International Journal for Parasitology 41 (2011) 363–372
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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 Department of Physiology, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, travessa 14, n321, CEP 05508-900, São Paulo, SP, Brazil 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 b c
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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 crucial to Plasmodium development, but the role of Ca2+ in their activity is not fully understood. Here we investigated the role of Ca2+ in protease modulation among rodent Plasmodium spp. 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) Ca2+ chelators were used to investigate the role played by Ca2+ in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these 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 thapsigargininduced proteolysis in P. berghei but decreased it in P. yoelii. Conversely, E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca2+ between species of Plasmodium. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
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, Ca2+ 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 Ca2+ release from internal pools and thereby the synchronisation of Plasmodium falciparum and Plasmodium chabaudi (Hotta et al., 2000; Beraldo et al., 2007). Ca2+ also modulates physiological features in Plasmodium sexual stages, as xanthurenic acid induces ⇑ Corresponding author. Tel.: +55 11 3091 7518; fax: +55 11 3091 7422. E-mail address:
[email protected] (C.R.S. Garcia).
gamete exflagelation by increasing the cytosolic Ca2+ concentration (Billker et al., 2004). Ca2+-mediated-signalling depends on the maintenance of low cytosolic Ca2+ during erythrocytic stages (Gazarini et al., 2003), with sequestration of Ca2+ in intracellular organelles such as mitochondria (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). Ca2+ is also regulated by upstream molecular mechanisms at the membrane level (Uyemura et al., 2000; Sibley, 2004; Ginsburg 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 Ca2+ is fundamental to modulate activation of plasmodial proteins. It is now well established that Plasmodium utilises proteases for a number 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;
0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2010.10.009
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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 expression in P. falciparum but is still poorly understood (Dahl and Rosenthal, 2005; Maier et al., 2009). Some proteases, such as falcipains 2 and 3, are believed to be synthesised as membrane-bound proteins, reaching the food vacuole via the ER/Golgi apparatus. These proteases undergo auto-activation and are processed in compartments with variable inhibitor access (Dahl and Rosenthal, 2005). The effects of Ca2+ modulation on protease activity of P. falciparum and P. chabaudi have been investigated by Farias and colleagues (2005), who studied the proteolysis of fluorescence resonance energy transfer (FRET) peptides, for which fluorescence is increased by increasing the spacing of the donor/quenching receptor pair Abz/EDDnp (which are ortho-aminobenzoic acid and ethylene diamine-2-4-dinitrophenyl, respectively) after hydrolysis of any intervening peptide bond. Farias and colleagues (2005) reported for P. chabaudi that increased intracellular Ca2+ 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
Ca2+-triggered proteolysis. However, the mechanism of protease activation and inactivation by Ca2+ signalling are poorly described in Plasmodium (Koyama et al., 2009), and a better understanding of the physiology of these processes will contribute to our understanding of plasmodial proteases and the development of new antimalarial drugs. In the present work, we investigated the importance of Ca2+ in modulation of proteolysis in different Plasmodium spp. We analysed the protease activity induced by increased Ca2+ using the FRET peptides Abz-KLRSSKQ-EDDnp (KLR) and Abz-AIKFFARQEDDnp (AIK), comparing the effects of releasing Ca2+ 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 Ca2+ (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 isolated Plasmodium berghei and Plasmodium yoelii parasites. (A, B, D and E) Representative tracing of Fluo4/AM (green-fluorescent calcium indicator) changes over time by addition of thapsigargin (Thg) (10 lM) and nigericin (Nig) (10 lM) in P. berghei and P. yoelii, respectively. (C) Analyses of Ca2+ concentration in P. berghei isolated parasites labelled with Fluo4/AM (5 lM) after Thg (10 lM) (3.809 arbitrary units (a.u.) ± 0.379, n = 9) and Nig (10 lM) (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 isolated P. yoelii loaded with Fluo4/AM (5 lM) by addition of different concentration of Thg (5, 10 and 25 lM) (2.422 a.u. ± 0.344, n = 8; 2.942 a.u. ± 0.144; n = 14 and 6.375 a.u. ± 0.498, n = 8, respectively) and Nig (10 lM) (2.247 a.u. ± 0.170, n = 9). Arb., arbitrary.
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(3-(N-morpholino)propanesulfonic acid), probenecid, L-polylysine, EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N-tetraacetic acid) and dihydroethidium (DHT) were purchased from Sigma– Aldrich (St. Louis, MO, USA). BAPTA/acetoxymethyl ester (AM), Fluo4/AM, DAPI and BODIPYÒ FL-Thapsigargin were from Molecular Probes Inc. (Eugene, OR, USA). The peptides AIK and KLR were analytical grade and synthesised according to Hirata and colleagues (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 asynchronous 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
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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 animal guidelines (COBEA). Filtration of the infected rodent blood through a cellulose column (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 lg ml1 saponin, and membranes were removed by centrifugation (10,000g for 10 min at 4 °C) in MOPS (3-(N-morpholino)propanesulfonic 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) in Plasmodium berghei and Plasmodium yoelii. (A) Localization of ER in Plasmodium berghei parasites stained with thapsigargin-FL (2.5 lM) and DAPI observed by phase contrast confocal microscopy, thapsigargin-FL, DAPI and merged images. (B) Localization of ER in P. yoelii parasites stained with thapsigargin-FL (2.5 lM) and observed by phase contrast confocal microscopy, thapsigargin-FL and merged images.
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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 lM) and controls (DMSO or peptide) in P. berghei and P. yoelii parasites. Isolated parasites (108 cells ml1) 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 lM). (C) Proteolytic activity in uninfected erythrocytes. Graphical representation of FRET peptide hydrolysis by thapsigargin (10 lM) treatment. The fluorescence was measured continuously 1 min after addition of the peptide AIK (10 lM). (D) Permeation of fluorescence peptides into free parasites. Plasmodium yoelii parasites were loaded with fluorescence AIK peptides (10 lM) and observed by phase contrast confocal microscopy, AIK peptide and merged images. Arb., arbitrary.
Fig. 4. Proteolytic activity induced by thapsigargin treatment in Plasmodium 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 lM) and thapsigargin (Thg) (10 lM). 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 lM) treatment (1.768 arbitrary units (a.u.) ± 0.081, n = 26) in infected P. yoelii erythrocytes incubated with the peptide AIK (10 lM). (C) Fluorescence imaging of infected erythrocytes. Erythrocytes infected by P. yoelii were loaded with AIK peptides (10 lM) and imaged before (control) and after addition of Thg (10 lM). Phase contrast, AIK peptide fluorescence and merged images are indicated. Arb., arbitrary.
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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 M1 cm1 at 365 nm. FRET peptide stock solution was used at 2 mM. 2.4. Spectrofluorimetric determinations Spectrofluorimetric measurements were performed in a Shimadzu RF-5301 PC at 37 °C with isolated parasites (108 cells ml1) incubated with MOPS buffer in a 1 ml cuvette. The fluorescence was measured continuously beginning 1 min after addition of the FRET peptides (10 lM). Excitation/emission wavelengths were adjusted to 320/420 nm for Abz. For experiments with protease inhibitors, parasites were preincubated with PMSF (10 or 15 lM), E64 (5, 10 or 20 lM) or Pepstatin A (10 or 15 lM) for 15 or 20 min at room temperature. For experiments with the extracellular Ca2+ chelator EGTA (5 mM) parasites were pre-incubated for 5 min at room temperature and for those with the intracellular Ca2+ chelator BAPTA/AM (25, 50, 100,
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200 and 500 lM), parasites were pre-incubated for 40 min at room temperature. All incubations were performed before the addition of the FRET substrate. Ca2+ measurements were performed in P. berghei and P. yoelii isolated parasites loaded with Fluo4-AM (5 lM) 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 lM) and nigericin (10 lM) were added during time course experiments and excitation/emission wavelengths 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 immersion 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 in Plasmodium berghei and Plasmodium yoelii. (A) Treatment of monensin (10 lM) and nigericin (10 lM) loaded with Abz-AIKFFARQ-EDDnp (AIK) had no effect in fluorescence resonance energy transfer (FRET) peptide hydrolysis in P. berghei (0.6791 ± 0.116, n = 3, P = 0.0005 and 0.9130 ± 0.321, n = 3, P = 0.252, respectively; P values 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 lM) or Abz-KLRSSKQ-EDDnp (KLR) (10 lM). Treatment of thapsigargin (Thg) (10 lM) with AIK or KLR (10 lM) peptides induced proteolysis activation (3.222 ± 0.28, n = 12, P < 0.0001, respectively; P values were compared with the ctr for AIK data 1.158 ± 0.0314, n = 7 and 3.264 ± 0.151, n = 15, P < 0.0001, P values 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 lM), monensin (10 lM) or nigericin (10 lM) loaded with AIK (10 lM) increased FRET peptide hydrolysis in P. 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; P values were compared with the ctr data 1.146 ± 0.033, n = 7). Isolated parasites (108 cells ml1) were incubated with MOPS buffer and fluorescence measured continuously after addition of the peptide AIK (10 lM). Bar graph represent mean with SEM of at least three different experiment days. Arb., arbitrary.
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ER was visualised in P. berghei and P. yoelli parasites stained with BODIPYÒ FL-Thapsigargin (2.5 lM) and nuclei were visualised by DAPI (1:1000) staining after 30 min incubation at room temperature 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 incubated 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 105 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 individual experiments. Student’s t-test was used for comparisons between 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 statistical tests. 3. Results 3.1. Induction of proteolysis by thapsigargin but not by monensin or nigericin Ca2+ 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 Ca2+-ATPase-SERCA-inhibitor) nigericin (K+/H+ exchanger) or monensin (Na+/H+ exchanger) to selectively discharge Ca2+ pools. Here we have investigated protease activity modulation by Ca2+ release from the ER using the SERCA inhibitor thapsigargin. Ca2+ release from the ER and from acidic pools using pharmacological compounds were reported in P. berghei and P. yoelii (Bagnaresi et al., 2009). In this study, we confirmed, using Ca2+ dyes, that addition of thapsigargin (10 lM) and nigericin (10 lM) induces an increase in the cytosolic Ca2+ concentration in P. berghei (Fig. 1A–C). The amount of Ca2+ released with thapsigargin is higher than that induced with nigericin in P. berghei and P. yoelii parasites (Fig. 1). A dose-dependent thapsigargin effect (5, 10 and 25 lM) in P. yoelii isolated parasites confirms the ability of this inhibitor to induce Ca2+ release in these parasites (Fig. 1F). A fluorescent thapsigargin was used in experiments with P. berghei and P. yoelii parasites which were then imaged by confocal microscopy (Fig. 2A and B). These results confirm the previous finding that P. berghei and P. yoelii ERs store Ca2+ (Bagnaresi et al., 2009). Treatment of P. berghei (Fig. 3A) and P. yoelii (Fig. 3B) with 10 lM thapsigargin (Ca2+ATPase-SERCA inhibitor) to induce Ca2+ release from the ER led to increased proteolysis of the FRET peptide AIK. In control experiments, addition of thapsigargin (10 lM) to the erythrocyte lysate or non-infected erythrocytes (Fig. 3C) 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. 3D). Fluorescence enhancement is related to proteolysis activity within parasites in infected erythrocytes and were not observed in the intact erythrocytes once thapsigargin (10 lM) was added (Fig. 4). We also investigated the effects of depleting acidic Ca2+ pools with nigericin 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 of Plasmodium berghei is different from Plasmodium yoelii. (A) Protease activity inhibited by intra and extracellular Ca2+ chelation in P. berghei. Effects of thapsigargin (Thg) (10 lM) on fluorescence resonance energy transfer (FRET) peptide Abz-AIKFFARQ-EDDnp (AIK) (10 lM) in P. berghei isolated parasites (108 cells ml1) and incubation with intracellular Ca2+ chelators BAPTA/AM (25, 50, 100, 200 and 500 lM), 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). All P values were compared with Thg data (3.222 ± 0.28, n = 12). (B) Proteases inhibited by Ca2+ play a major role in peptide hydrolysis in P. yoelii. Effects of Thg (10 lM) on FRET peptide AIK (10 lM) in P. yoelii isolated parasites (108 cells ml1) and incubation with intracellular Ca2+ chelators BAPTA/AM (25, 200 or 500 lM) 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). P values were compared with Thg data (2.975 ± 0.275, n = 15). The effect of the inhibitor BAPTA/AM (25 or 200 lM) without the presence of Thg on FRET AIK (10 lM) hydrolysis in P. yoelii isolated parasites was also verified (1.750 ± 0.064, n = 9, P < 0.0001 and 1.909 ± 0.112, n = 7, P < 0.0007, respectively; P values were compared with control (ctr) data 1.146 ± 0.033, n = 7). Bar graphs represent mean with SEM of at least three different experiment days. Arb., arbitrary.
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pools. Treatments of P. berghei parasites with nigericin (10 lM) and monensin (10 lM) had no effect on proteolysis of AIK, whilst thapsigargin (10 lM) treatment increased proteolysis of AIK and KLR peptide (Fig. 5A). Thus, it appears that Ca2+ from the ER induces P. berghei proteolysis. In contrast, in P. yoelii both nigericin (10 lM) and monensin (10 lM) led to a marginal increase in proteolysis of AIK peptide compared with proteolysis activation triggered by thapsigargin (Fig. 5B). The influence of Ca2+ levels on proteolytic activity was also investigated by incubation of parasites in buffers containing intracellular (BAPTA/AM) or extracellular (EGTA) Ca2+ chelators. Fig. 6 shows that lower concentrations of BAPTA/AM (25, 50 and 100 lM) had no effect; however 200 and 500 lM of the intracellular Ca2+ chelator inhibited protease activity in P. berghei (Fig. 6A). The role of intracellular Ca2+ in protease activation in P. yoelii differs from that in P. berghei. First, protease activation in P. yoelii increased after incubation with BAPTA/AM (25 and 200 lM), but this activation was suppressed by a higher concentration of BAPTA/AM (500 lM) (Fig. 6B). Second, treatment with EGTA (5 mM) inhibited protease activity in P. berghei but increased activity in P. yoelii (Fig. 6A and B, respectively).
have a tendency to induce reticulocytosis and to invade reticulocytes. 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 parasitemia (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 staining and flow cytometry. Fig. 8 shows that P. yoelii parasites at the beginning and end (3 h later) of FRET experiments showed no statistical difference in DHT fluorescence. Both methods confirmed that ex vivo experiments for measuring protease activity were performed in ideal conditions.
3.2. The Ca2+-induced proteolytic response is protease class-specific
4. Discussion
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. 7A), pre-incubation with the aspartic protease inhibitor Pepstatin A (15 lM) or the serine protease inhibitor PMSF (15 lM) followed by thapsigargin (10 lM) treatment increased peptide hydrolysis, whilst incubation with the cysteine protease inhibitor E64 (15 lM) reduced proteolysis. In contrast, results were markedly different in P. yoelii (Fig. 7B), with peptide hydrolysis stimulated by E64 and inhibited by Pepstatin A. FRET control experiments without thapsigargin and in the presence of either E64 (15 lM) or Bapta/AM (25 and 200 lM) show a very small change in peptide hydrolysis compared with control experiments (Figs. 6B and 7B). Plasmodium berghei and P. yoelii are relatively asynchronous, compared with P. falciparum and P. chabaudi, and these species
Our results show that release of Ca2+ from the ER by thapsigargin 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 Ca2+ 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 Ca2+ release from the ER in stimulating protease activity in different Plasmodium spp. In contrast, P. berghei release of Ca2+ from acidic pools by nigericin or monensin did not activate proteolysis (Fig. 5A). These data indicate that cellular proteases from P. berghei are sensitive to pH, since changes in cytosolic Ca2+ 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 berghei has different classes of proteases modulated by endoplasmic reticulum (ER) Ca2+ release. Effect of thapsigargin (Thg) (10 lM) on fluorescence resonance energy transfer (FRET) peptide KLRSSKQ-EDDnp (10 lM) hydrolysis in P. berghei. Isolated parasites (108 cells ml1) were incubated with Pepstatin A (15 lM) or phenylmethylsulphonyl fluoride (PMSF) (15 lM) for 20 min and E64 (15 lM) 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; P values 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 lM) on FRET peptide KLRSSKQ-EDDnp (10 lM) hydrolysis in P. yoelii. Isolated parasites (108 cells ml1) were incubated with Pepstatin A (10 lM), PMSF (10 lM) or E64 (15 lM) 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; P values were compared with Thg data 2.51 ± 0.198, n = 8). The effect of E64 (15 lM) on FRET hydrolysis without Thg addition in P. yoelii isolated parasites was also verified (1.748 ± 0.128, n = 8, P = 0.0009; P values were compared with control (ctr) data 1.146 ± 0.033, n = 7). Bar graphs represent mean with SEM of at least three different experiment days. All incubations were performed before addition of FRET substrate in MOPS buffer with CaCl2 (1 mM) and pH 7.2. Arb., arbitrary.
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Fig. 8. Viability was assessed by flow cytometry using dihydroethidium (DHT) staining in Plasmodium yoelii parasites. (A) Histogram distribution of fluorescence in nonlabelled parasites (control), parasites labelled at the beginning (dashed line) and 3 h later (solid line) in the same buffer. (B) Bar graph analyses of viability in P. 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, n = 3; P = 0.7495, respectively).
2007). Likewise, the effect of Ca2+ release from acidic pools in P. yoelii was marginal (Fig. 5B), suggesting that acidification of cytosol might interfere with protease activation in both parasites. We also performed experiments with intracellular (BAPTA/AM) and extracellular (EGTA) Ca2+ chelators to investigate the role played by Ca2+ in protease activation. We found that in P. berghei both EGTA and BAPTA block protease activation (Fig. 6A), whilst in P. yoelii (Fig. 6B) blocking Ca2+-dependent enzymes resulted in activation of proteases. Protease activity in P. yoelii in the absence of Ca2+ was higher than in the presence of Ca2+, indicating that the basal cytosolic Ca2+ was already modulating the activity (see control of basal protease activity before activation with Ca2+ in Fig. 3A and B) of the two classes of enzymes, Ca2+ activated and Ca2+ inhibited. Our results show that modulation of protease activation differs between P. berghei and P. yoelii. Understanding how these important 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 thapsigargininduced Ca2+ release (Fig. 3A 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. 7A), 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. 7B), 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 falcipains, 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 Ca2+ activation in P. berghei. In P. yoelii Ca2+ 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 plasmepsins (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 Ca2+ in modulating plasmodial biology are limited. We have shown that Plasmodium senses the environment and modulates its cell cycle through synchronisation of its erythrocytic forms. The plasmodial cell cycle is affected in a Ca2+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 Ca2+ release from P. falciparum. Melatonin initiated a complex signalling pathway resulting in increases of cAMP and intracellular Ca2+ concentration [Ca2+]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 Ca2+ during the erythrocytic cycle was not clear from these studies. We now report that Ca2+ 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 physiologies. Interestingly, rodent malarial species are known to differ in related features. Plasmodium chabaudi parasites are more synchronous 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 hormone has clear effects on cytosolic Ca2+ 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 activated by Ca2+. Additional work with different Plasmodium spp. will be needed to better understand the role of Ca2+ in the biology of malaria parasites. The parasite molecular machinery for signalling is quite complex 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 fundamental to understanding when and how second messengers are put into action to modulate protease activity.
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