Microbes and Infection 9 (2007) 915e921

Review

Phytomonas serpens: immunological similaritieswith the human trypanosomatid pathogens

Andre L.S. Santos a,*, Claudia M. d’Avila-Levy b, Camila G.R. Elias a,Alane B. Vermelho a, Marta H. Branquinha a

a Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goes (IMPPG), Centro de Ciencias da Saude (CCS),Bloco I, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fund~ao, Rio de Janeiro, RJ 21941-590, Brazil

b Departamento de Bioquımica e Biologia Molecular, Fundac~ao Oswaldo Cruz, Rio de Janeiro, Brazil

Received 5 January 2007; accepted 30 March 2007

Available online 12 April 2007

Abstract

The present review provides an overview of recent discoveries concerning the immunological similarities between Phytomonas serpens, a to-mato parasite, and human trypanosomatid pathogens, with special emphasis on peptidases. Leishmania spp. and Trypanosoma cruzi express pep-tidases that are well-known virulence factors, named leishmanolysin and cruzipain. P. serpens synthesizes two distinct classes of proteolyticenzymes, metallo- and cysteine-type peptidases, that share common epitopes with leishmanolysin and cruzipain, respectively. The leishmano-lysin-like and cruzipain-like molecules from P. serpens participate in several biological processes including cellular growth and adhesion to thesalivary glands of Oncopeltus fasciatus, a phytophagous insect experimental model. Since previous reports demonstrated that immunization ofmice with P. serpens induced a partial protective immune response against T. cruzi, this plant trypanosomatid may be a suitable candidate forvaccine studies. Moreover, comparative approaches in the Trypanosomatidae family may be useful to understand kinetoplastid biology, bio-chemistry and evolution.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Phytomonas serpens; Trypanosomatid; Peptidase; Trypanosoma cruzi; Leishmania spp; Immunological similarities; Leishmanolysin; Cruzipain

www.elsevier.com/locate/micinf

1. Introduction

The family Trypanosomatidae of the order Kinetoplastida ischaracterized by flagellated protozoa that present a characteris-tic structure named kinetoplast, a distinct region of the singlelong mitochondrion that contains a mass of coiled DNA fila-ments compacted in minicircles and maxicircles, which con-stitutes approximately 5% of the parasite DNA [1,2]. Mostof the recognized genera are parasitic protozoa and, dependingon host range, have been classified as either monoxenoustrypanosomatids including Leptomonas, Crithidia, Blastocri-thidia, Herpetomonas and Rhynchoidomonas or heteroxenoustrypanosomatids represented by the genus Trypanosoma,

* Corresponding author. Tel.:þ55 21 2562 6740; fax: þ55 21 2560 8344.

E-mail address: andre@micro.ufrj.br (A.L.S. Santos).

1286-4579/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserve

doi:10.1016/j.micinf.2007.03.018

Leishmania, Endotrypanum and Phytomonas (Fig. 1). Mem-bers of the heteroxenous group comprises species such asTrypanosoma cruzi, Trypanosoma brucei and other Africantrypanosomes, and several Leishmania spp. that are the causa-tive agents responsible for Chagas’ disease, African sleepingsickness and human leishmaniasis, respectively. These trypa-nosomatid parasites have complex life cycles that involve mul-tiple hosts (Fig. 1). Inside the insect vector or mammalianhost, these parasites undergo differentiation and multiplicationphases [1,2].

In 1909, the name Phytomonas was introduced to designatetrypanosomatids originally found in the latex from Euphorbia-ceae and later in a wide variety of plant species. These trypa-nosomatids are etiologic agents of plant diseases found overa wide range of geographical areas including southern Brazil,North and Central Africa and, in the western hemisphere, inseveral European countries. Phytomonads are found in plants

d.

916 A.L.S. Santos et al. / Microbes and Infection 9 (2007) 915e921

of great economical importance including cashew, coffee, cas-sava, coconut and oil palms, infecting also edible fruits such astomato, orange, guava, grape, star fruit and many others. Theyalso act as parasites of lactiferous plants without any apparentpathogenicity [3e5]. These differences in pathogenicity couldbe linked to host-tissue distribution of the microorganism,which is restricted mainly to three environments: latex ducts,fruits/seeds and phloem. Only isolates from the latter grouphave been associated with severe pathogenesis [6].

The presence of trypanosomatids in plants of economic in-terest has attracted the attention of several research groups andthe genus Phytomonas is the most studied plant trypanoso-matid, although other genera including Leptomonas, Crithidiaand Herpetomonas had been isolated from plants [5] (Fig. 1).The life cycle of the parasite in the insect vector and plantswas demonstrated by Jankevicius and co-workers [7] whoshowed in controlled laboratory cage experiments that P.serpens, the tomato parasite, is transmitted by the bite of thecoreid insect Phthia picta. However, this mode of transmissionproved to be difficult to verify experimentally for other Phyto-monas species [5]. The possibility of a life cycle with alternat-ing plant and insect hosts raises questions about thebiochemical and molecular adaptations involved, probably es-tablished during the evolutive process.

Different groups have studied some peculiarities of Phyto-monas metabolism in more detail [5,8,9]. Investigations of theculture form of Phytomonas spp. isolated from Euphorbiaplants showed that the parasites, which are found in carbohy-drate-rich tissues of plants, lack a cytochrome-mediated respi-ratory chain and Krebs cycle. They excrete hydrolases thatefficiently degrade polysaccharides to monosaccharides andpossess a high rate of glycolytic ATP production [8,9]. Littleis known about the Phytomonas metabolism in the insect vec-tor stage, where it has been postulated that amino acids wouldbe the main energy source [8,9].

Phytomonas

plant

invertebratevertebrate

RRR

LeishmaniaTrypanosoma

Endotrypanum

CrithidiaLeptomonas

BlastocrithidiaHerpetomonas

RhynchoidomonasWallaceinaAngomonasStrigomonas

Fig. 1. The life cycles of the trypanosomatid genera. In the heteroxenous ones,

trypanosomatids alternate between an invertebrate host and vertebrates (red

arrows) or plants (green arrows). The development of monoxenous genera

occurs in a single invertebrate host (dark blue arrow), although insect trypano-

somatids can be found in plants and recent reported cases describe the pres-

ence of these trypanosomatids in vertebrates (light blue arrows). The insect

trypanosomatid genera highlighted in grey were recently proposed (reviewed

in [11]).

The knowledge on a number of facets of the Phytomonasfundamental biology has been hampered by the difficulty inobtaining pure cultures in vitro [5]. Therefore, compared toother trypanosomatids, a vast information lag is in place forseveral essential issues such as life cycle, transmission, patho-genicity, immunogenicity, treatment and prophylaxis. The pur-pose of this review is to provide an overview of recentdiscoveries concerning the immunological similarities be-tween P. serpens and human trypanosomatid pathogens, withespecial attention to the proteolytic enzymes produced byT. cruzi and Leishmania spp.

2. Leishmanolysin-like molecules in P. serpens

Promastigotes of all Leishmania species examined to datepossess on their surface a membrane glycoprotein known asleishmanolysin, promastigote surface peptidase (PSP), majorsurface peptidase (MSP), EC 3.4.24.36 or gp63. This proteinis a zinc-metallopeptidase mainly bound to the membrane bya glycosylphosphatidylinositol (GPI) anchor and is highly con-served among species of Leishmania. The abundance of leish-manolysin coupled with its apparent conservation among thedifferent Leishmania species suggests that leishmanolysinserves some important functions for the parasite. Therefore,leishmanolysin molecules on the surface of promastigotesform a significant part of the interface between the invadingparasite cell and the mammalian or insect host at the time ofinfection. Alternatively, a significant proportion of leishmano-lysin is released through auto-proteolysis by Leishmania pro-mastigotes into the extracellular medium [reviewed in 10].

The functions performed by leishmanolysin on the interac-tion with the vertebrate host are covered by recent reviews[10,11]. The enzyme seems to be involved in extracellularmatrix degradation, resistance of promastigotes to comple-ment-mediated lysis, intracellular amastigote survival withinmacrophage phagolysosomes, and either a direct or indirectrole in receptor-mediated uptake of Leishmania. Thus, leish-manolysin participates in the interaction of both parasitestages with the mammalian host, although the exact molecularmechanisms have not been fully elucidated [10].

Leishmanolysin might also act before mammalian infection,ensuring that maximum numbers of parasites are produced andreleased from the insect vector. Nevertheless, leishmanolysinprecise role in the life cycle of the parasite during the insectcolonization remains under speculation. In this context, leish-manolysin is predominantly present on the surface of promas-tigotes residing in the midgut of the phlebotomine sandflyvector [12]. However, data from leishmanolysin knockouts inL. major do not support a major role for leishmanolysin in par-asite survival in the insect, since the number of metacyclic pro-mastigotes in the anterior gut of Phlebotomus dubosquiinfected with either the knockouts or the wild type was compa-rable [13]. Conversely, Hajmova and co-workers [14] reportedthat the down-regulation of leishmanolysin in a L. amazonensisclone adversely affects its early development in the neotropicalLutzomyia longipalpis sand fly. The possibility exists that leish-manolysin may function differently for these two distinct

917A.L.S. Santos et al. / Microbes and Infection 9 (2007) 915e921

Leishmania species in their interactions with different inverte-brate vector species.

Some insights on the functions performed by leishmanoly-sin on the insect stage might be gained by analyzing leishma-nolysin homologues in lower trypanosomatids (reviewed in[11]). Although direct evidence had never been obtained untilrecently, it has been well accepted that the leishmanolysin-like molecules from lower trypanosomatids might fulfill a nutri-tional role in the insect midgut (reviewed in [11]), since insectcolonization is believed to be the only life cycle stage commonto the monoxenous and heteroxenous flagellate trypanosoma-tids (Fig. 1). However, there is increasing evidence indicatingthat lower trypanosomatids can infect and survive in vertebratecells reviewed in [15]. Homologues of leishmanolysin were de-scribed either on the cell surface or being released by lower try-panosomatids belonging to the genera Crithidia, Herpetomona,Blastocrithidi, Leptomonas and Phytomonas (reviewed in [11]).

The characterization of the released peptidases by P.serpens through sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) co-polymerized with gelatin, ca-sein, bovine serum albumin or hemoglobin indicated that thisplant trypanosomatid releases exclusively metallopeptidases,migrating at 70, 90 and 94 kDa [16]. The propagation of theplant flagellates in the insect hosts would require the abilityto utilize amino acids as a major source of energy, since theKrebs cycle is inactivated and sugars are not abundant in theinsect vector [8,9]. Therefore, the extracellular metallopepti-dase activities detected in P. serpens could be involved in pro-tein degradation for amino acid uptake by the parasite [16].

The ubiquity of cysteine and metallopeptidases has beenshown in a wide range of trypanosomatids [17,18]. Curiously,no cell-associated metallopeptidase activity was identified inP. serpens even when a five-fold excess of parasite extractwas loaded on gelatin-SDS-PAGE using a modified lysis buffersupplemented with the cysteine peptidase inhibitor E-64[19,20]. Although a cell-associated metallopeptidase activityhas not been detected, two cellular polypeptides migrating inthe 63e52 kDa range cross-reacted with anti-leishmanolysinantibodies [19]. Additionally, a 60 kDa polypeptide was shownto be GPI-anchored to the plasma membrane and to be releasedby the parasite to the spent culture medium [19]. The surface lo-cation of the leishmanolysin-like molecules in P. serpens wasalso demonstrated by agglutination assay [11], flow cytometryand immunofluorescence microscopy analyses [19] (Fig. 2).

Since very little is known about the role of leishmanolysinin the interaction with the invertebrate hosts, our researchgroup has been using the phytophagous insect Oncopeltusfasciatus as an experimental model for the study of the roleof peptidases on the interaction of Phytomonas with the insectvector [19,20]. O. fasciatus (Hemiptera: Lygaeidae) is a goodmodel for studies of hosteparasite relationships, since it is thenatural host of Crithidia acidophili, Leptomonas oncopelti,Phytomonas elmassiani (reviewed in [1]) and Leptomonas wal-lacei [21], which are found in its intestinal tract. O. fasciatushas also been experimentally infected with other trypanoso-matids isolated from their original insect hosts [22], includingP. serpens [19,20].

The analysis of the adhesion of anti-leishmanolysin-treatedor control P. serpens to explanted salivary glands of O. fascia-tus indicated that this molecule is relevant to the interaction ofthe plant flagellate with the vector epithelium [19]. The searchfor a receptor of leishmanolysin in the salivary glands of O.fasciatus revealed the presence of a protein of 50 kDa with af-finity to the purified leishmanolysin-like molecule from P.serpens promastigotes (C.M. d’Avila-Levy et al., unpublisheddata). The identification of receptors responsible for the para-site attachment to the insect is of interest since proteinaceousepitopes are potential universal disease transmission blockingtargets. The isolation and sequencing of these insect cellularreceptors is an open field.

control +anti-leishmanolysin

A

B 512

0

100 101 102 103

Cel

l cou

nt

Fluorescence intensity

a

c

ba b

Fig. 2. (A) Binding of anti-leishmanolysin antibodies to the cell surface of P.

serpens promastigote cells. Live parasites were incubated in the presence of

the rabbit pre-immune serum (control ) or rabbit anti-leishmanolysin poly-

clonal antibody (þanti-leishmanolysin) at 1:100 dilution for 1 h. Then, cells

were fixed and experimental systems were analyzed under phase contrast im-

ages. Note the agglutination of parasites by the anti-leishmanolysin antibody.

(B) Flow cytometry analysis showing the anti-leishmanolysin antibody bind-

ing to the cell-surface of P. serpens. Paraformaldehyde-fixed cells were treated

(c) or not (a,b) with phospholipase C (0.1 U/ml for 16 h) and subsequently

incubated in the absence (a) or in the presence (b,c) of anti-leishmanolysin

antibody, and then analyzed by flow cytometry. Note the reduction in fluores-

cence intensity (c) due to the release of GPI-anchored leishmanolysin by phos-

pholipase. The inset shows the differential interferential contrast images (a)

and the corresponding immunofluorescence labeling (b) of P. serpens by

anti-leishmanolysin antibody. The bars represent 5 mm.

918 A.L.S. Santos et al. / Microbes and Infection 9 (2007) 915e921

3. Trypanosoma cruzi-like molecules in P. serpens

The lack of specificity and extensive cross-reactivity be-tween flagellates of different genera and species of trypanoso-matids was previously observed. In those studies, theimmunological cross-reactivity between T. cruzi and insect try-panosomatids that belong to the genera Crithidia, Herpetomo-nas, Leptomonas and Blastocrithidia as well as between T.cruzi and Leishmania spp. was observed. Bregano andco-workers [23] clearly demonstrated that P. serpens is highlyimmunogenic and presents antigenic cross-reactivity with T.cruzi. Sera from patients with Chagas’ disease presenteda strong reactivity with P. serpens antigens by serological as-says, although the titers were lower than those observed withT. cruzi. After absorption with P. serpens culture forms, humanserum from Chagas’ disease patients presented a significant re-duction of indirect immunfluorescence titers when tested withT. cruzi antigens. In addition, rabbit and mouse hyperimmuneserum raised against living forms of T. cruzi or P. serpenswas able to recognize both protozoa antigens in immunofluo-rescence and agglutination assays. Strikingly, BALB/c miceimmunized by the intraperitoneal or oral route with P. serpensand later challenged with a lethal inoculum of T. cruzi trypo-mastigote forms showed a significant decrease in parasitemiaand mortality. These results demonstrated that the previous im-munization of mice with P. serpens induces a partial protectiveimmune response against T. cruzi. As pointed out by the au-thors, these findings must be correlated to the possible inges-tion by humans or animals of living plant trypanosomatidspresent in naturally infected edible fruits, a fact that could po-tentially prime the immune response to T. cruzi antigens andinterfere with the development of T. cruzi infection [24].

Oral administration of antigens can induce antigen-specificperipheral immune tolerance. Accumulated evidence showsthat oral tolerance can be mediated by orally activated humoraland cellular factors. Oral tolerance therapy is a promising treat-ment for several autoimmune disorders. In animal and humanmodels, the development of several T cell-mediated autoim-mune diseases, such as multiple sclerosis, rheumatoid arthritis,uveitis and type-1 diabetes can be inhibited by oral immuniza-tion of the respective antigens. In allergy, oral administration ofcertain allergens can prevent or reduce both contact and atopicdermatitis [25]. Despite these encouraging results, the useful-ness of this approach for an alternative immunotherapy in Cha-gas’ disease needs to be investigated further.

In view of the fact that the protective mechanisms induced bythe immunization with parasites non-pathogenic to vertebrates,such as phytomonads, are unknown, there is a need to better un-derstand the relationships between P. serpens and the develop-ment of anti-T. cruzi immunological responses. In this sense,Pinge-Filho and co-workers [24] investigated the effect oforal immunization with living forms of P. serpens in either ge-netically manipulated mice lacking the inducible nitric oxidesynthase (iNOS) gene or wild type mice treated with inhibitorsof nitric oxide production, such as aminoguanidine, that weresubsequently intraperitoneally infected with T. cruzi. Asdemonstrated earlier by the same group [23], a reduction in

parasitemia and an increase in survival were observed in T.cruzi-infected wild type mice previously immunized with P.serpens. In contrast, iNOS knockout mice immunized andwild type mice immunized and treated with aminoguanidinepresented parasitemia and mortality rates comparable to thoseof infected and non-immunized mice. Wild type mice immu-nized with P. serpens showed fewer amastigotes nests in theheart following an acute T. cruzi infection. In addition, prior im-munization resulted in greater infiltration of inflammatory cellsduring the acute phase of a T. cruzi infection, which may explainthe reduction of parasitemia in this tissue observed during thepeak of infection. The authors concluded that protective immu-nity against T. cruzi infection induced by immunization with P.serpens is dependent upon enhanced NO production in an ex-perimental Chagas’ disease model [26]. Moreover, NO wasable to inhibit cruzipain, the major T. cruzi peptidase, by nitro-sylation of the active site cysteine residue [26].

3.1. Cruzipain-like molecules in P. serpens

Recently, our group described the production of two distinctcell-associated cysteine peptidases of 38 and 40 kDa by pro-mastigotes of P. serpens [20]. These peptidases possess severalcommon features with the cruzipain of T. cruzi, such as prefer-ential cytoplasmic location, best activity at acidic pH range,requirement of a reducing agent (such as dithiothreitol), andinhibition profile. Intriguingly, the 40 kDa cysteine peptidasewas also detected in the hydrophobic phase (membrane rich-fraction) of P. serpens cells after Triton X-114 extraction.Additionally, we demonstrated through immunoblotting thatanti-cruzipain polyclonal antibodies recognized two majorpolypeptides in P. serpens, including a 40 kDa surface compo-nent. Flow cytometry and ultrastructural immunocytochemicalanalyses confirmed that this cruzipain-like protein has a loca-tion on the cell surface [20] (Fig. 3A). In T. cruzi, cruzipainmolecules were also detected on the surface of the differentmorphological parasite stages [27]. Interestingly, phospholipaseC-treated P. serpens showed a reduction on the binding of anti-cruzipain antibody when compared to the non-treated parasites,suggesting that these molecules are expressed on the cell surfacethrough a GPI anchor (Elias et al., unpublished results).

With reference to the possible functions of cruzipain, in ad-dition to its obvious role in parasite nutrition as its major lyso-somal T. cruzi peptidase, there is indirect evidence fora possible participation of the enzyme in penetration of the try-pomastigotes into the host cell, in the defense of the parasiteagainst the immune system of the mammal, and in the differen-tiation processes leading from one stage to the next in the lifecycle of the parasite [27]. These results lead us to investigatethe involvement of cysteine peptidases of P. serpens in the in-teraction with explanted salivary glands of the phytophagousinsect O. fasciatus. When P. serpens cells were pre-treatedwith either cysteine peptidase inhibitors (antipain, cystatin, leu-peptin, iodoacetamide and E-64) or anti-cruzipain antibodies,a significant reduction of the interaction process was observed.Furthermore, we showed that the cysteine peptidase inhibitorsat 10 mM were able to arrest cellular growth as well as to

A.L.S. Santos et al. / Microbes a

A

B

living cells

dead cells

P. serpenspromastigote

leupeptin / antipainiodoacetamidesurface cysteine peptidase

Fig. 3. (A) Ultrastructure immunocytochemical analysis of P. serpens incu-

bated in the presence of anti-cruzipain antibody (1:50) and subsequently in

the presence of gold-labeled antibody. Gold particles (arrows) were detected

in the cell surface and in the intracellular compartment. Bar, 10 mm. (B) Pro-

posed representation of cysteine peptidase inhibitors (CPIs) action on P.

serpens promastigote cells in vitro. Two distinct events were observed: CPIs

with reversible or irreversible action. The first mechanism resulted first in

the binding of leupeptin/antipain on the cell surface cysteine peptidase (prob-

ably the 40 kDa component) (1) and then in shedding of membrane fragments

(2), containing the inhibitor-enzyme complex (3), as a defense mechanism

against the drug that is toxic to the parasite cells, promoting cellular growth

retardation. However, when parasites were washed and re-incubated in

a drug-free medium, the cells reestablished their normal growth [20]. The

promote alterations on the P. serpens morphology, includingthe parasites becoming short and round. Additionally, iodoace-tamide induced ultrastructural alterations, such as disintegra-tion of cytoplasmic organelles, swelling of the nucleus andkinetoplast-mitochondrion complex, which culminated in theparasite death, in a mechanism similar to necrosis (Fig. 3B).Conversely, leupeptin and antipain induced the appearance ofmicrovillar extensions and blebs on the cytoplasmic mem-brane, resembling a shedding process [20] (Fig. 3B). Since atleast one cysteine peptidase activity (40 kDa) was identifiedon the cell surface of P. serpens, we postulated that the shed-ding of the cell surface membrane detected in parasites treatedwith leupeptin and antipain could be a defense mechanismfrom the parasites, which released the complex of cysteine pep-tidase bound to the inhibitor to the extracellular environmentfor neutralizing the toxic effects of the cysteine peptidase in-hibitors (Fig. 3B). This hypothesis was corroborated by the ul-trastructure immunocytochemical analysis, which showed thepresence of the cruzipain-like molecule on the cell surfaceand in small membrane fragments released by the parasites[20]. In T. cruzi, peptidomimetic cysteine peptidase inhibitorsprevent the normal autocatalytic processing and trafficking ofcruzipain within the Golgi apparatus, which leads to massiveaccumulation of precursor protein in the Golgi complex, culmi-nating with peripheral distention of cisternae. These major al-terations of the Golgi complex parallel the death of T. cruziepimastigotes [28]. Mechanisms previously associated with de-creased sensitivity or resistance to chemotherapy in parasiticprotozoa include decreased drug uptake, increased export ofdrugs, decrease in drug activation, and alterations of the targetenzyme to decrease drug-binding [29]. Cysteine peptidase in-hibitors are an emerging therapy amenable for the treatmentof parasite protozoan infections [27,30]. Taken together, theseresults suggest that cysteine peptidases participate in several bi-ological processes in P. serpens, including cellular growth andinteraction with the invertebrate vector.

4. Conclusions

The studies of trypanosomatid peptidases are merited be-cause they may serve as a model for understanding the functionand evolution of peptidases in general. As there is little Phyto-monas genomic information, the study of immunological crossreactivity to demonstrate evolutionary similarity is appropriate.In this respect, P. serpens synthesizes two distinct classes ofproteolytic enzymes, metallo- and cysteine-type peptidases,that shares common epitopes with leishmanolysin and cruzi-pain, respectively. The evolutionary proximity of P. serpensto important human pathogens [31] is possibly reflected inthe similarity of some aspects of the basic cellular machinery.It can be speculated that similar peptidases are subjected to theselective pressures of similar environments (invertebrate host)and it is expected to be structurally and functionally conserved

second mechanism of CPI action was observed with iodoacetamide (4), which

is very toxic, causing a complete destruction of intracellular organelles, includ-

ing nucleus and mitochondrion, culminating with cell death (5).

919nd Infection 9 (2007) 915e921

920 A.L.S. Santos et al. / Microbes and Infection 9 (2007) 915e921

in distinct trypanosomatids. Comparison of amino acid se-quences, three-dimensional structures and the biochemicalmechanism of action of peptidases could assist in decipheringof their course of evolution.

Moreover, efforts to generate a safe and effective means ofimmunizing vertebrates, including humans, against T. cruziand Leishmania spp. infection have been ongoing for manyyears with only limited success. The use of live parasites gen-erally presents a risk of active infection if virulent microorgan-isms are used, because some may escape the attenuationprocess. P. serpens is a trypanosomatid isolated from toma-toes, a fruit usually consumed in natura by humans, that ex-press homologues of Leishmania spp. and T. cruzi virulencefactors, and does not cause disease in humans. Therefore,more attention should be given to this plant trypanosomatid,which is useful not only as a model for the biochemical andimmunological studies among the Trypanosomatidae family,but also as a candidate for vaccine studies.

Acknowledgements

The authors whish to thank Ms Ieda C.M. de Castro e Silva,Geralda R. Almeida, Fernanda M. Pereira, Ana C. Nogueira deMelo, Fernanda M. Araujo, Fernanda A. Marinho and Lıvia O.Santos for their technical assistance; and Felipe A. Dias, DrAngela H.C.S. Lopes and Dr Elvira M.B. Saraiva (Institutode Microbiologia Prof. Paulo de Goes, Universidade Federaldo Rio de Janeiro) for the help with the insect-trypanosomatidinteraction experiments. The authors are grateful to DrKwang-Poo Chang (University of Health Sciences, ChicagoMedical School, USA) and Peter Overath (Max-Planck-Insti-tut fur Biologie, Abteilung Membranbiochemie, Germany)for kindly providing the valuable anti-gp63 antibodies; DrJuan-Jose Cazzulo (Instituto de Investigaciones Biotecnologi-cas, Universidad Nacional de General San Martin, BuenosAires, Argentina) for supplying the anti-cruzipain antibody;Laboratorio de Fisiologia e Controle de Vetores (Departa-mento de Entomologia, Instituto Oswaldo Cruz, Rio de Ja-neiro, Brazil) for kindly providing Aedes aegypti; and DrPatrıcia Azambuja (Instituto Oswaldo Cruz, Rio de Janeiro,Brazil) for providing Oncopeltus fasciatus. We also wish tothank Dr Tha€ıs Souto-Padron and Rachel Ribeiro for the trans-mission electron microscopy analysis. This study was sup-ported by the Brazilian agencies: MCT/CNPq (ConselhoNacional de Desenvolvimento Cientıfico e Tecnologico),CEPG/UFRJ (Conselho de Ensino para Graduados e Pesqui-sas), FAPERJ (Fundac~ao de Amparo a Pesquisa do Estadodo Rio de Janeiro), FUJB (Fundac~ao Universitaria Jose Boni-facio) and CAPES (Coordenac~ao de Aperfeicoamento de Pes-soal de Nıvel Superior).

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