .DVDVRVPS. .AELTIKQIQ DLIPAKYFER MLPKQQMGGS VCNASIETVN TEATDPSEAK KIVLNAGRSE .KVNVYVPP. .STLMVRDIQ EQIPAEYFQR .......... .......... ...MGGGGRM PVPTKGKKSK SDVFQRVPSE KPPFTVGDLK KVIPPHCFQR 55 124 NTTRSVMFLL RDLAQVALTY AIMYAVALPL ATSMEVSAAR TVADGGALSL MAGTAMTTAA WLLKGVLWAV SAVWSMFYVF RDICQLLVVY CIMYRAVMPA LAGLES...R MYEAAGAT.. WASWSLVSA. ..VKLAVWNV SMWRSFSYLS RDMFQLFLTF VIMYNFVLPM LDSSLLN... .......... ....AVPPVA WLSRAAAWMI SVLHSFSYVV YDLVIAALFF YTASRYIHLQ PHPLSY.... .......... .......... .....VAWPL 125 194 FWFVQGLNGT ALWVLAHECG HQAFSPMKGL NDAVGMILHS ALLVPYHSWR ITHGGHHKHT NHLTKDLVFV FWFVQGLNGT ALWVLAHECG HQAFSPYRSL NNAVGLLLHS SVLVPYHSWR ITHGNHHKHT NHLTKDTVFV YWFIQGLNGT ALWVLAHECG HQAFCNSRRV NNAVGMILHS ALLVPYHSWR LTHGTHHKHT NHLTKDLVFV YWFCQGSVLT GVWVIAHECG HHAFSDYQWL DDTVGLLLHS ALLVPYFSWK YSHRRHHSNT GSLERDEVFV 195 262 PEKRNSVVEL VEDA..PLVS LIQTLIIFLF GWPAHLLFNA SGQEFGRLAC HFDPGAPFFR SEDRHDVVLS PRKESKVIDL LEET..PLVM VSNMLIMFIF GWPGYLLFNV ASQDYGRRTN HFEPSSPLFR KEDAHDIVLS PVQRSAVGEA VEEA..PIVM LWNMALMFLF GWPMHLLVNV GGQKFDRFTS HFDPNAPFFR RADYNNVMVS PKKRSGISWS SEYLNNPPGR VLVLLVQLTL GWPLYLMFNV SGRPYDRFAC HFDPKSPIYN DRERLQIYIS 263 332 NVGIVSALFV IFSSVYRFGF TNVFYWYIVP YLWVNFWLVY ITYLQHTDIR IPHYTHEHWT FVRGALAAVD DIGIFAALTV LGLCTYQYGA YNVLCWYVVP YMWVNFWLLY ITYLQHTDIR IPHYNHEHWT FVRGALAAVD NMGVLLTLSI LGACSWSFGF AVVVRWYLIP YLWVNFWLVY ITYMQHSDVR LPHYTHDHWT YVRGAVAAVD DAGIVAVMYG LYRLVAAKGV AWVVCYYGVP LLVVNGFLVL ITYLQHTQPS LPHYDSSEWD WLKGALATVD 333 402 RDYGFVLNTW LHHIKIPTPS HQDSHVVHHL FSKMPHYNAI QVTRKYIREI LGATYITDER SLWKMLWEQR RDYGFLINNW LHHI...... .HDSHVVHHL FSQMPFYHAI EVTRHHIKAI LGDNYVEDRR PLIDALCTSW RDFGPLLNSW LHHIN..... ..DSHVVHHL FSQMPHYNAI EVTRKHIRDI LGDLYVTDAK PLLKSLVHTW RDYG.FLNKV LHNIT..... ..DTHVAHHL FSTMPHYHAM EAT.KAIKPI LGDYYQCDRT PVFKAMYREV 403 419 KECRYVVPAE G...VCVFHG ...... KECAYVVPSE G...VSVFYG FNKKRA RECRYVVPSE G...ICIYRS ...... KECIYVEADE GDNKKGVFWY KNKL..
Homologous expression of ODTc fused to GFP
gDNA5`-Xba I
Xba I -5`
Xba I Xba IODTc
PCR
Neor
AMPr
GFPODTc
Xba IXba I Eco RV
pTEX
Electroporation
LIT
G418 (100 g/mL)
1% blood
Selection
Transfected parasites
50 g plasmid
1 x 106 epimastigotes/ 200 L
Co-localization of ODtc in the Endoplasmic Reticulum
ER-TrackerBlue-White DPX
OD-GFP
Phase Merge
Molecular characterization of OD in other trypanosomatids
HmBc
La LsCf
2.5
1.5
1.0
KbCf Hm Ls Tf Tr Tl
10.05.03.02.5
1.51.0
Kb
Southern - gDNA Southern - cDNA
Chromosomal localization
Tc Es Ls M Tc Es Ls
0.450.610.820.95
1.1
Mb
Tc Cf Hm Tcon Tf M Tc Cf Hm TconTf
Mb
0.380.450.610.680.750.820.95
Biochemical studies of the native OD
activity in different trypanosomatids
Freeze-dried
1010 parasites
+ 5 Ci [14C]-oleic acid
16 h, at 28 or 37ºC
Metabolic labeling and lipid analysis
Washed
Lipid extraction (C:M, 2:1; C:M:W, 1:2:0.8)
TAG
C18:1
PI
PCLPC
PE/PME
IPC
ErgDAG
T. cruziEpimastigote
PA/MAG
LPC
LPI
TAG
C18:1
PE/PMEPI
PC
Erg DAG
T. cruziTrypomastigote
TAG
C18:1Erg
DAG
PI/IPC
PE/PME
PA/MAG
LPC
Cf La Hm Ls
TAG
PC
PE/PME
Cf La Hm Ls
Total lipids
HPTLC
Autoradiography
Fatty acid hydrogenation of Trypanosomatid phospholipids
Hydrogenation + - + - + - + - + - + -
C18:1
C18:0
Tc epi Tc tryp Cf La Hm Ls
C18:2
C18:3
Phospholipids (PC, PE, PI)
Alkaline hydrolysis
+/-Hydrogenation
RP-HPTLC
Autoradiography
Methylation
C18:1
C18:0
C18:2
C18:3
• 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.
CONCLUSIONS
The Spider Antimicrobial Peptide Gomesin
as a New Adjuvant
in the Immunotherapy against T. cruzi
• 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
Antimicrobial peptides (AMP)
Gomesin
• 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
Gomesin kills T. cruzi epimastigotes in vitro
Concentration (M)
Su
rviv
al (
%) DL50 = 6.5 M
0 1 2 3 4 5 6 7 8 9 10
1.5×107 Control
Liposomal Gomesin
Free Gomesin
Empty Liposome
Time (days after infection)
Par
asite
mia
(N
o. o
f pa
rasi
tes/
mL)
In vivo activity of gomesin in the experimental infection of mice BALB/c by T. cruzi
1.0×107
0.5×107
0
In vivo activity of gomesin in the experimental infection of mice BALB/c by T. cruzi
Sur
viva
l (%
)
0 10 20 30 40 50 60 700
20
40
100
0 10 20 30 40 50 60 70
Control
Liposomal Gomesin
Free Gomesin
Empty Liposome
Non Infected +Liposomal Gomesin
Control
Non Infected + Free Gomesin
Time (days after infection)
60
80
How 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.
METHODOLOGY
• 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 tosignal via TLR4
Activation of TLR4 by Gomesin
CD
25 e
xp
ress
ion
Fo
ld i
nc
rea
se
None Gom S.a. LPS0.0
2.5
5.0
7.5
10.0
CHO/CD14/mMD2
CHO/CD14/mTLR4
CHO/CD14/TLR2
CHO/CD14/TLR4
Stimulus
12 0.12 0.001 1.2x10-6
CD
25 e
xp
ress
ion
Fo
ld i
nc
rea
se
Gomesin concentration (nM)
Gomesin activates TLR4 at low
nanomolar to picomolar range
Gomesin activates Toll-like receptor 4
Cytoplasm
TLR-4
Plasmamembrane
Signaling cascade
Nucleus
NF-B
Activationof
immune responsegenes
Synthesis ofPro-inflammatory
Cytokinesand
Chemokines
MyD88
IRAK
IB P
CD14
?
2
GOMESIN3
4
56
MD-2
CONCLUSIONS
• 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.
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 DundeeAlan Fairlamb
Financial support
FAPESP/ CNPq WHO/TDRThe Wellcome Trust-UK
ACKNOWLEDGMENTS