Phytomonas serpens: cysteine peptidase inhibitors interfere

with growth, ultrastructure and host adhesion

Andre L.S. Santos *, Claudia M. d’Avila-Levy, Felipe A. Dias, Rachel O. Ribeiro,

Fernanda M. Pereira, Camila G.R. Elias, Thaıs Souto-Padron, Angela H.C.S. Lopes,

Celuta S. Alviano, Marta H. Branquinha, Rosangela M.A. Soares

Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goes (IMPPG), Centro de Ciencias da Saude (CCS),

Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Fundao, Cidade Universitaria, Rio de Janeiro, RJ, 21941-590, Brazil

Received 12 July 2005; received in revised form 8 September 2005; accepted 9 September 2005

Abstract

In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid

Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 mM were able to arrest cellular growth as well as promote alterations

in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as

disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast–mitochondrion complex, which culminated in parasite death.

Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process.

A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally,

we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a

40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural

immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released

from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary

glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine

peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest

that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector.

q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Phytomonas serpens; Trypanosomatids; Cysteine peptidase inhibitors; Peptidases; Cell surface; Growth; Cellular interaction; Ultrastructural alterations;

Cruzipain

1. Introduction

Trypanosomatids are flagellate parasites with a wide range

of hosts including insects, mammals, plants and other protists.

Plant trypanosomatids are parasites of several families of

plants with a wide geographical distribution, being present in

South America, Africa, Europe and Asia (Dollet, 1984;

Camargo et al., 1990). Infection of plants with trypanosomatids

has been known since 1909 (Lafont, 1909). Immediately after

their discovery, Donovan proposed the generic name

0020-7519/$30.00 q 2005 Australian Society for Parasitology Inc. Published by E

doi:10.1016/j.ijpara.2005.09.004

* Corresponding author. Tel.:C55 21 2562 6740; fax: C55 21 2560 8344.

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

Phytomonas to distinguish them from trypanosomes isolated

from animals (Donovan, 1909).

The plant flagellates inhabit the phloem (phloemicola), latex

(lacticola), flowers (floricola) and fruits (fructicola) of many

plant families (Dollet, 1984; Camargo et al., 1990; Camargo,

1999). In many cases, this parasitism exists without any

apparent pathogenicity (Cunha et al., 2000). However, these

flagellates have attracted attention because they can be

responsible for significant economic losses in agriculture. For

instance, phloem-dwelling phytomonads cause acute and

chronic yellowing of leaves in coffee plants, ‘hartrot’ in

coconut palms and ‘marchitez wilt’ in oil palms, while the

latex-associated Phytomonas francai causes ‘chochamento das

raızes’ (empty roots) in manioc (reviewed by Camargo, 1999).

These parasites are transmitted by phytophagous hemipteran

International Journal for Parasitology 36 (2006) 47–56

www.elsevier.com/locate/ijpara

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A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–5648

insects of the families Coreidae, Lygaeidae, Pyrrhocoridae and

Pentatomidae (Camargo, 1999). In addition, a recent report

showed that Phytomonas serpens, a tomato parasite, shares

antigens with Trypanosoma cruzi, which were strongly

recognised by human sera from patients with Chagas’ disease,

and induced a protective immunity in BALB/c mice (Bregano

et al., 2003).

The functionally diverse cysteine peptidases of various

pathogens have received special attention as potential targets

for chemotherapeutic intervention because they play important

roles in facilitating the survival and growth of the parasites in

hosts (Sajid and McKerrow, 2002). For instance, our research

group demonstrated that cysteine peptidases are preferentially

expressed in virulent as opposed to avirulent Leishmania

amazonensis promastigotes (Soares et al., 2003). Additionally,

Leishmania mexicana null mutants for the multicopy cysteine

peptidase gene family lmpcb have their infectivity to

macrophages reduced by 80% and produced subcutaneous

lesions in BALB/c mice at a slower rate than wild-type

parasites (Mottram et al., 1996). Proteolytic inhibitor

compounds are an interesting alternative to facilitate the

cost-effective development of new anti-parasitic chemotherapy

(Selzer et al., 1999; Cazzulo et al., 2001). In this sense, cysteine

peptidase inhibitors (CPIs) have been shown to kill T. cruzi

(Ashal et al., 1990; Franke de Cazzulo et al., 1994),

Trypanosoma congolense (Mbawa et al., 1992), Trypanosoma

brucei (Troeberg et al., 1999), Leishmania major (Selzer et al.,

1999) and Trichomonas vaginalis (Irvine et al., 1997).

In the present work, the susceptibility of P. serpens to a

panel of five different CPIs was determined by analyzing their

effects on the cellular growth pattern, on the ultrastructural

level and on the adhesion index to explanted salivary glands of

the phytophagous insect Oncopeltus fasciatus. In addition, the

cellular proteolytic profile was analysed including the

relationship of P. serpens proteins to cruzipain, the major

cysteine peptidase from T. cruzi (reviewed by Cazzulo et al.,

2001).

2. Material and methods

2.1. Chemicals

Media constituents, reagents used in electrophoresis, buffer

components, nitrocellulose membrane and reagents for

chemiluminescence detection were purchased from Amersham

Life Science (Little Chalfont, England). Low molecular mass

standards were acquired from GIBCO BRL (Grand Island, NY,

USA). Dithiothreitol (DTT), gelatin, all proteolytic inhibitors

(antipain, cystatin, leupeptin, trans-epoxysuccinyl L-leucy-

lamido-(4-guanidino) butane [E-64], iodoacetamide, 1,10-

phenanthroline and phenyl-methyl sulfonyl-fluoride [PMSF])

and the secondary antibodies were purchased from Sigma

Chemical Co. (St Louis, MO, USA). The anti-cruzipain

polyclonal antibody was a gift from Dr Juan-Jose Cazzulo

(Instituto de Investigaciones Biotecnologicas, Universidad

Nacional de General San Martin, Buenos Aires, Argentina).

All other reagents were analytical grade.

2.2. Microorganisms and culture conditions

Phytomonas serpens (isolate 9T; CT-IOC-189), isolated

from tomato (Lycopersicon esculentum), was provided by Dr

Maria Auxiliadora de Sousa (Colecao de Tripanosomatıdeos,

Instituto Oswaldo Cruz, Rio de Janeiro). The plant flagellate

was grown in 50 ml Erlenmeyer flasks containing 20 ml of

brain heart infusion (BHI) medium, pH 7.4, at 26 8C (Santos

et al., 2002). The flagellate T. cruzi (Dm28c strain) is part of

our laboratory collection, and epimastigote forms were

cultured in liver infusion trypticase (LIT) medium, for 72 h

at 26 8C (Souto-Padron et al., 1990). Both culture media were

supplemented with 10% (v/v) heat-inactivated FCS.

2.3. Effects of CPIs on the growth rate

The experiments were performed in 10!100 mm glass

tubes containing 1 ml of BHICFCS medium. The inoculum

consisted of 10% (v/v) of a 48 h culture containing about 2.0!106 P. serpens cells. All CPIs (antipain, cystatin, leupeptin,

E-64 and iodoacetamide) were dissolved in water at 1 mM, and

then filter-sterilised in 0.22 mm membranes (Millipore). The

parasites were grown at 26 8C for periods ranging from 24 to

96 h, in the absence or presence of each peptidase inhibitor at

10 mM. Alternatively, parasites grown for 48 h in the presence

of each peptidase inhibitor were washed five times in cold PBS

(150 mM NaCl, 20 mM phosphate buffer, pH 7.2) prior to

resuspension in a drug-free fresh medium and allowed to grow

for another 48 h. Cellular growth was estimated daily by

counting the flagellates in a Neubauer chamber (Santos et al.,

2003).

2.4. Effects of CPIs on the ultrastructure of the flagellates

Control and CPI-treated phytomonads were grown for 48 h

at 26 8C, collected by centrifugation for 15 min at 1500!g at

4 8C, washed three times with PBS and fixed for 2 h with 1.5%

glutaraldehyde and 4% paraformaldehyde in 0.05 M cacody-

late buffer pH 7.2 containing 1 mM CaCl2. The cells were then

rinsed in cacodylate buffer and postfixed for 1 h in a solution

containing 1% osmium tetroxide and 0.8% potassium

ferricyanide in 0.1 M cacodylate buffer pH 7.2 supplemented

with 1 mM CaCl2. After being rinsed in the same buffer, the

parasites were dehydrated in graded acetone and embedded in

Epon resin. Ultrathin sections were stained with uranyl acetate

and lead citrate and observed in a Zeiss Em-900 transmission

electron microscope operating at 80 kV (Souto-Padron et al.,

1990).

2.5. Detergent extraction of proteins

Phytomonas serpens cells (1.0!108) were harvested as

described before and then lysed in 200 ml of 2% (w/v)

precondensed Triton X-114 in TBS (150 mM NaCl, 10 mM

Tris, pH 7.4) in a vortex by alternating 1 min shaking and 2 min

cooling intervals. Insoluble material was precipitated (10,

000!g for 30 min at 4 8C) and the supernatant was incubated

A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–56 49

at 37 8C to induce detergent phase separation. After centrifu-

gation for 15 min at 10,000!g at 25 8C, the complete

separation of the detergent-rich and the aqueous phases was

achieved. The separated phases were washed three times with

TBS and 5% Triton X-114, respectively. Alternatively, the

washing steps were omitted, the hydrophilic/hydrophobic

interface was discharged and each phase was supplemented

with 10 mM 1,10-phenanthroline and 1 mM PMSF (Bran-

quinha et al., 1996). To obtain total cellular protein extracts,

1.0!108 cells were resuspended in 30 ml of PBS and lysed by

the addition of 70 ml of SDS-PAGE sample buffer (125 mM

Tris, pH 6.8, 4% SDS, 20% glycerol and 2% bromophenol

blue) (Santos et al., 2003).

2.6. Gelatin-SDS-PAGE

Peptidases were assayed and characterised by electrophor-

esis on 10% SDS-PAGE with 0.1% co-polymerised gelatin as

substrate (Heussen and Dowdle, 1980). The gels were loaded

with the equivalent to 1.0!107 cells per slot and following

electrophoresis at a constant voltage of 200 V at 4 8C they were

soaked for 1 h at 25 8C in 2.5% Triton X-100. The gels were

then incubated for 72 h at 37 8C in 50 mM sodium phosphate

buffer, pH 5.5, supplemented with 2 mM DTT in the absence

and in the presence of 10 mM E-64. The gels were stained for

1 h with 0.2% Coomassie brilliant blue R-250 in methanol-

acetic acid-water (50:10:40) and destained in the same solvent

(d’Avila-Levy et al., 2005). The molecular mass of the

peptidases was calculated by comparison with the mobility of

low molecular mass standards.

2.7. Effects of proteolytic inhibitors on cysteine

peptidase activity

Parasites (1.0!107 cells) were incubated for 30 min at

37 8C with each CPI at a final concentration of 10 mM in 500 ml

of PBS supplemented with 18.5 mg/100 ml BHI. The cells

were then washed three times in cold PBS, resuspended in

500 ml of PBS and disrupted by sonication lysis at 4 8C

following centrifugation at 5000!g for 10 min. The super-

natants obtained correspond to the parasite cellular extracts.

For assaying proteolytic activity, gelatin (0.5 mg/ml), 8 mM

DTT and 50 mg of the parasite cellular extract were added to a

microcentrifuge tube (350 ml) and incubated in 50 mM

phosphate buffer (pH 5.5) for 2 h at 26 8C. After this

incubation, three 100 ml aliquots of the reaction mixture were

transferred to wells on a microtiter plate containing 50 ml of

water and 100 ml of a Coomassie brilliant blue solution

(0.025% Coomassie brilliant blue G-250, 11.75% ethanol and

21.25% phosphoric acid). After 10 min to allow dye binding,

the plate was read on a Molecular Devices Thermomax

microplate reader at an absorbance of 595 nm. The results were

expressed as the relative percentage of activity of each CPI-

treated parasite cellular extract subtracted from the activity of a

non-treated cellular extract (Buroker-Kilgore and Wang,

1993).

2.8. Immunoblotting assay

Samples containing cellular extracts (equivalent to 1.0!107

cells) were added to SDS-PAGE sample buffer and mixed with

10% (v/v) b-mercaptoethanol, followed by heating at 100 8C

for 5 min. Thereafter, protein extracts were separated in 12%

SDS-PAGE and the polypeptides electrophoretically trans-

ferred at 4 8C at 100 V/300 mA for 2 h to a nitrocellulose

membrane. The membrane was blocked in 5% (w/v) low-fat

dried milk in TBS containing 0.5% Tween 20 (TBS/Tween) for

1 h at room temperature. Then, membranes were washed three

times (10 min each) with the blocking solution and incubated

with the anti-cruzipain polyclonal antibody at 1:1000 dilution

in TBS/Tween for 2 h. The secondary antibody used was

peroxidase-conjugated goat anti-rabbit Fc at 1:2500 followed

by immunodetection by chemiluminescence (d’Avila-Levy

et al., 2005).

2.9. Flow cytometry analysis

Parasites (1.0!107 cells) used for these experiments were

fixed at 4 8C in 2% (v/v) paraformaldehyde in PBS (pH 7.2) for

30 min, followed by extensive washing in the same buffer.

Alternatively, the fixed cells were permeabilised by 0.01%

(v/v) Triton X-100 in PBS for 15 min at room temperature and

then washed twice in PBS. The fixed and/or permeabilised cells

maintained their morphological integrity, as verified by optical

microscopic observation. After this step, the cells were

incubated for 1 h at room temperature with a 1:250 dilution

of rabbit anti-cruzipain polyclonal antibody and then incubated

for an additional hour with a 1:250 dilution of fluorescein

isothiocyanate (FITC)-labeled goat anti-rabbit IgG. Finally,

these cells were washed three times in PBS and examined in a

fluorescence-activated cell sorter (FACS) FACSCalibur (BD

Bioscience, USA) equipped with a 15 mW argon laser emitting

at 488 nm. Untreated cells and those treated only with the

secondary antibody were used as controls. The mapped

population (nZ10,000) was then analysed for log green

fluorescence by using a single-parameter histogram.

2.10. Immunocytochemistry

The parasites cultured for 48 h in the absence or presence of

10 mM leupeptin were collected by centrifugation, washed

twice in PBS and fixed in a solution containing 0.1%

glutaraldehyde, 2% formaldehyde in 0.1% cacodylate buffer,

pH 7.2. After 1 h fixation, cells were washed in PBS,

dehydrated in methanol and embedded in Lowicryl K4M

resin at K20 8C. Ultrathin sections were collected in nickel

grids and incubated in 0.1 M TBS followed by a 15 min

incubation in 50 mM ammonium chloride. After three

washings in TBS, the grids were incubated with the rabbit

anti-cruzipain antibody diluted at 1:50 in TBS, 1% BSA, 1%

Tween 20 (TBSBT). Grids were washed three times in TBSBT

and subsequently incubated with gold-labeled goat anti-rabbit

IgG (15 nm, dilution 1:10) for 60 min. Ultrathin sections were

stained with uranyl acetate and lead citrate and observed in

A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–5650

a Zeiss ETM 10 C transmission electron microscope (TEM)

(Souto-Padron et al., 1990).

2.11. Influence of CPIs and anti-cruzipain on the

P. serpens-salivary glands interaction

Adult O. fasciatus (Hemiptera: Lygaeidae) from the original

colony established by Dr N. Ratcliffe (Swansea University,

UK) were a gift of Dr Alexandre Romeiro (Instituto de

Biofısica Carlos Chagas Filho, UFRJ, Brazil). The insects were

kept at room temperature in plastic pitchers and fed with peeled

and toasted sunflower seeds and distilled water (Romeiro et al.,

2000). The parasites (1.0!107 cells) were incubated for

30 min at 26 8C with antipain, iodoacetamide, leupeptin,

cystatin and E-64 at a final concentration of 10 mM in 500 ml

of PBS supplemented with 18.5 mg/100 ml BHI. Alternatively,

the same number of parasites was treated for 1 h with anti-

cruzipain antibody (1:1000 and 1:2500) and with an irrelevant

antibody (1:1000) (kindly provided by Dr Marcio L.

Rodrigues, Universidade Federal do Rio de Janeiro, Brazil).

The viability of the organisms throughout the experiment was

assessed by mobility, trypan blue dye exclusion and by

monitoring lactate dehydrogenase, an intracellular enzyme, in

the supernatant from the incubation systems (d’Avila-Levy et

al., 2005). The cells were then washed three times in cold PBS,

resuspended in PBS (5.0!106 cells in 100 ml) and added to

dissected salivary glands (10 per group). The parasites were

allowed to bind for 1 h at room temperature in PBS. After the

interaction period the salivary glands were extensively washed

with PBS, individually transferred to microcentrifuge tubes

containing 50 ml of PBS and homogenised (Fampa et al., 2003).

The released trypanosomatids were counted in a Neubauer

chamber. The results are shown as the meanGstandard error of

the mean (SEM) of three experiments.

2.12. Statistical analysis

The experiments were performed in triplicate, in three

independent experimental sets. The data was analysed

statistically by means of Student’s t-test. Alternatively, the

analysis of variance between groups was made by means of

ANOVA test using EPI-INFO 6.04 (Database and Statistics

Program for Public Health) computer software. P-values of

0.05 or less were considered statistically significant.

3. Results

3.1. Effect of CPIs on the cellular growth rate

Five different CPIs (antipain, cystatin, E-64, iodoacetamide

and leupeptin) were added to replicating P. serpens promas-

tigote forms as a single dose, as previously determined by other

studies (Bonaldo et al., 1991; Engel et al., 1998; Franke de

Cazzulo et al., 1994; Harth et al., 1993; Troeberg et al., 1999;

Santos et al., 2003) and the cellular growth was monitored for

4 days. Our results showed that all CPIs arrested the growth of

P. serpens with different patterns of inhibition (Fig. 1A). For

instance, leupeptin, antipain, cystatin and E-64 promoted a

significant reduction on the cellular growth rate by approxi-

mately 50% after 72 h, while iodoacetamide-treated parasites

showed the strongest toxic effect, since this inhibitor

completely arrested P. serpens replication (Fig. 1A).

All drugs had a reversible effect on P. serpens growth since

parasites that were transferred to a drug-free fresh medium

were still capable of normal development, except for flagellates

pre-treated with iodoacetamide, which even after 2 days of re-

cultivation showed no increase in the cell number (Fig. 1B).

Corroborating these results, optical microscopy observations

showed lysis of promastigote cells after treatment of the

parasites with 10 mM iodoacetamide for 48 h (Fig. 1A, inset c).

The drastic reduction of P. serpens growth induced by

iodoacetamide showed to be concentration-dependent

(Fig. 1B, inset).

3.2. Effects of CPIs on the ultrastructure of P. serpens

All the CPIs at 10 mM, except for E-64, promoted some

alterations on the cell morphology at optical microscopy level

such as the flagellates becoming short and round after treatment

with antipain, cystatin and leupeptin (a representative image is

shown in Fig. 1A, inset b). Therefore, we carried out a study of

the cell morphology by ultrastructural analysis of P. serpens

following treatment of the flagellates with the CPIs at 10 mM

for 48 h (Fig. 2). The normal morphology and the ultrastructure

of the cellular membranes and organelles in a P. serpens

promastigote form is shown for comparison (Fig. 2A). The

inhibitors E-64 and cystatin did not promote any detectable

alteration at the ultrastructural level of P. serpens (data not

shown). However, antipain promoted the appearance of

discrete microvillar extensions of the cell surface membrane

(Fig. 2D), while leupeptin-treated flagellates showed micro-

villar projections all over the cell surface membrane (Fig. 2C)

and, in some cells, these projections were detected specifically

in the flagellar pocket region (Fig. 2B), the site where

endocytosis and exocytosis take place in trypanosomatids. In

some cases, blebs detaching from the entire cell surface were

observed, indicating a shedding of the parasite surface

(Fig. 2C). The parasites treated with 10 mM iodoacetamide

showed the most severe morphological alterations, resembling

a necrotic cell with disintegration of cytoplasmic organelles

and nucleus, as well as swelling of the kinetoplast-mitochon-

drion complex (Fig. 2E).

3.3. Partition of P. serpens cellular peptidases

after Triton X-114 extraction

The cell-associated proteolytic profile of P. serpens

comprised two major bands migrating at 40 and 38 kDa

(Fig. 3A, lane 1). The analysis of the cellular peptidases after

Triton X-114 extraction showed that both peptidases partitioned

in the aqueous phase (Fig. 3A, lane 2). However, a very faint

band was detected in the hydrophobic phase (Fig. 3A, lane 3).

In an attempt to improve the detection of this

Num

ber

of c

ells

x 1

07

Time (hours)

0

2

4

6

8

10

12

14

16

48 96

Iodoacetamide [µM]

% o

f co

ntro

l

0

20

40

60

80

100

10 1 0.1 0.01 0.001 0.0001

B

Num

ber

of c

ells

x 1

07

0

1

2

3

4

5

6

7control cells

cystatin

leupeptin

E-64

antipain

iodoacetamide

0 24 48 72 96

(a)

(b)

(c)

10 µm

A

Fig. 1. Effect of different cysteine peptidase inhibitors (CPIs) on the growth rate of Phytomonas serpens. (A) Growth pattern of P. serpens when cultivated at 26 8C in

the absence (control cells) or in the presence of CPIs (cystatin, leupeptin, E-64, antipain and iodoacetamide). The insets show the phase-contrast images of control

P. serpens (picture labeled (a) or cells treated with leupeptin (b) and iodoacetamide (c). For simplicity, the image of the parasite treated with E-64, which did not

show any morphological alterations, and the images of the flagellates treated with antipain or cystatin, which are similar to the parasites treated with leupeptin, are

not shown. (B) Mechanism of CPI action. The parasites were grown in the absence or in the presence of CPIs for 48 h, then harvested, washed and transferred to fresh

drug-free medium, followed by incubation for additional 2 days (total of 96 h). The inset shows the effect of different concentrations of iodoacetamide on the growth

rate of P. serpens cultivated in brain heart infusion supplemented with FCS at 26 8C for 48 h. Data shown are meansGstandard deviations of three independent

experiments, which were performed in triplicate and analysed using Student’s t-test (P!0.05 were considered statistically significant).

A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–56 51

membrane-associated proteolytic activity, we performed a

detergent extraction avoiding the washing steps after the

detergent phase separation. The hydrophilic/hydrophobic inter-

face was discharged in order to prevent contamination of both

phases and metallo and serine peptidase inhibitors were added to

each sample immediately after the separation of the phases. This

procedure improved the detection of the 40 kDa component in

the detergent-rich phase (Fig. 3B, lane 3). The inhibition profile

suggested that both proteolytic activities were cysteine

peptidases, based on their entire inhibition by E-64 (Fig. 3C).

3.4. Effect of peptidase inhibitors on the parasite

cysteine peptidases

The five CPIs used in the present study inhibited P. serpens

cysteine peptidases by approximately 90%. The residual

Fig. 2. Effect of cysteine peptidase inhibitors on the ultrastructural morphology of Phytomonas serpens. Parasites were cultivated for 48 h in the absence (A) or in the

presence of the following cysteine peptidase inhibitors at 10 mM: leupeptin (B–C), antipain (D) and iodoacetamide (E). Arrows in C indicate the microvillar

extension on the cell surface membrane; arrowheads in B indicate the small membrane fragments suggesting a shedding of the parasite surface; asterisk in E indicates

the swelling of the kinetoplast and large arrow indicates disintegration of nuclear membrane. n, nucleus; k, kinetoplast and f, flagellum.

A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–5652

activity of the proteolytic activity was 9.0, 7.0, 10.0, 9.0 and

10.0% after treating live parasites with cystatin, leupeptin, E-

64, antipain and iodoacetamide, respectively, at 10 mM for

30 min.

256

b1 (a)

4038

(b) (c)

3.5. Detection of a cruzipain-like molecule in P. serpens

Phytomonas serpens produced a 40 kDa cysteine peptidase

that possesses similar biochemical properties with cruzipain,

the major cysteine peptidase produced by T. cruzi. To explore

this fact, we verified the possible immunological cross-

reactivity between P. serpens and cruzipain from T. cruzi.

30

45

66

97

A B C

1 2 3 1 2 3 1 2 3

Fig. 3. Proteolytic enzymes in Phytomonas serpens cells analysed on gelatin-

SDS-PAGE. Fractions: total cellular extract (1), hydrophilic (2) and

hydrophobic (3) phases after Triton X-114 extraction. The Triton X-114

extraction was performed according to Branquinha et al. (1996) (A), or adapted

to enhance the detection of hydrophobic peptidases, as described under

Section 2 (B, C). Gel strips were incubated for 72 h at 37 8C in 50 mM sodium

phosphate, pH 5.5, supplemented with 2 mM dithiothreitol in the absence (A,

B) and in the presence of 10 mM E-64 (C). The numbers on the left indicate

relative molecular mass markers expressed in kilodaltons.

The anti-cruzipain strongly recognised a 50 kDa polypeptide in

the epimastigote forms of T. cruzi, which was included as a

positive control (data not shown). Two major polypeptides (38

and 40 kDa) from P. serpens were recognised by the anti-

cruzipain polyclonal antibodies through western blotting

analysis (Fig. 4, inset a). Curiously, one of them was

partitioned in the P. serpens membrane-rich fraction (Fig. 4,

0

100 101 102 103

Cel

l cou

nt

Fluorescence intensity

a c23

Fig. 4. Flow cytometric analysis showing the anti-cruzipain antibody binding to

Phytomonas serpens. Paraformaldehyde-fixed cells were permeabilised (3) or

not (1, 2) with Triton X-100, to permit the access of the antibody to the

intracellular environment of the parasite cells. These cells were subsequently

incubated in the absence (1) or in the presence (2, 3) of anti-cruzipain antibody

as described under Section 2 and then analysed by flow cytometry. The inset

demonstrates the reactive polypeptides detected in the total cellular extract (a),

hydrophilic (b) and hydrophobic (c) fractions of P. serpens through western

blotting analysis using anti-cruzipain antibody. The numbers on the left

indicate the relative molecular masses expressed in kilodaltons.

A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–56 53

inset c). Corroborating these results, flow cytometric analysis

showed cruzipain-like molecules on the cell surface of

P. serpens promastigotes (Fig. 4). Moreover, a significant

augment in the fluorescence intensity was observed when

P. serpens cells were previously permeabilised with Triton

X-100, showing that cruzipain-like molecules were preferen-

tially located in cytoplasmic compartments (Fig. 4), which is in

agreement with Triton X-114 extraction (Fig. 3).

3.6. Immunocytochemistry

Phytomonas serpens promastigote forms were immuno-

cytochemically labeled with anti-cruzipain antibodies associ-

ated with colloidal gold and analysed by TEM. The results

showed a faint labeling of the membrane lining the cell body

and the flagellum (Fig. 5A). Gold particles could be also

observed in the cytoplasm of the parasite (Fig. 5A). A similar

labeling pattern was observed in the CPIs-treated parasites

(data not shown). Shedding of cytoplasmic membrane

associated with cysteine peptidase was observed in CPIs-

treated P. serpens. Representative images of cell surface,

intracellular labeling and release of membrane fragments (from

leupeptin-treated parasites) are shown in the Fig. 5B–E.

Fig. 5. Electron microscopy immunocytochemical analysis of Phytomonas serp

promastigotes of P. serpens were incubated in the presence of anti-cruzipain (1:50) a

membrane enclosing the cell body and the flagellum as well as throughout the cyto

intracellular compartment (B) as well as in membrane fragments detached from le

4 mm.

3.7. Effect of CPIs and anti-cruzipain on

the P. serpens-salivary glands interaction

Based on the effects of CPIs on P. serpens, we tested the

influence of these inhibitors on the interaction of P. serpens

with O. fasciatus salivary glands in vitro. The parasites

maintained their viability after treatment for 30 min with each

CPI at 10 mM as judged by their mobility, trypan blue dye

exclusion (in which more than 99% of the parasites were

viable) and by the absence of lactate dehydrogenase, an

intracellular enzyme, in the supernatant from the incubation

systems (data not shown). After incubation of P. serpens

promastigotes with explanted salivary glands of adult O.

fasciatus, many parasites were seen adhered to the salivary

glands by their flagella (data not shown). Parasites pre-treated

with the CPIs showed a significant reduction (P!0.05) in their

capacity to adhere to the salivary glands (Fig. 6A), especially

after treatment with iodoacetamide (P!0.01) at 10 (Fig. 6A)

and 1 mM (Fig. 6A, inset). Similarly, pre-treatment of parasites

with anti-cruzipain antibody at 1:1000, a concentration that did

not promote parasite agglutination, also considerably dimin-

ished the interaction process (Fig. 6B). To test the anti-

cruzipain antibody specificity, an irrelevant antibody and

ens. Thin sections of untreated (control) (A) and leupeptin-treated (B–E)

nd subsequently in the presence of gold-labeled antibodies. Faint labeling of the

plasm was evident (A). Gold particles were also detected in cell surface and in

upeptin-treated parasites (arrows in C–E). k, kinetoplast and f, flagellum. Bar:

0 10 20 30 40

cystatin

leupeptin

E-64

antipain

iodoacetamide

control

Iodoacetamide [µm]

Inte

ract

ion

(% o

f co

ntro

l)

0

20

40

60

80

1 0.1 0.01 0.001

A

Protozoa x 103 / salivary glands

Protozoa x 103 / salivary glands

0 10 20 30 40

control

anti-cruzipain(1:1000)

anti-cruzipain(1:2500)

irrelevant IgG

B

Fig. 6. Effect of cysteine peptidase inhibitors and anti-cruzipain antibody in the

interaction process between Phytomonas serpens and explanted salivary glands

of Oncopeltus fasciatus. The parasites (1.0!107 cells) were treated for 30 min

at 26 8C with antipain, iodoacetamide, leupeptin, cystatin and E-64 at a final

concentration of 10 mM (A) or incubated for 1 h with the anti-cruzipain or an

irrelevant antibody (B). The viability of the parasites was not affected by the

treatments used in this set of experiments. Following interaction with salivary

gland cells, the released trypanosomatids were counted in a Neubauer chamber.

The results are shown as the meanGSEM. of three independent experiments.

The treatment with iodoacetamide at 10 mM significantly inhibited parasite

adhesion. The inset in A shows the percentage of adhesion after treatment for

30 min of parasite cells with different iodoacetamide concentrations. Parasites

treated with CPIs and anti-cruzipain antibody at 1:1000 dilution had an

adhesion index significantly different from untreated (control) cells using

ANOVA test (*, P!0.05, and **, P!0.01).

A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–5654

a higher dilution of anti-cruzipain were pre-incubated with

parasites and the interaction process was very similar to that

obtained with non-treated parasites (Fig. 6B).

4. Discussion

Plant-associated microorganisms play essential roles in

agriculture and food safety as well as contribute to the

environmental equilibrium (Mosolov et al., 2001). Phytopatho-

genic microorganisms produce, in addition to other enzymes,

active peptidases. In certain cases, a correlation was found

between the activity of the phytopathogen peptidases and the

severity of the host plant disease. Proteolytic enzymes not only

supply the parasites with peptides and amino acids required for

their growth and development, they also play important roles in

crossing the plant protective barriers. For instance, peptidases

of phytopathogenic microorganisms can cleave antimicrobial

proteins of plants and rapidly degrade their cell wall proteins

(reviewed by Mosolov et al., 2001).

Phytomonas serpens promastigote cells synthesised two

cysteine peptidases of 38 and 40 kDa active at acidic pH (5.5).

These activities were mainly detected in the hydrophilic

fraction after Triton X-114 extraction, suggesting a cyto-

plasmic location. Cysteine peptidases are frequently detected

in acidic cytoplasmic compartments, mainly lysosome or

lysosome-like organelles, in a vast number of trypanosomatids

(Souto-Padron et al., 1990; Branquinha et al., 1996; Coombs

and Mottram, 1997; Sajid and McKerrow, 2002). On the other

hand, cysteine peptidase activity associated to cell surface is

well characterised in T. cruzi by biochemical and ultra-

structural methodologies (Souto-Padron et al., 1990; Coombs

and Mottram, 1997; Cazzulo et al., 2001). Here, we

demonstrated that the 40 kDa cysteine peptidase was also

partitioned in the detergent fraction, showing a probable

interaction with membrane domains. Flow cytometry and

ultrastructure immunocytochemical analyses corroborated this

result. Recent evidence suggests that membrane-bound

peptidases are multifunctional proteins. It has become clear

that non-catalytic effects of membrane-bound peptidases are

also of great importance in some biological regulations,

including signal transduction and adhesion (Sedo et al., 1996).

A number of sophisticated studies have been conducted on

trypanosomatid cysteine peptidases because they are potential

drug targets. For instance, CPIs interfere with the differen-

tiation of T. cruzi (Fanke de Cazzulo et al., 1994), T. brucei

(Muttomba and Wang, 1998) and Herpetomonas samuelpes-

soai (Santos et al., 2003), are active against Leishmania

growing intracellularly in vitro (Mottram et al., 1996; Selzer

et al., 1999; Mahmoudzadeh-Niknam and McKerrow, 2004),

block Plasmodium falciparum invasion of host cell erythro-

cytes (Greenbaum et al., 2002) and interfere with Entamoeba

hystolytica trophozoite adhesion, thus making amoebas

deficient in substrate degradation and cell damage (Franco

et al., 1999). The CPIs used in our experiments arrested the

promastigote growth of P. serpens. E-64 showed the weakest

effect on the plant trypanosomatid growth. This strong cysteine

inhibitor is thought to be unable to cross membranes and so is

likely to be denied access to many cell compartments (Bonaldo

et al., 1991). However, it may be taken up by fluid phase

endocytosis and so enter lysosomes/phagosomes and inhibit

the peptidases within those compartments (Irvine et al., 1997).

In our study, we detected some peculiar alterations in the

cell morphology of plant flagellates when treated with CPIs

(exception for E-64), such as cells becoming round and short.

Ashall et al. (1990) and Troeberg et al. (1999) reported a

similar phenomenon and postulated that it indicates osmotic

stress caused by peptidase inhibition, although such changes

could also be the consequence of disruptions of the intracellular

scaffolding of the proteins (Fuchs and Cleveland, 1998).

Therefore, the killing activity of these CPIs suggests that a

cysteine peptidase is required for the viability of plant

trypanosomatid flagellates. Iodoacetamide showed the most

profound effects on the parasite growth rate, suggesting

A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–56 55

irreversible metabolic injury. These results were corroborated

by observations of the lysis of promastigote cells.

Some of the CPIs (antipain and leupeptin) caused major

alterations of the cellular membrane (including the flagelar

pocket region) in P. serpens cells, but no abnormalities were

noted in the Golgi complex or other intracellular organelles of

P. serpens when treated with cystatin, leupeptin, antipain and

E-64. Since at least one cysteine peptidase activity (40 kDa)

was identified on the cell surface of P. serpens, we postulated

that the shedding of the cell surface membrane detected in

parasites treated with leupetin and antipain could be a defense

mechanism from the parasites, which released the complex of

cysteine peptidase bound to the inhibitor to the extracellular

environment for neutralising the cysteine peptidase inhibitor

effects. This hypothesis was corroborated by the ultrastructure

immunocytochemical analysis, which showed the presence of

the cruzipain-like protein on the cell surface and in small

membrane fragments released by the parasites. Mechanisms

previously associated with decreased sensitivity or resistance

to chemotherapy in parasitic protozoa include decreased drug

uptake, increased export of drugs, decrease in drug activation,

and alterations of the target enzyme to decrease drug-binding

(Borst and Ouellette, 1995). Conversely, iodoacetamide

destroyed the parasite cellular structure, leading to a complete

disruption of nuclear and kinetoplast chromatin, followed by

cell lysis.

The involvement of cysteine peptidases of P. serpens in the

interaction with explanted salivary glands of the phytophagous

insect O. fasciatus was also observed in experiments using

CPIs and anti-cruzipain antibody. Our results indicate that all

CPIs as well as anti-cruzipain antibody significantly interfered

with the ability of the trypanosomatid to adhere to the insect

salivary gland epithelial cells, suggesting that the cysteine

peptidase are relevant to this process. Similarly, the treatment

of T. cruzi trypomastigotes with different CPIs resulted in a

considerable decrease in adhesion to Vero cells (Franke de

Cazzulo et al., 1994), fibroblasts (Piras et al., 1985) and heart

muscle cells (Meirelles et al., 1992). The CPIs may act

indirectly, inhibiting the intracellular processing of surface

precursor proteins possibly engaged in host cell recognition.

An extracellular function for the peptidase cannot be ruled out

because this cysteine peptidase is found on the P. serpens

surface and there are circumstantial data supporting the

involvement of peptidases in the penetration process (Piras

et al., 1985; Meirelles et al., 1992; Franke de Cazzulo et al.,

1994).

The biological functions filled by peptidases in plant

trypanosomatids are still largely unknown. Our study provided

clear evidence that cysteine peptidases from P. serpens might

be effective targets for some CPIs. Therefore, the results

described above add P. serpens to the list of trypanosomatids

whose cellular growth seems to be correlated with peptidase

expression. Consequently, these enzymes can be considered

potential targets for immunotherapeutic and chemotherapeutic

agents and as serodiagnostic reagents for detection of parasitic

diseases. Collectively, our results suggest that cysteine

peptidases participate in several biological processes in

P. serpens including cellular growth and interaction with the

invertebrate vector.

Acknowledgements

The authors thank Celina Monteiro Abreu for her technical

assistance. The authors are indebted to Dr. Juan-Jose Cazzulo

for donating the valuable anti-cruzipain antibody. This work

was supported by grants from the Brazilian Agencies:

Fundacao Universitaria Jose Bonifacio (FUJB), Financiadora

de Estudos e Projetos (FINEP), Conselho Nacional de

Desenvolvimento Cientıfico e Tecnologico (CNPq), Conselho

de Ensino para Graduados e Pesquisa (CEPG/UFRJ) and

Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro

(FAPERJ).

References

Ashall, F., Harris, D., Roberts, H., Healy, N., Shaw, E., 1990. Substrate

specificity and inhibitor sensitivity of a trypanosomatid alkaline peptidase.

Biochim. Biophys. Acta 1035, 293–299.

Bonaldo, M.C., D’Escoffier, L.N., Salles, J.M., Goldenberg, S., 1991.

Characterization and expression of protease during Trypanosoma cruzi

metacyclogenesis. Exp. Parasitol. 73, 44–51.

Borst, P., Ouellette, M., 1995. New mechanisms of drug resistance in parasitic

protozoa. Annu. Rev. Microbiol. 49, 426–460.

Branquinha, M.H., Vermelho, A.B., Goldenberg, S., Bonaldo, M.C., 1996.

Ubiquity of cysteine- and metalloproteinase activities in a wide range of

trypanosomatids. J. Eukaryot. Microbiol. 43, 131–135.

Bregano, J.W., Picao, R.C., Graca, V.K., Menolli, R.A., Jankevicius, S.I.,

Filho, P.P., Jankevicius, J.V., 2003. Phytomonas serpens, a tomato parasite,

shares antigens with Trypanosoma cruzi that are recognized by human sera

and induce protective immunity in mice. FEMS Immunol. Med. Microbiol.

39, 257–264.

Buroker-Kilgore, M., Wang, K.K.W., 1993. A Coomassie brilliant blue G-250-

based colorimetric assay for measuring activity of calpain and other

proteases. Anal. Biochem. 208, 387–392.

Camargo, E.P., 1999. Phytomonas and other trypanosomatid parasites of plants

and fruit. Adv. Parasitol. 42, 29–112.

Camargo, E.P., Kastelein, P., Roitman, I., 1990. Trypanosomatid parasites of

plants (Phytomonas). Parasitol. Today 6, 22–25.

Cazzulo, J.J., Stoka, V., Turk, V., 2001. The major cysteine proteinase of

Trypanosoma cruzi: a valid target for chemotherapy of Chagas disease.

Curr. Pharmacol. Des. 7, 1143–1156.

Coombs, G.H., Mottram, J.C., 1997. Parasite proteinases and amino acid

metabolism: possibilities for chemotherapeutic exploitation. Parasitology

114, S61–S80.

Cunha, M., Gomes, V.M., Xavier-Filho, J., Attias, M., de Souza, W., Miguens,

F.C., 2000. The laticifer system of Chamaesyce thymifolia: a closed host

environment for plant trypanosomatids. Biocell 24, 123–132.

d’Avila-Levy, C.M., Araujo, F.M., Vermelho, A.B., Soares, R.M.A., Santos,

A.L.S., Branquinha, M.H., 2005. Proteolytic expression in Blastocrithidia

culicis: influence of the endosymbiont and similarities with virulence

factors of pathogenic trypanosomatids. Parasitology 130, 413–420.

Dollet, M., 1984. Plant diseases caused by flagellate protozoa (Phytomonas).

Ann. Rev. Phytopathol. 22, 115–132.

Donovan, C., 1909. Kala azar in Madras especially with regard to its connection

with the dog and the bug (Conorrhinus). Lancet 177, 1495–1496.

Engel, J.C., Doyle, P.S., Palmer, J., Hsieh, I., Bainton, D.F., McKerrow, J.H.,

1998. Growth arrest of T. cruzi by cysteine protease inhibitors is

accompanied by alterations in Golgi complex and ER ultrastructure.

J. Cell Sci. 111, 597–606.

Fampa, P., Correa-da-Silva, M.S., Lima, D.C., Oliveira, S.M.P., Motta,

M.C.M., Saraiva, E.M.B., 2003. Interaction of insect trypanosomatids with

A.L.S. Santos et al. / International Journal for Parasitology 36 (2006) 47–5656

mosquitoes, sand fly and the respective insect cell lines. Int. J. Parasitol. 33,

1019–1026.

Franco, E., Soares, R.M.A., Meza, I., 1999. Specific and reversible

inhibition of Entamoeba histolytica cysteine-proteinase activities by

Zn2C: implications for adhesion and cell damage. Arch. Med. Res. 30,

82–88.

Franke de Cazzulo, B.M., Martınez, J., North, M.J., Coombs, G.H., Cazzulo,

J.J., 1994. Effects of proteinase inhibitors on the growth and differentiation

of Trypanosoma cruzi. FEMS Microbiol. Lett. 124, 81–86.

Fuchs, H., Cleveland, J., 1998. A structural scaffolding of intermediate

filaments in health and disease. Science 279, 514–519.

Greenbaum, D.C., Baruch, A., Grainger, M., Bozdech, Z., Medzihradszky,

K.F., Engel, J., DeRisi, J., Holder, A.A., Bogyo, M., 2002. A role for the

protease falcipain 1 in host cell invasion by the human malaria parasite.

Science 298, 2002–2006.

Harth, G., Andrews, N., Miles, A.A., Engel, J.C., Smith, R., McKerrow, J.H.,

1993. Peptide-fluoromethyl ketones arrest intracellular replication and

intracellular transformation of Trypanosoma cruzi. Mol. Biochem.

Parasitol. 58, 17–24.

Heussen, C., Dowdle, E.B., 1980. Electrophoretic analysis of plasminogen

activators in polyacrilamide gels containing sodium dodecyl sulphate and

copolymerized substrates. Anal. Biochem. 102, 196–202.

Irvine, J.W., North, M.J., Coombs, G.H., 1997. Use of inhibitors to identify

essential cysteine proteinases of Trichomonas vaginalis. FEMS Microbiol.

Lett. 149, 45–50.

Lafont, A., 1909. Sur la presence d’un parasite de la classe des flagelles dans le

latex de l’Euphorbia pilulifera. C. R. Soc. Biol. 66, 1011–1013.

Mahmoudzadeh-Niknam, H., McKerrow, J.H., 2004. Leishmania tropica:

cysteine proteases are essential for growth and pathogenicity. Exp.

Parasitol. 106, 158–163.

Mbawa, Z.R., Gumm, I.D., Shaw, E., Lonsdale-Eccles, J.D., 1992.

Characterisation of a cysteine protease from bloodstream forms of

Trypanosoma congolense. Eur. J. Biochem. 204, 371–379.

Meirelles, M.N.L., Juliano, L., Carmona, E., Silva, S.G., Costa, E.M.,

Murta, A.C.M., Scharfstein, J., 1992. Inhibitors of the major cysteinyl

proteinase (GP57/51) impair host cell invasion and arrest the

intracellular development of Trypanosoma cruzi in vitro. Mol.

Biochem. Parasitol. 52, 175–184.

Mosolov, V.V., Grigor’eva, L.I., Valueva, T.A., 2001. Involvement of

proteolytic enzymes and their inhibitors in plant protection. Appl. Biochem.

Microbiol. 37, 115–123.

Mottram, J.C., Souza, A.E., Hutchison, J.E., Carter, R., Frame, M.J., Coombs,

G.H., 1996. Evidence from disruption of the lmcpb gene array of

Leishmania mexicana that cysteine proteinases are virulence factors.

Proc. Natl Acad. Sci. USA 93, 6008–6013.

Mutomba, M.C., Wang, C.C., 1998. The role of proteolysis during

differentiation of Trypanosoma brucei from the bloodstream to the

procyclic form. Mol. Biochem. Parasitol. 93, 11–22.

Piras, M.M., Henriquez, D., Piras, R., 1985. The effect of proteolytic enzymes

and protease inhibitors on the interaction Trypanosoma cruzi-fibroblasts.

Mol. Biochem. Parasitol. 14, 151–163.

Romeiro, A., Sole-Cava, A., Sousa, M.A., De Souza, W., Attias, M., 2000.

Ultrastructural and biochemical characterization of promastigotes and cytic

form of Leptomonas wallacei n. sp. isolated from the intestine of its natural

host Oncopeltus fasciatus (Hemiptera: Lygaeidae). J. Eukaryot. Microbiol.

47, 208–220.

Sajid, M., McKerrow, J.H., 2002. Cysteine proteases of parasitic organisms.

Mol. Biochem. Parasitol. 120, 1–21.

Santos, A.L.S., Alviano, C.S., Soares, R.M.A., 2002. Detection of sialoglyco-

molecules in five plant trypanosomatids and in an insect phytophagous

isolate. FEMS Microbiol. Lett. 214, 19–23.

Santos, A.L.S., Rodrigues, M.L., Alviano, C.S., Angluster, J., Soares, R.M.A.,

2003. Herpetomonas samuelpessoai: dimethylsulfoxide-induced differen-

tiation is influenced by proteinase expression. Curr. Microbiol. 46, 11–17.

Sedo, A., Mandys, V., Krepela, E., 1996. Cell membrane-bound proteases: not

‘only’ proteolysis. Physiol. Res. 45, 169–176.

Selzer, P.M., Pingel, S., Hsieh, I., Ugele, B., Chan, V.J., Engel, J.C., Bogyo, M.,

Russell, D.G., Sakanari, J.A., McKerrow, J.H., 1999. Cysteine protease

inhibitors as chemotherapy: lessons from a parasite target. Proc. Natl Acad.

Sci. USA 96, 11015–11022.

Soares, R.M.A., Santos, A.L.S., Bonaldo, M.C., Andrade, A.F.B., Alviano, C.S.,

Angluster, J., Goldenberg, S., 2003. Leishmania (Leishmania) amazonensis:

differential expression of proteinases and cell-surface polypeptides in

avirulent and virulent promastigotes. Exp. Parasitol. 104, 104–112.

Souto-Padron, T., Campetella, O.E., Cazzulo, J.J., De Souza, W., 1990. Cysteine

proteinase in Trypanosoma cruzi: immunocytochemical localization and

involvement in parasite–host cell interaction. J. Cell Sci. 96, 485–490.

Troeberg, L., Morty, R.E., Pike, R.N., Lonsdale-Eccles, J.D., Palmer, J.T.,

McKerrow, J.H., Coetzer, T.H.T., 1999. Cysteine proteinase inhibitors kill

cultured bloodstream forms of Trypanosoma brucei brucei. Exp. Parasitol.

91, 349–355.