Platelet-activating factor-like activity isolated from Trypanosoma cruzi
Marta T. Gomesa,b, Robson Q. Monteiroc, Luciano A. Grilloa,c, Francisco Leite-Lopesa,
Heleni Stroederd, Antonio Ferreira-Pereiraa, Celuta S. Alvianoa, Eliana Barreto-Bergtera,
Hugo Castro-Faria Netoe, Narcisa L. Cunha e Silvab, Igor C. Almeidad,f,
Rosangela M.A. Soaresa, Angela H. Lopesa,*
aInstituto de Microbiologia, Prof. Paulo de Go ´es, Universidade Federal do Rio de Janeiro, Cidade Universita ´ria, CCS, Bloco I,
Ilha do Funda ˜o, 21941-590 Rio de Janeiro, RJ, Brazil
bInstituto de Biofı ´sica Carlos Chagas Filho, UFRJ, Rio de Janeiro, RJ, Brazil
cInstituto de Bioquı ´mica Me ´dica, UFRJ, Rio de Janeiro, RJ, Brazil
dDepartamento de Parasitologia, Instituto de Cie ˆncias Biome ´dicas, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, SP, Brazil
eLaborato ´rio de Imunofarmacologia, Departamento de Fisiologia e Farmacodina ˆmica, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brazil
fDepartment of Biological Sciences, University of Texas at El Paso (UTEP), El Paso, TX 79968-0519, USA
Received 23 May 2005; received in revised form 30 September 2005; accepted 30 September 2005
Platelet-activating factor is a phospholipid mediator that exhibits a wide variety of physiological and pathophysiological effects, including
induction of inflammatory response, chemotaxis and cellular differentiation. Trypanosoma cruzi, the etiological agent of Chagas’ disease, is
transmitted by triatomine insects and while in the triatomine midgut the parasite differentiates from a non-infective epimastigote stage into the
pathogenic trypomastigote metacyclic form. We have previously demonstrated that platelet activating factor triggers in vitro cell differentiation of
T. cruzi. Here we show a platelet activating factor-like activity isolated from lipid extract of T. cruzi epimastigotes incubated in the presence of
[14C]acetate. Trypanosoma cruzi-platelet activating factor-like lipid induced the aggregation of rabbit platelets, which was prevented by platelet
activating factor-acetylhydrolase. Mouse macrophage infection by T. cruzi was stimulated when epimastigotes were kept for 5 days in the
presence of T. cruzi-platelet activating factor, before interacting with the macrophages. The differentiation of epimastigotes into metacyclic
trypomastigotes was also triggered by T. cruzi-platelet activating factor. These effects were abrogated by a platelet activating factor antagonist,
WEB 2086. Polyclonal antibody raised against mouse platelet activating factor receptor showed labelling for T. cruzi epimastigotes using
immunoblotting and immunofluorescence assays. These data suggest that T. cruzi contain the components of an autocrine platelet activating
factor-like ligand–receptor system that modulates cell differentiation towards the infectious stage.
q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Keywords: Trypanosoma cruzi; Platelet-activating factor (PAF); PAF-like; Differentiation; Macrophage
Trypanosoma cruzi is a protozoan parasite that exhibits
developmental regulation of virulence and is transmitted by
triatomine (reduviid) insects. These insects become infected by
ingesting trypomastigotes from the blood of the mammalian
hosts; the parasites then multiply as epimastigotes and
differentiate into metacyclic trypomastigotes in the lumen of
the crop and midgut. Trypomastigotes infect mammalian cells,
where they transform into intracellular multiplicative amasti-
gotes and differentiate back into trypomastigotes that are then
released into peripheral blood (De Souza, 1984).
Trypanosoma cruzi molecules that modulate the interaction
of the parasite with both mammalian cells (Almeida et al.,
2000) and the reduviid insects (Pereira et al., 1981) have been
described. Mechanisms underlying the differentiation of the
parasite are poorly understood, although we do know that
platelet-activating factor (PAF) triggers the differentiation of
T. cruzi from epimastigotes into metacyclic trypomastigotes
(Rodrigues et al., 1996).
PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is
synthesised by a diverse array of cells, including neutrophilic
International Journal for Parasitology 36 (2006) 165–173
0020-7519/$30.00 q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Tel.: C55 21 2562 6710; fax: C55 21 2560 8344.
E-mail address: firstname.lastname@example.org (A.H. Lopes).
polymorphonuclear leukocytes, platelets, mast cells, mono-
cytes/macrophages, vascular endothelial cells and lympho-
cytes. PAF targets these and other cells via specific,
G-protein-coupled receptors to initiate intracrine, autocrine,
paracrine and juxtacrine cell activation (Prescott et al., 2000;
Ishii and Shimizu, 2000). The physiological role of PAF in
lower eukaryotes is still largely unknown, although it has been
implicated in several morphoregulatory functions (Kulikov
and Muzya, 1997) and in some physiological roles related to
cell differentiation in trypanosomatids (Lopes et al., 1997;
Rodrigues et al., 1999; Silva-Neto et al., 2002). In this study
we show that a PAF-like lipid stimulates, through receptor
activation, cell differentiation in T. cruzi and the ability of the
latter to infect mouse peritoneal macrophages.
2. Materials and methods
lyso-PAF, phosphatidylcholine, lyso-phosphatidylcholine,
monoacylglycerol (MAG), diacylglycerol (DAG), triacylgly-
cerol (TAG) and the calcium ionophore A23187 were
purchased from Sigma Chemical Co., St Louis, MO, USA.
High performance thin-layer chromatography plates and ether-
lipid standards were purchased from Merck, Darmstadt,
Germany. The aminopropyl columns (Supelclean LC-NH2)
were purchased from Supelco, Supelco Park Bellefonte, PA,
USA. The [14C]acetate was purchased from Amersham
Bioscience, NJ, USA. The antibodies and the developing
system for the Western blotting and immunofluorescence were
purchased from Santa Cruz Biotechnology, CA, USA, Pierce
Biotechnology, Rockford, IL, USA or Molecular Probes, OR,
USA. PAF acetylhydrolase was a gift from ICOS Corp.,
Bothel, WA, USA. WEB 2086 (3-[4-(2-clorfenil)-9-metil-6H-
tieno-[3,2-f] [1,2,4]triazolo-[4,3,a-] [1,4] diazepine-2-g1-1-(4-
morfoline-g1)-1-propanone), a competitive PAF antagonist,
was kindly provided Dr H. Heurer (Boehringer Ingelheim,
Germany). All other reagents were analytical grade.
2.2. Trypanosoma cruzi cultivation
Trypanosoma cruzi (Dm 28c clone) epimastigotes were
maintained by weekly transfers in LIT (liver infusion tryptone)
medium (Camargo, 1964) supplemented with 10% heat-
inactivated FCS, at 28 8C. Metacyclic trypomastigotes were
obtained by adding PBS-washed epimastigotes in a serum-free
chemically defined medium (TAUP), where they were kept for
up to 6 days at 28 8C (Contreras et al., 1988). In these
conditions we obtained about 48% epimastigotes and 52%
trypomastigotes. In this study all the experiments were
performed using parasites that were harvested by centrifu-
gation and washed three times with PBS (pH 7.2) before use,
unless otherwise specified.
2.3. Preparation and analysis of [14C]acetate-T. cruzi
Live T. cruzi culture epimastigotes were harvested by
centrifugation, washed three times in HEPES buffered saline,
pH 7.4, and resuspended in 1 ml of the same buffer containing
1.3 mM CaCl2plus 25 mCi [14C]acetate. After 5 min incu-
bation at 37 8C, the calcium ionophore A23187 (dissolved in
0.1% DMSO) was added to a final concentration of 2 mM and
the mixture was incubated for 30 min at room temperature. The
same volume of 0.1% DMSO was added to the control
parasites. Then 0.5 ml of a solution containing 50 mM acetic
acid in methanol was added to the mixture. The lipids were
then resuspended in 1.25 ml chloroform plus 1.25 ml glacial
acetic acid, followed by vortex mixing and centrifugation at
5000!g for 5 min. The lower phase was collected, dried under
N2gas stream and kept in liquid N2until use. The lipids were
resuspended in chloroform:methanol (9:1, v/v) and partially
purified using aminopropyl columns (Supelclean LC-NH2,
Supelco), as described (Kaluzny et al., 1985). This procedure
separates neutral from charged lipids. Briefly, the total lipid
fraction (lower phase) from T. cruzi was resuspended in 500 ml
and applied to the column, previously washed with 4 ml
hexane. The column was then sequentially eluted with 4 ml of
each of the solvents: A (chloroform:isopropanol, 2:1, v/v), B
(ethyl ether:acetic acid, 98:2, v/v), C (100% methanol) and D
(chloroform:methanol:water, 10:10:3, v/v/v). Neutral lipids
were preferentially eluted in solvent A, whereas charged lipids,
including PAF, were eluted in solvent C.
Both authentic PAF and T. cruzi lipid extracts were
hydrolysed by treatment with 40% hydrofluoric acid (HF) for
24 h at 0 8C (ice bath), for the release of phosphate-containing
head groups (Ferguson, 1992), as well as by treatment with
25% NH4OH for 12 h at 37 8C, for the release of acyl groups at
the lipid tails. To analyse their lipid content, samples were
separated by high performance thin-layer chromatography
(HPTLC) on silica gel-60 plates using two sequential solvent
systems: first, chloroform:methanol:water (65:35:6, v/v/v) to
separate polar lipids, allowed to run up to the middle of the
plate, and then hexane:diethyl ether:acetic acid (70:30:1, v/v/
v), allowed to run up to the top of the plate to separate neutral
lipids in the upper half (Bligh and Dyer, 1959; Florin-
Christensen et al., 1992). Plates were then dried and exposed
to iodine vapour (Sambasivarao and McCluer, 1963). Lipids
were identified by comparison with authentic standards. Each
lane was individually scraped at every 1 cm and the scraped
materials were placed in vials and analysed for radioactivity
content in a Liquid Scintillation Analyser (Tri-Carb 2100TR,
Packard) using Bray’s liquid scintillation mixture (100 ml
methanol, 4 g PPO (2, 5-diphenyloxazole), 60 g naphthalene,
20 ml ethylene glycol, 0.1 g POPOP (2-p-phenylenebis
5-phenyloxazole); diluted to 1 l with p-dioxan).
2.4. Estimation of Tc-PAF concentration
The biological experiments described in this paper were
performed using concentrations of Tc-PAF estimated by its
M.T. Gomes et al. / International Journal for Parasitology 36 (2006) 165–173 166
biological activity in a platelet aggregation assay, as compared
with authentic PAF.
2.5. Platelet aggregation assay
Platelet aggregation experiments were performed with a
Chronolog Aggregometer (Havertown, PA, USA), using
washed rabbit platelets (or rat platelets, as a negative control),
prepared from blood anticoagulated with 5 mM EDTA.
Platelets were isolated by centrifugation, washed and resus-
pended in a modified Tyrode’s buffer, pH 7.4 containing 2 mM
CaCl2at 300-400,000 cells/ml (Zingali et al., 1993). Rabbit and
rat platelets, as well as mouse peritoneal macrophages used in
this investigation were obtained following the guidelines for
animal experimentation of the USA National Institutes of
Health and the experimental protocol was approved by the
Instituto de Biofı ´sica Carlos Chagas Filho (Universidade
Federal do Rio de Janeiro) ethical committee for animal
2.6. PAF-acetylhydrolase and phospholipase A1 treatment
Bioactive HPTLC fractions were incubated with 2 mg of
recombinant human PAF acetylhydrolase for 1 h at 37 8C or
with phospholipase A1 (PLA1: phospholipase from Rhizopus
arrhizus) for 18 h at 30 8C, under constant shaking. In the latter
assays, 0.1 mg/ml PLA1 in reaction medium (0.1 M boric acid,
pH 6.5; 10 mM CaCl2; 1 mg/ml deoxycholate; 0.4 mg/ml
bovine serum albumin) was used. The PAF–acetylhydrolase-
treated and the PLA1-treated fractions were then assayed for
platelet aggregation as described above.
2.7. Evaluation of cell differentiation
Epimastigotes were added to TAUP medium where they
were maintained at 28 8C for periods ranging from 1 to 6 days,
in the absence or in the presence of the following compounds:
Tc-PAF (10K8M) and/or anti-PAF receptor antibodies (1:1000
dilution), WEB 2086 (10K7M), or vehicle (Tc-PAF was
dissolved at 1 mM in ethanol, and WEB 2086 in 0.1 N HCl; the
final concentration of each compound was adjusted with
culture medium). The percentage of epimastigote and
trypomastigote forms was daily determined by using Giemsa-
stained preparations. At least 200 cells were examined by
phase-contrast microscopy in each preparation.
2.8. Infection of macrophages
Resident peritoneal macrophages from female BALB/c
mice were collected in 0.9% saline and allowed to adhere to
coverslips, placed in 24-wells culture plates, for 30 min at
37 8C in a 4% CO2atmosphere. Before the interaction assays,
epimastigotes were added to TAUP medium where they
were maintained for 5 days at 28 8C, in the absence or in the
presence of the following compounds: Tc-PAF (10K8M)
and/or anti-PAF receptor antibodies (1:1000 dilution), WEB
2086 (10K7M), or vehicle (Tc-PAF was dissolved at 1 mM in
ethanol, and WEB 2086 in 0.1 M HCl; the final concentration
of each compound was adjusted with culture medium).
Adherent macrophages were cultured for 24 h in RPMI culture
medium supplemented with 10% FCS. The parasites were then
washed with PBS and allowed to interact with the macrophages
(10:1 ratio) for 4 h, after which the coverslips were fixed,
stained with Giemsa and the percentage of infected macro-
phages was determined by counting 600 cells in triplicate
coverslips, as described (Rosa et al., 2001). Each experiment
was repeated at least three times. The association indices were
determined by multiplying the percentage of infected
macrophages by the mean number of parasites per cell.
2.9. Western blotting
Whole protein extracts of T. cruzi culture epimastigotes
[104/ml], T. cruzi intermediate cultures containing 48%
epimastigotes plus 52% trypomastigotes [104/ml], rat platelets
[104/ml] and rabbit platelets [104/ml] were separated by SDS-
PAGE (Laemmli, 1970). The proteins were transferred to
nitrocellulose membrane, probed with a polyclonal antibody
raised against mouse PAF receptor (1:2000 and 1: 8000
dilutions) and developed using the alkaline phosphatase system
(Santa Cruz Biotechnology) or Super Signal West Pico kit
Trypanosoma cruzi culture epimastigotes were harvested by
centrifugation and fixed in 4% formaldehyde in PBS for
30 min. Washed epimastigotes were adhered on 0.1% poly-L-
lysine coated glass coverslips, incubated with 50 mM NH4Cl in
PBS and permeabilised or not with 1% Triton X-100 for 5 min.
After blockage in 3% teleostean fish gelatine in PBS for 1 h at
room temperature, epimastigotes were incubated without
(negative control) or with a polyclonal antibody raised against
mouse PAF receptor diluted 1:100 in blocking buffer, for 2 h,
washed and incubated with 1:400 Alexa 546 donkey anti-goat
IgG (Molecular Probes) for 1 h. Coverslips were mounted in
0.2 M n-propylgallate in glycerol:PBS (9:1) and observed in a
Zeiss LSM 310 Confocal Laser Scanning microscope,
operating at conventional mode.
All results are presented as the mean and standard error of
the mean (SEM). Normalised data were analysed by a one-way
analysis of variance (ANOVA), and differences between
groups were assessed by using the Student–Newman–Keuls
post-test. A P value of !0.05 was considered significant.
A lipid extract from A23187-treated [14C]acetate-labelled
T. cruzi epimastigotes was submitted to high performance thin-
layer chromatography (HPTLC). Almost all the radioactivity
obtained was present in the fraction that co-migrated with
M.T. Gomes et al. / International Journal for Parasitology 36 (2006) 165–173167
authentic PAF (Fig. 1a). The [14C]acetate-labelled lipid extract
from T. cruzi was hydrolysed with 40% HF, which gave rise to
a product that co-migrated with diacylglycerol (DAG)
(Fig. 1b), as this treatment should release phosphate, choline
and 1-alkyl-2-acetyl-glycerol and this last resultant molecule
would have a similar behaviour to diacylglycerol by HPTLC.
The [14C]acetate-labelled lipid extract from T. cruzi was also
hydrolysed with NH4OH, which reduced drastically the
amount of the lipid that co-migrated with PAF (Fig. 1c), as
this treatment releases the acetate from the PAF molecule,
transforming it into lyso-PAF.
Fractions obtained by thin-layer chromatography containing
component(s) that co-migrated with PAF were assayed with
rabbit platelets to test for biological activity. In a set of
experiments platelets were pre-incubated for 30 min with
10K8M WEB 2086, final concentration. WEB 2086 com-
pletely abolished platelet aggregation induced by Tc-PAF and
authentic PAF (both at 10K9M, final concentration) (Fig. 2a).
Pre-incubation of either authentic PAF or Tc-PAF with 2 mg
of recombinant human PAF-acetylhydrolase abolished their
ability to stimulate platelet aggregation, whereas it did not
affect the aggregation induced by a-thrombin (2!10K9M,
final concentration) (Fig. 2b). On the other hand, Tc-PAF was
insensitive to phospholipase A1 from R. arrhizus (Table 1).
When epimastigotes were kept in TAUP medium for
periods ranging from 1 to 6 days in the presence of Tc-PAF
(10K8M), there was an enhancement of the percentage of
trypomastigotes forms, as compared with the condition where
epimastigotes were kept in the absence of Tc-PAF (Fig. 3a).
Also, mouse peritoneal macrophage infection by T. cruzi was
stimulated when epimastigotes were added to TAUP medium
Fig. 1. Analysis and quantification of [14C]acetate-Tc-PAF (Trypanosoma cruzi
platelet activating factor). The arrows indicate the distance from the origin of
the high performance thin-layer chromatography plate to each spot of the
standards (PAF, lyso-PAF, monoacylglycerol (MAG), diacylglycerol (DAG)
and triacylglycerol (TAG)) verified by iodine vapour. (a) [14C]acetate-Tc-PAF
without any treatment; (b) [14C]acetate-Tc-PAF treated with hydrofluoric acid
(HF) (40% HF for 24 h in ice bath); (c) [14C]acetate-Tc-PAF treated with
NH4OH (25% NH4OH for 12 h at 37 8C). In the three set of experiments (a, b
and c), the bars represent the meanCSE of at least three independent
experiments. * means the amount of radioactivity measured in the indicated
sample was significantly different from the other samples (P!0.05, two-tailed
ANOVA, Student–Newman–Keuls post-test).
Fig. 2. Effect of Trypanosoma cruzi platelet activating factor (Tc-PAF) on the
aggregation ofrabbit platelets.Plateletaggregationassays were performedwith
a Chronolog Aggregometer (Havertown, PA, USA), using 300–400,000/ml
washed rabbit platelets (or rat platelets, as a negative control). (a) Platelets
(control or pre-treated for 30 min with 10K8M WEB 2086) were assayed in the
absence or in the presence of authentic PAF (10K9M) or Tc-PAF (10K9M).
All concentrations of Tc-PAF used in this work were estimated by its biological
activity in a platelet aggregation assay, as compared with authentic PAF. (b)
Aliquots of both authentic PAF (10K9M PAF) and Tc-PAF (10K9M) were
incubated with 2 mg of recombinant human PAF acetylhydrolase for 1 h at
37 8C. PAF-acetylhydrolase-treated and control samples were then assayed for
platelet aggregation. Platelets were also assayed in the presence of 2!10K9M
M.T. Gomes et al. / International Journal for Parasitology 36 (2006) 165–173168
and kept for 5 days in the presence of 10K8M Tc-PAF before
interacting with the macrophages (Fig. 3b). In both assays
Tc-PAF effects were abrogated by the PAF antagonist WEB
2086 and partially inhibited by anti-PAF receptor polyclonal
antibodies (Fig. 3a and b).
Polyclonal antibody raised against mouse PAF receptor
recognised proteins from both T. cruzi epimastigotes (Fig. 4, E)
and intermediate cultures containing 48% epimastigotes plus
52% trypomastigotes (Fig. 4, Tr), through immunoblotting. A
65 kDa protein showed a strong reaction with this antibody
(Fig. 4, Tr), while two other faint bands (40 and 200 kDa,
respectively) were also observed in that lane of the blot, when
intermediate cultures and anti-PAF receptor antibody at 1:2000
dilution were used (Fig. 4, Tr). The 200 kDa component was
the only band observed when the proteins were obtained from
culture epimastigotes and the anti-PAF receptor antibody was
used at 1:8000 dilution (Fig. 4a, E). When the blots were pre-
incubated with WEB 2086, there was a significant reduction in
the signal obtained from the antigen–antibody reaction, using
proteins from epimastigotes and anti-PAF receptor antibody at
1:8000 dilution (Fig. 4, WE). WEB 2086 was not able to block
the antigen–antibody reaction when this antibody was used at
1:2000 dilution, neither for proteins obtained from epimasti-
gotes or metacyclic trypomastigotes (data not shown). The
anti-PAF receptor antibody recognised a 68 kDa rabbit platelet
protein (Fig. 4, rab), but did not react with rat platelet protein
extract (Fig. 4, rat).
Immunofluorescence was performed in order to provide a
localisation of T. cruzi PAF receptor (Fig. 5). A weak but clear
reaction against PAF receptor polyclonal antibodies is shown
on all the surface of the epimastigotes, including cell body and
flagellum (Fig. 5a, c and e). A strong reaction is observed in the
interior of the cells, especially in the permeabilised epimas-
tigotes (Fig. 5a and c), but also in the non-permeabilised ones
Effect of phospholipase A1 (PLA1) and platelet-activating factor acetylhy-
drolase (PAF-AH)aon the ability of authentic PAF and Tc-PAF aggregateb
Enzyme treatmentAuthentic PAFTc-PAF
aPlatelet-activating factor acetylhydrolase (PAF-AH) is a unique type of
phospholipase A2 (PLA2).
Fig. 3. Effect of Trypanosoma cruzi platelet activating factor (Tc-PAF) on the
differentiation and infectivity of T. cruzi against mouse peritoneal macro-
phages. (a) Differentiation of T. cruzi from epimastigote to metacyclic
trypomastigote forms. Epimastigotes were maintained in the absence (control)
or in the presence of the following compounds: Tc-PAF (10K8M) and/or WEB
2086 (10K7M), anti-PAF receptor antibody (1:1000 dilution) or vehicle. (b)
Trypanosoma cruzi-macrophage interaction. Epimastigotes were maintained in
the absence (control) or in the presence of the following compounds: Tc-PAF
(10K8M) and/or WEB 2086 (10K7M), anti-PAF receptor antibody (1:1000
dilution) or vehicle. In both sets of experiments (a and b) the bars represent the
meanCSE of at least three independent experiments, which were performed
in triplicate. * means the results are significantly different from the control and
** means the results are significantly different from the Tc-PAF system
(P!0.05, one-way ANOVA, Student–Newman–Keuls post-test).
Fig. 4. Western blot analysis revealing reaction of anti-platelet activating factor
(PAF) receptor antibody with protein extract of Trypanosoma cruzi
epimastigotes (E) and intermediate cultures containing 48% epimastigotes
plus 52% trypomastigotes (Tr). Proteins obtained from epimastigotes (E) and
epimastigotes treated with WEB 2086 (WE) were probed with the anti-PAF
antibody. Protein extracts from T. cruzi intermediate cultures (Tr), rat platelets
(rat) and rabbit platelets (rab) were probed with the anti-PAFreceptor antibody.
MM: molecular mass. Asterisks show the 200 kDa protein and the arrowhead
indicates the 40 kDa protein.
M.T. Gomes et al. / International Journal for Parasitology 36 (2006) 165–173169
Synthesis of PAF has been found in some bacteria,
protozoa, yeasts, plants, marine invertebrates, lower ver-
tebrates, and mammals (Kulikov and Muzya, 1997).
However, all methods for PAF identification described to
date may be inadequate for samples containing extremely
low amounts of PAF (Owen et al., 2005), so that studies on
the biology of PAF have been limited by measurements
using bioassays, which was the case in the present study.
Here we show that T. cruzi synthesises a PAF-like lipid
(Tc-PAF) extracted from epimastigotes and labelled with
[14C]acetate, which co-migrated with authentic PAF in
HPTLC. Chemical hydrolysis of this lipid gave rise to
molecules that presented predicted behaviour by HPTLC.
This set of results is highly suggestive that T. cruzi parasites
synthesise a PAF-like molecule.
Ether lipids in general have been losing their role as main
structural molecule, while functions of PAF, a potent ether
lipid mediator, have been changing and enlarging. The
regulatory role of PAF is suggested to already appear in
protozoa and later be maintained during the subsequent
evolution of living organisms (Kulikov and Muzya, 1997). In
the present study, we show that Tc-PAF was able to promote
aggregation of rabbit platelets, which was prevented by pre-
incubation of the platelets with WEB 2086, a competitive
PAF antagonist that binds specifically to PAF receptors
(Chao and Olson, 1993). Similar results were obtained using
authentic PAF. Interestingly, neither authentic PAF nor
Tc-PAF displayed pro-aggregating activity when assayed
Fig. 5. Immunolocalisation in Trypanosoma cruzi epimastigotes of platelet activating factor (PAF) receptor. Immunofluorescence for PAF receptor (a, c and e);
phase-contrast (b, d and f). The epimastigotes were permeabilised (a–d) or not (e and f) with 1% Triton X-100 for 5 min. BarZ5 mm.
M.T. Gomes et al. / International Journal for Parasitology 36 (2006) 165–173170
using rat platelets (not shown), cells that lack PAF receptors
on their surface (Gomez-Cambronero et al., 1984).
A recombinant PAF-acetylhydrolase, which hydrolyses
PAF and related phospholipids containing modified snK2
acyl chains (Tjoelker et al., 1995), was used in order to further
explore the similarities between authentic PAF and Tc-PAF.
Here we show that a recombinant human PAF-acetylhydrolase
abolished the ability of both PAF and Tc-PAF to stimulate
rabbit platelet aggregation, reinforcing our idea that Tc-PAF
bears structural resemblance to authentic PAF. This obser-
vation is of special relevance since one of the most remarkable
aspects of PAF and related phospholipids is the strict structural
requirement for their recognition as substrates by this
degrading enzyme (Prescott et al., 2000). Also, Tc-PAF was
insensitive to phospholipase A1 from R. arrhizus, which is one
of the features of authentic PAF (Michel et al., 1988).
Autocrine activities related to cell growth and differen-
tiation have already been described both for free-living
protozoa (Vallesi et al., 1995) and for pathogenic protozoan
parasites (Vassella et al., 1997; Coppi et al., 2002).
Accordingly, the differentiation of epimastigotes into meta-
cyclic trypomastigotes was triggered by Tc-PAF. Similar
results have been reported for authentic PAF, both in T. cruzi
and Herpetomonas muscarum muscarum (Rodrigues et al.,
1996; Lopes et al., 1997). Also, here we show that peritoneal
mouse macrophage infection by T. cruzi was stimulated when
epimastigotes were pre-treated with Tc-PAF. Intriguingly, PAF
was shown to induce nitric oxide (NO) secretion by T. cruzi-
infected macrophages and the secreted NO inhibited intra-
cellular parasite growth, both in macrophages from wild and
PAF receptor-deficient mice (Aliberti et al., 1999; Talvani et
al., 2003). Nevertheless, several features in their approach are
different from those employed in the present study. For
instance, they used T. cruzi Y strain and inflammatory
macrophages, while we employed here macrophages from
naı ¨ve mice and T. cruzi Dm 28c clone, which is considered to
be from a different phylogenetic lineage (Souto et al., 1996).
However, the most important difference is that in the present
report the parasites were treated with Tc-PAF, prior to the
parasite–macrophage interaction, while in the other studies the
macrophages were pre-treated with PAF (Aliberti et al., 1999;
Talvani et al., 2003). In fact, both types of experiments were
performed in macrophage infection by Leishmania amazonen-
sis, where similar results were obtained for authentic PAF. A
decrease in the number of amastigotes was found in the
macrophages, when those were pre-treated with PAF, while
PAF-treated promastigotes induced an increase in the
macrophage infection (Rosa et al., 2001).
PAF effects described for several species of trypanosoma-
tids were all abrogated by WEB 2086, suggesting the presence
of PAF-receptors in these parasites (Dutra et al., 1998;
Rodrigues et al., 1999; Rosa et al., 2001). Actually, considering
PAF induction of secreted phosphatase activity in T. cruzi, PAF
effects were only observed in live cells, but not when
membrane enriched fractions were used, which is highly
suggestive that they occur through transduction of signals from
PAF receptors to the interior of the cells (Rodrigues et al.,
1999). Accordingly, PAF effects on the trypanosomatid H. m.
muscarum rely on the activation of casein kinase 2 (CK2),
which is modulated by several kinases and phosphatases. In
addition, WEB 2086 abrogated PAF-enhanced CK2 activity,
which is directly related to PAF-induced cell differentiation of
this trypanosomatid parasite (Silva-Neto et al., 2002).
Similarly, Tc-PAF effects described here were partially
inhibited by a polyclonal antibody raised against mouse PAF
receptor and/or abrogated by the PAF antagonist WEB 2086,
which highly suggests that Tc-PAF acts through a receptor.
This hypothesis was tested by probing whole protein extract
of T. cruzi intermediate cultures containing 48% epimastigotes
plus 52% trypomastigotes against mouse PAF receptor
polyclonal antibodies, through immunoblotting, and a 65 kDa
protein was recognised. This result is consistent with
mammalian PAF receptor, which molecular mass typically
ranges from 39 to 69 kDa (Chao and Olson, 1993; Ishii et al.,
2002). Two other lighter bands (40 and 200 kDa, respectively)
were also observed. Intriguingly, polymerisation of PAF-
receptor proteins has been described, where monomers of
40 kDa, dimers of 83 kDa and multimers of 200 kDa have been
demonstrated (Perron et al., 2003). Only the 200 kDa band was
observed when the proteins were obtained from culture
epimastigotes. In fact, expression of human PAF receptor of
different molecular mass has been described in keratinocytes,
which also seem to be related to cell differentiation (Bayerl
et al., 2003). Moreover, we show here that when the blots were
pre-incubated with WEB 2086, there was a fading in the signal
obtained from the antigen–antibody reaction. Interestingly,
monoclonal anti-idiotypic antibodies, which interact with PAF
receptors, stimulate rabbit platelets to aggregate and this
aggregation was totally blocked by the specific PAF receptor
antagonist WEB 2086 (Wang and Tai, 1991).
Viral infections have been described in several parasitic
protozoa, which could be an explanation for the acquisition of
mammalian genes, including those that code for membrane
receptors (Yu et al., 1996). Some receptors for mammalian
ligands have been described for a few trypanosomatids, such as
Leishmania (Barcinski et al., 1992). Here we show that the
T. cruzi putative PAF receptor was located on the surface of
culture epimastigotes, but primarily at the flagellar pocket
region. This result agrees with the hypothesis that the 200 kDa
element, here observed by immunoblotting, is a result of
multimerisation of PAF-receptor proteins, which influence
their internalisation, as described for human PAF-receptor
(Perron et al., 2003). On the other hand, similar localisation has
been described for T. cruzi TcRho1, a Rho family orthologue,
and for TcRAB7, a mammalian RAB7 homologue, being the
latter proven to be localised at the Golgi apparatus in
epimastigotes (De Melo et al., 2004; Araripe et al., 2004).
Actually, in contrast to most vertebrate cells, PAF produced by
Dictyostellium discoideum is not released to the extracellular
medium (Bussolino et al., 1991) and an intracellular role for
PAF involving signal transduction was postulated, as there are
evidences for the presence of PAF receptors inside those cells
(Sordano et al., 1993). Further studies need to be done in order
M.T. Gomes et al. / International Journal for Parasitology 36 (2006) 165–173171
to establish the internal location of the putative PAF receptor in
In conclusion, in this study we demonstrate that stimulated
T. cruzi synthesises a lipid with PAF-like activity that is both
recognised by the mammalian PAF receptor and by the
metabolising enzyme PAF-acetylhydrolase. Also, we show that
Tc-PAF stimulates cell differentiation and infectivity of these
parasites towards mouse peritoneal macrophages. Whether
these activities correspond to authentic PAF or to structurally
related phospholipids that also stimulate the PAF receptor is
still under investigation. We believe this is the first report of an
intrinsic protozoan parasite lipid molecule regulating, through
receptor interaction, the differentiation and infectivity of this
We thank Drs Thomas M. McIntyre, Renato Cordeiro,
Patricia Bozza, Rafael Linden, Ma ´rio Silva-Neto, Felipe Dias,
Danielle Vieira, Carla Polycarpo, Maria H. Villas-Bo ˆas,
Lucianne Madeira, Luciana Zimmermann, Alan Carneiro and
Norton Heise, for valuable discussions. This work was
supported by grants from the Brazilian Agencies CNPq,
FINEP, FAPERJ, FAPESP, CAPES and PRONEX. ICA is
supported by a grant from BBRC/Biology/UTEP (NIH No.
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