- Open Access
Intracellular proteolysis of kininogen by malaria parasites promotes release of active kinins
- Piero Bagnaresi†1,
- Nilana MT Barros†1, 2,
- Diego M Assis1,
- Pollyana MS Melo1,
- Raphael G Fonseca1,
- Maria A Juliano1,
- João B Pesquero1,
- Luiz Juliano1,
- Philip J Rosenthal3,
- Adriana K Carmona1Email author and
- Marcos L Gazarini4Email author
© Bagnaresi et al.; licensee BioMed Central Ltd. 2012
- Received: 14 February 2012
- Accepted: 7 May 2012
- Published: 7 May 2012
The malaria burden remains a major public health concern, especially in sub-Saharan Africa. The complex biology of Plasmodium, the apicomplexan parasite responsible for this disease, challenges efforts to develop new strategies to control the disease. Proteolysis is a fundamental process in the metabolism of malaria parasites, but roles for proteases in generating vasoactive peptides have not previously been explored.
In the present work, it was demonstrated by mass spectrometry analysis that Plasmodium parasites (Plasmodium chabaudi and Plasmodium falciparum) internalize and process plasma kininogen, thereby releasing vasoactive kinins (Lys-BK, BK and des-Arg9-BK) that may mediate haemodynamic alterations during acute malaria. In addition, it was demonstrated that the P. falciparum cysteine proteases falcipain-2 and falcipain-3 generated kinins after incubation with human kininogen, suggesting that these enzymes have an important role in this process. The biologic activity of peptides released by Plasmodium parasites was observed by measuring ileum contraction and activation of kinin receptors (B1 and B2) in HUVEC cells; the peptides elicited an increase in intracellular calcium, measured by Fluo-3 AM fluorescence. This effect was suppressed by the specific receptor antagonists Des-Arg9[Leu8]-BK and HOE-140.
In previously undescribed means of modulating host physiology, it was demonstrated that malaria parasites can generate active kinins by proteolysis of plasma kininogen.
- Plasmodium Falciparum
- Infected Erythrocyte
- Plasmodium Parasite
Multiple proteolytic enzymes have been identified in Plasmodium species and appear to be involved in several important aspects of parasite biology [1, 2]. Notably, cysteine and aspartic proteases are implicated in the hydrolysis of haemoglobin [3, 4] to provide free amino acids for protein synthesis by intraerythrocytic parasites. Cysteine and aspartic protease inhibitors block this process  and kill parasites at nanomolar concentrations . Recent studies demonstrated that the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 act with similar specificity in haemoglobin degradation, not via an ordered hydrolytic pathway, but through rapid hydrolysis at multiple sites . Falcipain-2 knockout parasites showed reduced haemoglobin degradation while falcipain-3 knockouts were not viable, suggesting that late expression of falcipain-3 rescued falcipain-2 knockouts and that falcipain-3 is essential for intraerythrocytic development [8, 9]. Considering the important involvement of proteases in Plasmodium biology, the roles of these enzymes in cellular and biochemical events are targets of active investigation.
The pathophysiology of malaria is poorly understood, but endothelial cell activation and adherence of infected erythrocytes to endothelial cells appear to be important features in pathogenesis [10, 11]. The contact of adherent infected red blood cells with endothelium can induce vessel wall shear stress, stimulating the release of the potent vasodilator nitric oxide . However, it is unclear if nitric oxide is beneficial or harmful in malaria . In fact, when endothelium is disturbed, cells can exhibit a wide range of biochemical responses , including bradykinin (BK) release . Kinins are biologically active peptides released from a multifunctional plasma protein, kininogen (HK). They can induce vasodilatation, stimulate the production of nitric oxide, activate endothelial cells, enhance microvascular permeability and modulate the metabolism of different tissues [15–19]. It has been reported that cysteine proteases from some pathogenic microorganisms, including Trypanosoma cruzi[20, 21], Porphyromonas gingivalis and Fasciola hepatica, release kinins. During the Trypanosoma cruzi infection, immature dendritic cells (DC) sense the presence of the parasite in the peripheral and lymphoid tissues by B2R stimulation mediated by bradykinin, which is generated by action of its major cysteine protease, cruzipain. These now activated DC trigger a cascade of immune cells activation, culminating in the generation of immunoprotection by IFN-γ- producing T cells . The B2R activation also potentiates Trypanosoma cruzi invasion of host cells . Porphyromonas gingivalis is also able to release kinin by its own proteolytic activity. These kinins promote B2R pathway activation that, in conjunction with TLR2 activation by the bacterial LPS, modulates effector T cells commitment in the pathology .
However, kinin formation in Plasmodium infection has been poorly explored. Older reports suggested that kinins may be involved in malaria pathology [27, 28] and a reduction of plasma kininogen was observed in mice infected by Plasmodium berghei. Nonetheless, the mechanisms by which kinins are generated and the biological effects of released kinins have not previously been reported. In the present study, it is shown that malaria parasites take up plasma proteins from the host and process these proteins to generate biologically active peptides.
Plasmodium chabaudi (clone AS) was maintained in Balb/C mice by weekly transfers from previously infected mice. Animals infected at the trophozoite stage (parasitaemia ~60%) were sacrificed, blood was collected, and leukocytes and platelets were removed from whole blood by filtration through a powdered cellulose column (Whatman CF11). Trophozoite-infected erythrocytes were then washed three times by centrifugation at 1,500 g for 5 min in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO3, 1.4 mM Na2HPO3, pH 7.4). Parasites (107 cells/ml) were isolated by lysis of erythrocyte membranes with 10 mg/ml saponin in PBS. After pelleting to remove red cell membranes, the parasites were washed twice in PBS by centrifugation at 2,000 g at 4°C.
Plasmodium falciparum (3D7 strain) was cultured in RPMI 1640 medium supplemented with 10% human serum as previously described  and parasites were isolated from erythrocytes when cultures reached ~5% parasitaemia using the same procedure employed for P. chabaudi.
Kinin generation by Plasmodium parasites
Isolated parasites (P. chabaudi and P. falciparum) (104 cells/ml) were incubated with 0.35 μM of human high molecular weight kininogen (HK) or with the synthetic fluorogenic peptide Abz-MISLMKRPPGFSPFRSSRI-NH2 (30 μM; Abz = ortho-aminobenzoic acid) for 5 min, 10 min, 30 min, 1 and 2 h at 37°C in Tris–HCl 50 mM, pH 7.4. After incubation, the samples were centrifugated (2,000 rpm, 5 min) and the supernatant was analysed by MALDI-TOF/MS as follows. Aliquots (1 μL) were added to 1 μL of alpha-ciano-4-hydroxycinnamic acid (10 mg/mL) matrix solution, spotted onto a stainless steel MALDI target plate and dried at room temperature before analysis. Mass spectra were obtained with a Bruker Daltonics Microflex LT instrument operating in linear, positive ion mode previously calibrated with angiotensin I, angiotensin II, somatostatin and bradykinin. For the analysis of peptide release, mass spectra were acquired using the following instrument parameters: pulsed ion extraction delay of 30 ns, ion source voltage one, 20 kV, ion source voltage two, 18.65 kV, and ion source lens voltage 7.1 kV. For each sample, mass spectra were acquired by accumulating 50 laser shots at 50% laser power in the m/z range of 800–2600 Da.
SDS-PAGE analysis of biotin-HK hydrolysis by falcipain-2 and falcipain-3
Human single chain HK was biotinylated (Biotin-HK) as previously described . Biotin-HK(0.4 μM) was incubated with falcipain-2 (0.2 μM) and falcipain-3 (0.2 μM) in sodium acetate buffer 100 mM, pH 5.5, for 1 h at 37°C and the reactions were stopped by boiling. Samples containing only biotin-HK, falcipain-2 or falcipain-3 were also analysed as experimental controls, and were assayed in the same conditions. The samples were submitted to western blotting and nitrocellulose transblots were washed with PBS and incubated with PBS / Tween 0.01% / BSA 1% solution. After extensively washes with PBS, the membrane was incubated with streptavidin–peroxidase conjugated (1:1000) for 1 h at 4°C and the results were obtained by degradation of peroxidase substrate 4-Chloro-1-naphthol (SIGMA) (30 mg 4-Chloro-1-naphthol solution in 10 mL of methanol to 50 mL of PBS) at room temperature.
Guinea pig ileum contraction induced by kinin peptide generation
The biological activity of the released kinins was measured as isotonic contraction on isolated guinea pig terminal ileum. The isolated organ was suspended in a 5 mL bath in Tyrode’s solution (135 mM NaCl, 2.7 mM KCl, 1.36 mM CaCl2, 0.5 mM MgCl2, 0.36 mM NaH2PO4, 11.9 mM NaHCO3, 5.04 mM glucose) at 37°C, pH 7.4. The sensitivity of the response was calibrated with standard solutions of bradykinin (1–10 nM). HK (0.33 μM) was incubated with isolated P. chabaudi trophozoites (104 cells) and with recombinant falcipain-2 (46 nM) or falcipain-3 (18 nM). After 10 min, parasites, when present, were pelleted by centrifugation, and supernatants were added to the bath and the isotonic contraction recorded as described previously . The volume of supernatant was adjusted so that the released kinin fit inside the dose–response curve for bradykinin. Similar experiments were performed in the presence of 20 nM of the bradykinin B2 receptor antagonist HOE-140 (D-Arg-Arg-Pro-Hyp-Gly-b-[2-thienyl]Ala-Ser-D-tetrahydroisoquiniline-3-carboxylicacid-octahydroindole-2-carboxylic acid-Arg), which was pre-incubated for 2 min. Falcipain-2 (46 nM) and falcipain-3 (18 nM) were pre-activated with 2 mM DTT for 5 minutes and then incubated with HK (0.48 μM) in sodium acetate buffer 0.1 M, pH 5.5 at 37°C for six hours. The reaction was then kept in ice. Afterwards, 100μL of the solution was added to the system containing the guinea pig ileum in Tyrode’s solution. As a control, falcipains were also assayed in the same system after 10 min incubation with the specific cysteine protease inhibitor E-64 (10 μM).
Confocal Microscopy of FITC labelled HK
FITC (Sigma) was conjugated to HK according to manufacturer’s specifications. To investigate the permeability of infected erythrocytes to HK, Plasmodium chabaudi and Plasmodium falciparum infected cells were ressuspended in PBS and 104 cells were placed on microscopy plates pre-incubated with poly-L-lysine (1 h at room temperature) to enhance cell adhesion. Afterwards, labelled HK (1 μg) was added to the plates and incubated for 1 h at 37°C. Fluorescence images were obtained with a confocal microscope (Carl Zeiss LSM-510 META) with excitation wavelength of 488 nm and emission of 505–550 nm.
Intracellular calcium measurement in HUVEC cells stimulated with kinin peptides
Human umbilical vein endothelial cells were cultured in RPMI 1640 (GIBCO) containing 10% heat-inactivated fetal bovine serum (FBS), 24 mM sodium bicarbonate, 40 mg/mL gentamicin, 10 mM HEPES, pH 7.4 at 37°C in a 5% CO2 humidified atmosphere. 5 × 104 cells were plated on 60 mm dishes (Costar 3260), and incubated in culture medium containing 5 μM Fluo-3 AM (Invitrogen Corporation, CA) for 1 h at 37°C, followed by 3 washes with culture medium to discard remaining extracellular probe. The cells were then placed into a culture chamber at 37°C on the stage of a confocal microscope (Zeiss LSM 510 META) with excitation at 488 nm and emission at 505–530 nm. After calcium probe incubation, assays were performed with the antagonists Des-Arg9[Leu8]-BK (4 μM) and HOE-140 (10 μM). The antagonists were incubated for 10 min before the addition of: 20 μg kininogen (HK), 10 µL of culture supernatant after culturing 104P. falciparum for 30 minutes without HK (Pf - HK); 10 μL of culture supernatant after culturing 104P. falciparum for 30 minutes with HK (1.9 μg/μL) (Pf + HK); or 5 μM THG. Fluorescence data were normalized as F/Fmax, where Fmax represents maximal intracellular fluorescence obtained with Ca2+ released from ER with addition of THG (Ca2+ATPase inhibitor).
Kinin peptides are generated by Plasmodium parasites
Mass spectrometry analysis of the BK-related peptide by Plasmodium falciparum parasites
Plasmodium parasites take up HK
Falcipains mediate HK cleavage
Fragments generated by falcipains and Plasmodium chabaudi possess biological activity in guinea pig ileum
Calcium signalling by kinin peptides generated by Plasmodium falciparum
The results reported here show that Plasmodium can process the plasma protein kininogen and generate peptides that may modulate hemodynamics in the host. P. falciparum and P. chabaudi are able to internalize and process kininogen, with subsequent release of kinins (des-Arg9-BK, Lys-BK and BK). This processing is inhibited by the cysteine protease inhibitor E-64 and reproduced in vitro by isolated falcipain-2 and falcipain-3, suggesting roles for these proteases in kininogen processing. Kininogen peptides were biologically active, as demonstrated by their ability to induce ileum contraction and activate endothelial cells via calcium signaling, both principally through B2 receptor activation.
The processing of plasma macromolecules by the malaria parasites has already been reported [32–35]. However, parasites have not previously been shown to modulate host physiology by these events. Kininogen is a multipurpose plasma protein that plays essential roles in modifying vascular haemodynamics, controlling vascular tone and maintaining blood pressure . The results here reported suggest that parasites can metabolize peptides and proteins from plasma or from the endothelial cell surface, releasing active peptides into the extracellular milieu and thereby impacting upon host haemodynamics.
The processing of a plasma protein by falcipains in vitro suggests a second physiologic function for these proteases in addition to the hydrolysis of haemoglobin . Interestingly, falcipain-2 and falcipain-3 generated distinct proteolytic profiles, suggesting that the activities were not redundant. However, the participation of other proteases in this process cannot be excluded. Recently, the Plamodium falciparum aminopeptidase PfAPP was shown to localize to the food vacuole and cytosol of the parasite , suggesting potential roles in the hydrolysis of both haemoglobin and HK. Interestingly, recombinant PfAPP was able to hydrolyse bradykinin . The mass spectrum data showed the removal of amino acid residues from the N-termini of proteins, supporting the involvement of aminopeptidases in kinin processing.
It was of interest to determine if peptides generated by parasites and isolated falcipains possess biological activity and exert a specific physiologic effect. Alternatively, they might be generated simply for acquisition of nutrients, as appears to be the case for haemoglobin. Indeed, generated peptides promoted guinea pig ileum contraction, which was abolished in the presence of the B2 receptor antagonist HOE-140. Further, the peptides stimulated Ca2+ release in endothelial cells and this process was also blocked by HOE-140. Thus, proteolysis of kininogen by malaria parasites generates biologically active peptides. This effect may explain clinical features of malaria, which is commonly accompanied by alterations in vascular haemodynamics.
Another potential role for BK and B2 receptor stimulation is attenuation of host systemic damage during infection by anti-apoptotic or other protective effects. Similar protective effects were observed in cardiac, renal and liver damage models . For instance, B2 activation induces a hypertensive hepatic response , which could play a role in malaria liver stage physiology, with modulation of hepatic blood flow and parasite distribution in the tissue.
In the late 60s, some studies suggested that kinins could be involved in the pathological manifestations of malaria [27–29]. However, these works were not conclusive and, despite the evidences, no more reports correlating kinins with malaria could be found in literature. This new set of evidences points out to the participation of the kinins in the pathophysiology on Plasmodium infection. This process appears to depend on endothelial cell activation, which may be promoted by proinflammatory agents such as cytokines (e.g. Tumor Necrosis Factor), chemokines and infected erythrocytes [33, 35, 38]. This activation leads to enhanced expression of genes that are involved in inflammation and apoptosis . Nitric oxide production is induced by kinin receptors, has been shown to mitigate brain haemorrhages and vascular damage in a murine cerebral malaria model  and may modulate host haemodynamics during malarial infection. The modulation of vessel caliber by vasoactive peptides might be important as a mechanism to prevent thrombosis by the enhanced cytoadherence and promote better blood flow in the microcirculation, which is impaired by the malarial infection, in a process that confers some protection to the host, and consequently ensuring that the pathogen can continues its life cycle.
Piero Bagnaresi, Nilana MT de Barros, contributed equally to this work
This work was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). PJR is a Distinguished Clinical Scientist of the Doris Duke Charitable Foundation. MLG receives fellowship from FAPESP (09/54598-9) and FADA (UNIFESP). We thank Prof. Gerhard Wunderlich (ICB-USP) and Prof. Célia R. S. Garcia (IB-USP) for kindly provided P. falciparum lineage; the Multiuser Multiphoton Confocal Microscopy Laboratory (INFAR-UNIFESP) for fluorescence images. We also thank Dr. Thaysa Paschoalin for kindly providing HUVEC cells for the confocal microscopy assays.
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