FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages

of the malaria parasites

Plasmodium berghei

and

Plasmodium yoelii

Laura Nogueira da Cruz

a,b

, Eduardo Alves

a

, Mônica Teixeira Leal

b

, Maria A. Juliano

c

, Philip J. Rosenthal

d

,

Luiz Juliano

c

_{, Celia R.S. Garcia}

b,

_⇑

a_{Department of Parasitology, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374 Edifício Biomédicas II, CEP 05508-900, São Paulo, SP, Brazil} b_{Department of Physiology, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, travessa 14, n321, CEP 05508-900, São Paulo, SP, Brazil}

c_{Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100 Vila Clementino, CEP 04044-020, São Paulo, SP, Brazil} d_{Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143, USA}

a r t i c l e

i n f o

Article history:

Received 8 July 2010

Received in revised form 26 October 2010 Accepted 27 October 2010

Available online 17 December 2010

Keywords:

Malaria

Plasmodium berghei Plasmodium yoelii

Protease activity Ca2+_modulation

FRET

a b s t r a c t

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca2+_{is a ubiquitous signalling ion that} appears to regulate protease activity through changes in its intracellular concentration. Proteases are cru-cial toPlasmodiumdevelopment, but the role of Ca2+_{in their activity is not fully understood. Here we} investigated the role of Ca2+_{in protease modulation among rodentPlasmodiumspp. Using fluorescence} resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca2+ _{from the} endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca2+_{-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K}+_/H+ exchanger) or monensin (a Na+_/H+_{exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca}2+ chelators were used to investigate the role played by Ca2+_{in protease activation. InPlasmodium berghei} both EGTA and BAPTA blocked protease activation, whilst inPlasmodium yoeliithese compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis inP. bergheibut decreased it inP. yoelii. Conversely, E64 reduced proteolysis inP. bergheibut stimulated it inP. yoelii. The data point out key differences in proteolytic responses to Ca2+ between species ofPlasmodium.

Ó2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd.

1. Introduction

Malaria is one of the most important infectious diseases in the

world, responsible for more than 1.5 million deaths each year

(

Snow et al., 2005

). The

Plasmodium

cell cycle is very complex

and displays several structural and biochemical changes during

the erythrocytic cycle (

Bannister and Mitchell, 1989; Garcia et al.,

1997; Blackman, 2004; Bozdech et al., 2008

). It has been well

established by several laboratories that the second messenger,

Ca

2+

_{can modulate}

_Plasmodium

_{cellular processes (}

_Blackman,

2000; Alleva and Kirk, 2001; Billker et al., 2004; Sibley, 2004;

Docampo et al., 2005; Nagamune and Sibley, 2006; Maier et al.,

2009

). The hormone melatonin and its derivatives induce Ca

2+

re-lease from internal pools and thereby the synchronisation of

Plasmodium falciparum

and

Plasmodium chabaudi

(

Hotta et al.,

2000; Beraldo et al., 2007

). Ca

2+

_{also modulates physiological}

fea-tures in

Plasmodium

sexual stages, as xanthurenic acid induces

gamete exflagelation by increasing the cytosolic Ca

2+

_{concentration}

(

Billker et al., 2004

).

Ca

2+

_{-mediated-signalling depends on the maintenance of low}

cytosolic Ca

2+

_{during erythrocytic stages (}

_{Gazarini et al., 2003}

_),

with sequestration of Ca

2+

_{in intracellular organelles such as}

mito-chondria (

Gazarini and Garcia, 2004

), endoplasmic reticulum (ER)

(

Passos and Garcia, 1997, 1998; Marchesini et al., 2000; Varotti

et al., 2003; Lew and Tiffert, 2007

), and acidic compartments

(

Garcia et al., 1998; Docampo et al., 2005; Moreno and Docampo,

2009

). Ca

2+

_{is also regulated by upstream molecular mechanisms}

at the membrane level (

Uyemura et al., 2000; Sibley, 2004;

Gins-burg and Stein, 2005; Vaid and Sharma, 2006; Vaid et al., 2008

)

and cAMP can stimulate kinase activity (

Beraldo et al., 2005; Billker

et al., 2009; Koyama et al., 2009

). Understanding the role of Ca

2+

_is

fundamental to modulate activation of plasmodial proteins. It is

now well established that

Plasmodium

utilises proteases for a

num-ber of processes during the erythrocytic cycle, including entry into

and exit from its host erythrocyte and feeding intracellularly on

erythrocytic

haemoglobin

(

Klemba

and

Goldberg,

2002;

Rosenthal, 2004; O’Donnell and Blackman, 2005; Liu et al., 2006;

0020-7519Ó2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. doi:10.1016/j.ijpara.2010.10.009

⇑

Corresponding author. Tel.: +55 11 3091 7518; fax: +55 11 3091 7422.

E-mail address:cgarcia@usp.br(C.R.S. Garcia).

Contents lists available at

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International Journal for Parasitology

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Open access under the Elsevier OA license.

Sinnis and Coppi, 2007

). All classes of proteases are represented

in the

Plasmodium

genome (

Florens et al., 2002; Klemba and

Goldberg, 2002; Wu et al., 2003; McKerrow et al., 2006

), and for

Plasmodium

survival, correct protein trafficking and targeting must

be regulated. Protein transport has been studied using GFP

expres-sion in

P. falciparum

but is still poorly understood (

Dahl and

Rosen-thal, 2005; Maier et al., 2009

). Some proteases, such as falcipains 2

and 3, are believed to be synthesised as membrane-bound

pro-teins, reaching the food vacuole via the ER/Golgi apparatus. These

proteases undergo auto-activation and are processed in

compart-ments with variable inhibitor access (

Dahl and Rosenthal, 2005

).

The effects of Ca

2+

modulation on protease activity of

P.

falcipa-rum

and

P. chabaudi

have been investigated by

Farias and

colleagues (2005

), who studied the proteolysis of fluorescence

res-onance energy transfer (FRET) peptides, for which fluorescence is

increased by increasing the spacing of the donor/quenching

recep-tor pair Abz/EDDnp (which are

ortho-aminobenzoic acid and

ethyl-ene diamine-2-4-dinitrophenyl, respectively) after hydrolysis of

any intervening peptide bond.

Farias and colleagues (2005)

re-ported for

P. chabaudi

that increased intracellular Ca

2+

_{elicited by}

the addition of pharmacological agents or the hormone melatonin

(

Hotta et al., 2000

) led to an increase in proteolytic activity, and

that the specific cysteine protease inhibitor E64 decreased such

Ca

2+

_{-triggered proteolysis. However, the mechanism of protease}

activation and inactivation by Ca

2+

_{signalling are poorly described}

in

Plasmodium

(

Koyama et al., 2009

), and a better understanding of

the physiology of these processes will contribute to our

under-standing of plasmodial proteases and the development of new

antimalarial drugs.

In the present work, we investigated the importance of Ca

2+

_in

modulation of proteolysis in different

Plasmodium

spp. We

ana-lysed the protease activity induced by increased Ca

2+

_{using the}

FRET peptides Abz-KLRSSKQ-EDDnp (KLR) and

Abz-AIKFFARQ-EDDnp (AIK), comparing the effects of releasing Ca

2+

_{from either}

the ER or acidic compartments. We also examined the classes of

protease involved in these responses and the effects of chelating

internal or external Ca

2+

_{(BAPTA/AM or EGTA, respectively) in the}

rodent malaria parasites

Plasmodium berghei

and

Plasmodium yoelii.

2. Materials and methods

2.1. Reagents

Nigericin, monensin, thapsigargin, phenylmethylsulphonyl

fluoride (PMSF), Pepstatin A, E64, saponin, probenecid and MOPS

Fig. 1.Ca2+_{mobilization from endoplasmatic reticulum and acidic pools in isolatedPlasmodium bergheiandPlasmodium yoeliiparasites. (A, B, D and E) Representative tracing}

of Fluo4/AM (green-fluorescent calcium indicator) changes over time by addition of thapsigargin (Thg) (10

l

M) and nigericin (Nig) (10

l

M) inP. bergheiandP. yoelii,

respectively. (C) Analyses of Ca2+_{concentration inP. bergheiisolated parasites labelled with Fluo4/AM (5}

l

M) after Thg (10

l

M) (3.809 arbitrary units (a.u.) ± 0.379,n= 9) and

Nig (10

l

M) (2.055 a.u. ± 0.225,n= 14) treatment. Bar graph represents maximum Ca2+concentration obtained after Thg and Nig treatment, and SEM of at least three

different experiments. (F) Dose dependent Ca2+_{response by Thg in isolatedP. yoeliiloaded with Fluo4/AM (5}

l

M) by addition of different concentration of Thg (5, 10 and

(3-(N-morpholino)propanesulfonic acid), probenecid,

L

-polylysine,

EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N

0

_{,N-tetraacetic}

acid) and dihydroethidium (DHT) were purchased from Sigma–

Aldrich (St. Louis, MO, USA). BAPTA/acetoxymethyl ester (AM),

Fluo4/AM, DAPI and BODIPY

Ò

FL-Thapsigargin were from

Molecu-lar Probes Inc. (Eugene, OR, USA). The peptides AIK and KLR were

analytical grade and synthesised according to Hirata and

col-leagues (

Hirata et al., 1994

;

Carmona et al., 2009

).

2.2. Plasmodium berghei (strain NK65) and P. yoelii (strain 17X)

parasites

Plasmodium berghei

and

P. yoelii

were maintained in

asynchro-nous parasitemia in mice (Balb/c strain) by transfer every 4 days

and parasitemias

determined from Giensa-stained smears.

To assess parasitemia forms in Balb/c mice and

Plasmodium

morphology, no less than 1000 erythrocytes were counted in

Giemsa-stained smears. All animal procedures were approved by

the São Paulo University Ethics Committee for Animal Experiments

(CEEA) according to the Colégio brasileiro de experimentação

ani-mal guidelines (COBEA).

Filtration of the infected rodent blood through a cellulose

col-umn (Whatman CF11) removed leucocytes and platelets. The

erythrocytes were washed twice in PBS (137 mM NaCl, 2.7 mM

KCl, 4.3 mM Na2HPO4, 1.4 mM NaH2PO4) by centrifugation at

1500g

for 5 min. Erythrocytes were then lysed in PBS with

60

l

g ml

1

saponin, and membranes were removed by

centrifuga-tion (10,000g

for 10 min at 4

°

_{C) in MOPS}

(3-(N-morpholino)pro-panesulfonic acid) buffer (116 mM NaCl, 5.4 mM KCl, 0.8 mM

MgSO4, 5.5 mM

D

-glucose, 50 mM MOPS and 1 mM CaCl2, pH

7.2). After erythrocyte lysis, the parasites were kept in MOPS

buffer. It is well established by previous work in our laboratory

that the parasites remain viable after this lysis treatment (

Beraldo

et al., 2005; Farias et al., 2005

).

Fig. 2.Cellular localization of endoplasmatic reticulum (ER) inPlasmodium bergheiandPlasmodium yoelii. (A) Localization of ER inPlasmodium bergheiparasites stained with thapsigargin-FL (2.5

l

M) and DAPI observed by phase contrast confocal microscopy, thapsigargin-FL, DAPI and merged images. (B) Localization of ER inP. yoeliiparasites

Fig. 3.Intracellular protease activity dependent on Ca2+_{release from the endoplasmic reticulum of Plasmodium berghei (A) and Plasmodium yoelii(B). Graphical}

representation of fluorescence resonance energy transfer (FRET) peptide hydrolysis increased by thapsigargin (10

l

M) and controls (DMSO or peptide) inP. bergheiand

P. yoeliiparasites. Isolated parasites (108_{cells ml} 1_{) were incubated in MOPS buffer in a 1 ml cuvette. The fluorescence was measured continuously 1 min after addition of the}

peptide Abz-AIKFFARQ-EDDnp (AIK) (10

l

M). (C) Proteolytic activity in uninfected erythrocytes. Graphical representation of FRET peptide hydrolysis by thapsigargin (10

l

M) treatment. The fluorescence was measured continuously 1 min after addition of the peptide AIK (10

l

M). (D) Permeation of fluorescence peptides into free parasites.

Plasmodium yoeliiparasites were loaded with fluorescence AIK peptides (10

l

M) and observed by phase contrast confocal microscopy, AIK peptide and merged images. Arb., arbitrary.

Fig. 4.Proteolytic activity induced by thapsigargin treatment inPlasmodium yoelii-infected and uninfected erythrocytes (RBC). (A) Graphical representation of confocal microscopy of infected and uninfected erythrocyte fluorescence after addition of the peptide Abz-AIKFFARQ-EDDnp (AIK) (10

l

M) and thapsigargin (Thg) (10

l

M). Note there

was no change in fluorescence in uninfected erythrocytes. (B) Rate of fluorescence in infected erythrocytes. Bar graph of increase in fluorescence rate after Thg (10

l

M)

treatment (1.768 arbitrary units (a.u.) ± 0.081,n= 26) in infectedP. yoeliierythrocytes incubated with the peptide AIK (10

l

M). (C) Fluorescence imaging of infected

erythrocytes. Erythrocytes infected byP. yoeliiwere loaded with AIK peptides (10

l

M) and imaged before (control) and after addition of Thg (10

l

M). Phase contrast, AIK

2.3. Peptide loading

The FRET peptides KLR and AIK have a fluorescent group, Abz,

and a quencher group, EDDnp. Both peptides are able to access free

malaria parasites after 1 min incubation in MOPS buffer. Stock

solutions were prepared in DMSO/water (1:1), and concentrations

were measured spectrophotometrically using a molar absorption

coefficient of 17,300 M

1

_cm

1

_{at 365 nm. FRET peptide stock}

solu-tion was used at 2 mM.

2.4. Spectrofluorimetric determinations

Spectrofluorimetric measurements were performed in a

Shima-dzu RF-5301 PC at 37

°

_{C with isolated parasites (10}

8

_{cells ml}

1

₎

incubated with MOPS buffer in a 1 ml cuvette. The fluorescence

was measured continuously beginning 1 min after addition of the

FRET peptides (10

l

M). Excitation/emission wavelengths were

ad-justed to 320/420 nm for Abz.

For experiments with protease inhibitors, parasites were

pre-incubated with PMSF (10 or 15

l

M), E64 (5, 10 or 20

l

M) or

Pep-statin A (10 or 15

l

M) for 15 or 20 min at room temperature. For

experiments with the extracellular Ca

2+

_{chelator EGTA (5 mM)}

par-asites were pre-incubated for 5 min at room temperature and for

those with the intracellular Ca

2+

_{chelator BAPTA/AM (25, 50, 100,}

200 and 500

l

M), parasites were pre-incubated for 40 min at room

temperature. All incubations were performed before the addition

of the FRET substrate.

Ca

2+

_{measurements were performed in}

_{P. berghei}

_and

_{P. yoelii}

isolated parasites loaded with Fluo4-AM (5

l

M) and probenecid

(2.8 mM) to minimise indicator extrusion in MOPS buffer for

30 min at room temperature. Parasite suspensions were then

washed twice with MOPS buffer before addition of the substrates.

Thapsigargin (5, 10 or 25

l

M) and nigericin (10

l

M) were added

during time course experiments and excitation/emission

wave-lengths adjusted to 505/530 nm for Fluo4-AM.

2.5. Confocal microscopy

Imaging was performed with an LSM 510 laser scanning

microscope (Carl Zeiss) using LSM 510 software, version 2.5. The

Axiovert 100M microscope was equipped with a 63X water

immer-sion objective. Parasites were plated onto microscopy coverslips

(MatTek Corp., USA) pre-treated for 1 h with

L

-polylysine solution

and excited at 351 nm. Emitted light was collected through a band

pass filter at 387–470 nm. Experiments were performed with free

P. yoelii

parasites, infected erythrocytes and intact erythrocytes

loaded with FRET peptide AIK after 1 or 10 min of incubation time

at room temperature in MOPS buffer.

Fig. 5.Intracellular protease activity is not modulated by Ca2+_{released from acidic pools inPlasmodium bergheiandPlasmodium yoelii. (A) Treatment of monensin (10}

l

M)

and nigericin (10

l

M) loaded with Abz-AIKFFARQ-EDDnp (AIK) had no effect in fluorescence resonance energy transfer (FRET) peptide hydrolysis inP. berghei(0.6791 ± 0.116,

n= 3,P= 0.0005 and 0.9130 ± 0.321,n= 3,P= 0.252, respectively;Pvalues were compared with the control (ctr) for AIK data 1.158 ± 0.0314,n= 7). Isolated parasites (108

cells ml1_{) were incubated with MOPS buffer and fluorescence measured continuously after addition of the peptide AIK (10}

l

M) or Abz-KLRSSKQ-EDDnp (KLR) (10

l

M). Treatment of thapsigargin (Thg) (10

l

M) with AIK or KLR (10

l

M) peptides induced proteolysis activation (3.222 ± 0.28,n= 12,P< 0.0001, respectively;Pvalues were compared with the ctr for AIK data 1.158 ± 0.0314,n= 7 and 3.264 ± 0.151,n= 15,P< 0.0001,Pvalues were compared with the ctr of KLR data 1.05 ± 0.121,n= 5). Bar graph represents mean with SEM of at least three different experiment days. (B) Intracellular protease activity is marginally activated by Ca2+_{released from acidic pools in}

Plasmodium yoelii. Treatment of Thg (10

l

M), monensin (10

l

M) or nigericin (10

l

M) loaded with AIK (10

l

M) increased FRET peptide hydrolysis inP. yoelii. (2.975 ± 0.275,

n= 15,P= 0.0002; 1.39 ± 0.047,n= 11,P= 0.0016; 1.5 ± 0.071,n= 8,P= 0.0009, respectively;Pvalues were compared with the ctr data 1.146 ± 0.033,n= 7). Isolated parasites (108_{cells ml}1_{) were incubated with MOPS buffer and fluorescence measured continuously after addition of the peptide AIK (10}

l

M). Bar graph represent mean with SEM of

ER was visualised in

P. berghei and P. yoelli

parasites stained

with BODIPY

Ò

FL-Thapsigargin (2.5

l

M) and nuclei were visualised

by DAPI (1:1000) staining after 30 min incubation at room

temper-ature in MOPS buffer. Erythrocytes were plated onto microscopy

coverslips (MatTek Corp.) pre-treated for 1 h with

L

-polylysine

solution and excited at 488 nm. Emitted light was collected

through a band pass filter at 505–550 nm.

2.6. Cell viability

To verify cell viability at the beginning and at the end of the

experiments, isolated

P. berghei

and

P. yoelii

parasites were

incu-bated with Trypan Blue (1:1) for 30 min at room temperature

and at least 1000 cells were counted. Viability was also assessed

in

P. yoelii

by DHT (1:200) staining for 20 min at 37

°

_{C and analysed}

by dot plots (side scatter versus fluorescence) of 10

5

_{cells acquired}

on a FACSCalibur cytometer using CELLQUEST software (Becton

Dickinson). Initial gating was carried out with unstained, isolated

parasites to account for parasite autofluorescence.

2.7. Statistical analyses

All results are expressed as mean ± SEM of at least three

indi-vidual experiments. Student’s

t-test was used for comparisons

be-tween two groups, whereas repeated measures ANOVA was used

for comparisons among larger groups. A

P

value less than 0.05

was considered indicative of a statistically significant difference.

GraphPad Prism software (San Diego, CA, USA) was used for all

sta-tistical tests.

3. Results

3.1. Induction of proteolysis by thapsigargin but not by monensin or

nigericin

Ca

2+

_{homeostasis in}

_Plasmodium

_{was reported to be regulated}

by the ER, mitochondria (

Gazarini and Garcia, 2004

) and acidic

pools (

Passos and Garcia, 1997; Garcia et al., 1998; Varotti et al.,

2003; Docampo et al., 2005

). These studies were based on confocal

microscopy and spectrofluorimetry using pharmacological tools

such as thapsigargin (Sarco/ER Ca

2+

_{-ATPase-SERCA-inhibitor)}

nige-ricin (K

+

_/H

+

_{exchanger) or monensin (Na}

+

_/H

+

_{exchanger) to}

selec-tively discharge Ca

2+

_{pools. Here we have investigated protease}

activity modulation by Ca

2+

_{release from the ER using the SERCA}

inhibitor thapsigargin.

Ca

2+

_{release from the ER and from acidic pools using}

pharmaco-logical compounds were reported in

P. berghei

and

P. yoelii

(

Bagnaresi et al., 2009

). In this study, we confirmed, using Ca

2+

dyes, that addition of thapsigargin (10

l

M) and nigericin (10

l

M)

induces an increase in the cytosolic Ca

2+

_{concentration in}

_{P. berghei}

(

Fig. 1A–C

). The amount of Ca

2+

_{released with thapsigargin is}

high-er than that induced with nighigh-ericin in

P. berghei

and

P. yoelii

para-sites (

Fig. 1

). A dose-dependent thapsigargin effect (5, 10 and

25

l

M) in

P. yoelii

isolated parasites confirms the ability of this

inhibitor to induce Ca

2+

_{release in these parasites (}

_{Fig. 1}

_F).

A fluorescent thapsigargin was used in experiments with

P. berghei

and

P. yoelii

parasites which were then imaged by

confo-cal microscopy (

Fig. 2

A and B). These results confirm the previous

finding that

P. berghei

and

P. yoelii

ERs store Ca

2+

₍

_{Bagnaresi et al.,}

2009

).

Treatment of

P. berghei

(

Fig. 3

A) and

P. yoelii

(

Fig. 3

B) with

10

l

M thapsigargin (Ca

2+

ATPase-SERCA inhibitor) to induce Ca

2+

release from the ER led to increased proteolysis of the FRET peptide

AIK. In control experiments, addition of thapsigargin (10

l

M) to

the erythrocyte lysate or non-infected erythrocytes (

Fig. 3

C) led

to no change in the base line level of fluorescence, confirming that

the fluorescence changes induced by thapsigargin required intact

P. berghei

or

P. yoelii

parasites. By confocal microscopy we observed

that free parasites were permeable to FRET peptides (

Fig. 3

D).

Fluo-rescence enhancement is related to proteolysis activity within

par-asites in infected erythrocytes and were not observed in the intact

erythrocytes once thapsigargin (10

l

M) was added (

Fig. 4

). We also

investigated the effects of depleting acidic Ca

2+

_{pools with}

nigeri-cin and monensin, classical ionophores that exchange K

+

_–H

+

_and

Na

+

–H

+

, respectively, from internal compartments such as acidic

Fig. 6.Influence of intra and extracellular Ca2+_{on proteolytic activity ofPlasmodium bergheiis different fromPlasmodium yoelii. (A) Protease activity inhibited by intra and}

extracellular Ca2+_{chelation inP. berghei. Effects of thapsigargin (Thg) (10}

l

M) on fluorescence resonance energy transfer (FRET) peptide Abz-AIKFFARQ-EDDnp (AIK) (10

l

M)

inP. bergheiisolated parasites (108_{cells ml} 1_{) and incubation with intracellular Ca}2+_{chelators BAPTA/AM (25, 50, 100, 200 and 500}

l

M), for 40 min (2.696 ± 0.379,n= 10,

P= 0.268; 2.846 ± 0.25,n= 7,P= 0.378; 2.571 ± 0.318,n= 9,P= 0.142; 2.112 ± 0.154,n= 9;P= 0.0052,; 1.455 ± 0.089,n= 7;P= 0.0002, respectively) or the extracellular Ca2+

chelator EGTA (5 mM) for 5 min (1.578 ± 0.199,n= 19;P< 0.0001, respectively). AllPvalues were compared with Thg data (3.222 ± 0.28,n= 12). (B) Proteases inhibited by Ca2+_{play a major role in peptide hydrolysis inP. yoelii. Effects of Thg (10}

l

M) on FRET peptide AIK (10

l

M) inP. yoeliiisolated parasites (108_{cells ml}1_{) and incubation with}

intracellular Ca2+_{chelators BAPTA/AM (25, 200 or 500}

l

M) for 40 min (5.69 ± 0.372,n= 9,P< 0.0001; 3.95 ± 0.348,n= 12,P= 0.035; 2.713 ± 0.175,n= 15,P= 0.429,

respectively) or the extracellular Ca2+_{chelator EGTA (5 mM) for 5 min (5.136 ± 0.475,n= 6;P= 0.0006).Pvalues were compared with Thg data (2.975 ± 0.275,n= 15). The}

effect of the inhibitor BAPTA/AM (25 or 200

l

M) without the presence of Thg on FRET AIK (10

l

M) hydrolysis inP. yoeliiisolated parasites was also verified (1.750 ± 0.064,

pools. Treatments of

P. berghei

parasites with nigericin (10

l

M) and

monensin (10

l

M) had no effect on proteolysis of AIK, whilst

thapsigargin (10

l

M) treatment increased proteolysis of AIK and

KLR peptide (

Fig. 5

A). Thus, it appears that Ca

2+

_{from the ER}

in-duces

P.

berghei

proteolysis. In contrast, in

P. yoelii

both nigericin

(10

l

M) and monensin (10

l

M) led to a marginal increase in

pro-teolysis of AIK peptide compared with propro-teolysis activation

trig-gered by thapsigargin (

Fig. 5

B).

The influence of Ca

2+

_{levels on proteolytic activity was also}

investigated by incubation of parasites in buffers containing

intra-cellular (BAPTA/AM) or extraintra-cellular (EGTA) Ca

2+

_chelators.

_{Fig. 6}

shows that lower concentrations of BAPTA/AM (25, 50 and

100

l

M) had no effect; however 200 and 500

l

M of the

intracellu-lar Ca

2+

_{chelator inhibited protease activity in}

_{P. berghei}

₍

_{Fig. 6}

_A).

The role of intracellular Ca

2+

_{in protease activation in}

_{P. yoelii}

dif-fers from that in

P. berghei. First, protease activation in

P. yoelii

in-creased after incubation with BAPTA/AM (25 and 200

l

M), but this

activation was suppressed by a higher concentration of BAPTA/AM

(500

l

M) (

Fig. 6

B). Second, treatment with EGTA (5 mM) inhibited

protease activity in

P. berghei

but increased activity in

P. yoelii

(

Fig. 6

A and B, respectively).

3.2. The Ca

2+

_{-induced proteolytic response is protease class-specific}

Protease inhibitors were used to investigate the specific classes

of proteases involved in substrate hydrolysis in

P. berghei

and

P. yoelii. In

P. berghei

(

Fig. 7

A), pre-incubation with the aspartic

pro-tease inhibitor Pepstatin A (15

l

M) or the serine protease inhibitor

PMSF (15

l

M) followed by thapsigargin (10

l

M) treatment

in-creased peptide hydrolysis, whilst incubation with the cysteine

protease inhibitor E64 (15

l

M) reduced proteolysis. In contrast,

re-sults were markedly different in

P. yoelii

(

Fig. 7

B), with peptide

hydrolysis stimulated by E64 and inhibited by Pepstatin A.

FRET control experiments without thapsigargin and in the

pres-ence of either E64 (15

l

M) or Bapta/AM (25 and 200

l

M) show a

very small change in peptide hydrolysis compared with control

experiments (

Figs. 6B and 7

B).

Plasmodium berghei

and

P. yoelii

are relatively asynchronous,

compared with

P. falciparum

and

P. chabaudi, and these species

have a tendency to induce reticulocytosis and to invade

reticulo-cytes. Using Giensa-stained smears, we observed that the vast

majority of parasites in our experiments with both

P. berghei

and

P. yoelii

were at the trophozoite stage (most physiologically active

state). Distribution of

P. berghei

and

P. yoelii

life forms and

parasi-temia (45.62% ± 3.55,

n

= 8; 31.03 ± 2.58,

n

= 14, respectively) in

proportion to rings (6.16% ± 1.38,

n

= 8; 5.34% ± 1.06,

n

= 14,

respectively), trophozoites (28.57% ± 1.79,

n

= 8; 21.82% ± 1.86,

n

= 14, respectively) and schizonts (10.89% ± 2.2,

n

= 8; 3.86 ±

0.55,

n

= 14, respectively) were constant among the experiments.

Plasmodium berghei

and

P. yoelii

parasite viability was assessed

by Trypan Blue staining at the beginning and at the end of the

experiment and showed no statistical difference (98.92 ± 0.12

n

= 9 and 98.75 ± 0.25,

n

= 5;

P

= 0.5048 and 98.16 ± 0.18,

n

= 5

and 98.17 ± 0.21,

n

= 6,

P

= 0.9864, respectively).

Finally, we have performed viability assays by using DHT

stain-ing and flow cytometry.

Fig. 8

shows that

P. yoelii

parasites at the

beginning and end (3 h later) of FRET experiments showed no

sta-tistical difference in DHT fluorescence. Both methods confirmed

that ex vivo experiments for measuring protease activity were

per-formed in ideal conditions.

4. Discussion

Our results show that release of Ca

2+

_{from the ER by}

thapsigar-gin treatment increases the rate of hydrolysis of FRET peptides in

P. berghei

and

P. yoelii

(

Figs. 3–5

). These results are similar to those

of

Farias and colleagues (2005)

for two other

Plasmodium

spp.,

P. falciparum

and

P. chabaudi. The ER, is an important compartment

for intracellular Ca

2+

_{storage in eukaryotic cells (}

_{Thastrup et al.,}

1987; Sagara et al., 1992

), including protozoan parasites (

Sibley,

2004; Berridge, 2006

). These results indicate the importance of

Ca

2+

_{release from the ER in stimulating protease activity in}

differ-ent

Plasmodium

spp.

In contrast,

P. berghei

release of Ca

2+

_{from acidic pools by}

nige-ricin or monensin did not activate proteolysis (

Fig. 5

A). These data

indicate that cellular proteases from

P. berghei

are sensitive to pH,

since changes in cytosolic Ca

2+

_{with nigericin and monensin should}

also lead to a change in the pH (

Rohrbach et al., 2005; Kuhn et al.,

Fig. 7.The Ca2+_{-induced proteolytic response is protease class-specific. (A)Plasmodium bergheihas different classes of proteases modulated by endoplasmic reticulum (ER)}

Ca2+_{release. Effect of thapsigargin (Thg) (10}

l

M) on fluorescence resonance energy transfer (FRET) peptide KLRSSKQ-EDDnp (10

l

M) hydrolysis inP. berghei. Isolated parasites (108_{cells ml} 1_{) were incubated with Pepstatin A (15}

l

M) or phenylmethylsulphonyl fluoride (PMSF) (15

l

M) for 20 min and E64 (15

l

M) for 15 min (3.96 ± 0.2,

n= 8,P= 0.0119; 4.55 ± 0.31,n= 11,P= 0.0005; 1.5 ± 0.16,n= 7,P< 0.0001, respectively;Pvalues were compared with the Thg data 3.26 ± 0.15,n= 15). (B)Plasmodium yoelii

has multiple proteases modulated by ER Ca2+_{release. Effects of Thg (10}

l

M) on FRET peptide KLRSSKQ-EDDnp (10

l

M) hydrolysis inP. yoelii. Isolated parasites (108cells

ml1_{) were incubated with Pepstatin A (10}

l

M), PMSF (10

l

M) or E64 (15

l

M) for 15 min (1.10 ± 0.237,n= 6,P= 0.0007; 1.83 ± 0.128,n= 3,P= 0.0791; 5.55 ± 0.647,n= 9,

P= 0.0007, respectively;Pvalues were compared with Thg data 2.51 ± 0.198,n= 8). The effect of E64 (15

l

M) on FRET hydrolysis without Thg addition inP. yoeliiisolated

2007

). Likewise, the effect of Ca

2+

_{release from acidic pools in}

P. yoelii

was marginal (

Fig. 5

B), suggesting that acidification of

cytosol might interfere with protease activation in both parasites.

We also performed experiments with intracellular (BAPTA/AM)

and extracellular (EGTA) Ca

2+

_{chelators to investigate the role}

played by Ca

2+

_{in protease activation. We found that in}

_{P. berghei}

both EGTA and BAPTA block protease activation (

Fig. 6

A), whilst

in

P. yoelii

(

Fig. 6

B) blocking Ca

2+

_{-dependent enzymes resulted in}

activation of proteases. Protease activity in

P. yoelii

in the absence

of Ca

2+

_{was higher than in the presence of Ca}

2+

_{, indicating that the}

basal cytosolic Ca

2+

_{was already modulating the activity (see}

con-trol of basal protease activity before activation with Ca

2+

_in

_{Fig. 3}

_A

and B) of the two classes of enzymes, Ca

2+

_{activated and Ca}

2+

inhibited.

Our results show that modulation of protease activation differs

between

P. berghei

and

P. yoelii. Understanding how these

impor-tant classes of enzymes are activated or inhibited will help us to

better understand their role in parasite biology. Although

P. berghei

and

P. yoelii

both showed peptide hydrolysis upon

thapsigargin-induced Ca

2+

_{release (}

_{Fig. 3}

_{A and B), the nature of the proteolysis}

induced by thapsigargin differed in the two species. In

P. berghei

proteolysis was inhibited by E64 and stimulated by Pepstatin A

and PMSF (

Fig. 7

A), suggesting cysteine protease activity, as was

also seen with

P. chabaudi

(

Farias et al., 2005

). In

P. yoelii

proteolysis was inhibited by Pepstatin A and stimulated by E64

(

Fig. 7

B), suggesting aspartic protease activity. It is not clear

why these two similar parasites utilise different proteolytic

mechanisms.

The best known cysteine proteases in

Plasmodium

are the

falci-pains, in particular falcipain-2 and -3 (

Rosenthal, 2004; Sijwali and

Rosenthal, 2004

) which are food vacuole haemoglobinases, but

also may play other roles in erythrocytic parasites (

Rosenthal,

2004

). Further work is needed to determine whether one or more

of this family is responsible for the observed Ca

2+

_{activation in}

P. berghei. In

P. yoelii

Ca

2+

activated proteolysis might be principally

due to activity of plasmepsin aspartic proteases (

Eggleson et al.,

1999

). Malaria parasites express a large set of aspartic proteases,

known as plasmepsins (

Dame et al., 2003; Omara-Opyene et al.,

2004; Liu et al., 2005; Ersmark et al., 2006

). Some of the

plasmep-sins (in

P. falciparum

plasmepsins I–IV) are also food vacuole

haemoglobinases but others play different roles, including

plasmepsin V, which processes the PEXEL motif in the

P. falciparum

ER (

Klemba and Goldberg, 2005; Boddey et al., 2010; Russo et al.,

2010

).

Studies on the role of Ca

2+

_{in modulating plasmodial biology are}

limited. We have shown that

Plasmodium

senses the environment

and modulates its cell cycle through synchronisation of its

erythro-cytic forms. The plasmodial cell cycle is affected in a Ca

2+

-dependent manner by melatonin (

Hotta et al., 2000, 2003

), its

precursor

N-acetylserotonin, and tryptamine, serotonin and

N(1)-acetyl-N(2)-formyl-5-methoxykynuramine-AFMK (

Beraldo et al.,

2005; Budu et al., 2007

). These molecules all induced Ca

2+

_release

from

P. falciparum. Melatonin initiated a complex signalling

path-way resulting in increases of cAMP and intracellular Ca

2+

concen-tration [Ca

2+

_]i

_{with the two second messengers interacting with}

each other in a synergistic manner (

Beraldo et al., 2005

). However,

the physiological significance of the parasite’s ability to increase

cytosolic Ca

2+

_{during the erythrocytic cycle was not clear from}

these studies.

We now report that Ca

2+

_{modulates protease activity in rodent}

malaria parasites, but that different classes of proteases appear to

be activated in different plasmodial species. These data suggest

that different

Plasmodium

spp. have evolved unique cellular

physi-ologies. Interestingly, rodent malarial species are known to differ

in related features.

Plasmodium chabaudi

parasites are more

syn-chronous than

P. berghei

and

P. yoelii, and we have recently

reported that

P. berghei

and

P. yoelii

do not respond to the hormone

melatonin (

Bagnaresi et al., 2009

), whilst in

P. chabaudi

the

hor-mone has clear effects on cytosolic Ca

2+

_{and the cell cycle (}

_Hotta

et al., 2000

). Curiously, even the similar species

P. berghei

and

P.

chabaudi

differed in the class of protease that appears to be

acti-vated by Ca

2+

_{. Additional work with different}

_Plasmodium

_{spp. will}

be needed to better understand the role of Ca

2+

_{in the biology of}

malaria parasites.

The parasite molecular machinery for signalling is quite

com-plex and includes heptahelical receptors (

Garcia et al., 2008;

Madeira et al., 2008

) and a receptor for activated-C kinase (

Madeira

et al., 2003

). Interestingly, it has been recently reported that the

second messenger cADPr modulates

Plasmodium

invasion into

erythrocytes (

Jones et al., 2009

). Further investigation will be

fun-damental to understanding when and how second messengers are

put into action to modulate protease activity.

Fig. 8.Viability was assessed by flow cytometry using dihydroethidium (DHT) staining inPlasmodium yoeliiparasites. (A) Histogram distribution of fluorescence in non-labelled parasites (control), parasites non-labelled at the beginning (dashed line) and 3 h later (solid line) in the same buffer. (B) Bar graph analyses of viability inP. yoelii. Mean of three independent experiments shows no statistical difference in DHT fluorescence at the beginning or at the end of the experiment (99.42 ± 0.22,n= 3 and 99.29 ± 0.29,

Acknowledgments

This work was funded by Fundação de Amparo à Pesquisa do

Estado de São Paulo (FAPESP), Brazil, to CRSG and LJ. LNC received

a fellowship from FAPESP. CRSG and LJ are research fellows of

Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq), Brazil. CRSG also thanks INCT (CNPq)-INBqmed and

Malaria Pronex-MS-CNPq for funding. We thank Lawry Bannister

for critically reading the manuscript.

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