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Erythrocyte calpain is dispensable for malaria parasite invasion and growth
Molecular & Biochemical Parasitology 122 (2002) 227 /229 www.parasitology-online.com
Short communication
Erythrocyte calpain is dispensable for malaria parasite invasion and growth Manjit Hanspal *, Vikas K. Goel, Steven S. Oh, Athar H. Chishti Departments of Medicine, Anatomy, and Cell Biology, St. Elizabeth’s Medical Center, Tufts University School of Medicine, Biomedical Research, ACH 406, Boston, MA 02135, USA Received 20 February 2002; received in revised form 23 April 2002; accepted 3 May 2002 Keywords: Plasmodium falciparum ; Malaria; mu-Calpain; Cysteine proteases
Plasmodium falciparum causes the most severe form of human malaria and is responsible for nearly all malaria-related mortality in the holoendemic areas. Clinical disease results from asexual replication of the blood-stage malaria parasite in circulating erythrocytes of the human host. The erythrocytic life cycle of the parasite begins when free merozoites invade erythrocytes in the peripheral circulation. The intraerythrocytic parasites mature from ring forms to trophozoites and then to the schizonts. Mature segmented schizonts rupture the host erythrocytes and release merozoites, which rapidly invade other erythrocytes to reinitiate the infection cycle. Mounting evidence now suggests a pivotal role of parasite-derived proteases at all stages of the parasite development within erythrocytes, including parasite invasion, hemoglobin degradation, host cytoskeleton remodeling, and the rupture of the erythrocyte membrane [1 /3]. Cysteine proteases have been implicated during the parasite invasion and release from the red blood cells [4,5]. Specifically, the erythrocyte calpain, a calciumdependent cysteine protease, has been reported to play an important role in the parasite invasion of the human red blood cells [6]. In this previous report, two peptidebased inhibitors of cysteine proteases were used to block P. falciparum invasion into red blood cells prompting the authors to suggest that host calpain plays a role in the invasion process [6]. This view was further supported by the demonstration that the influx of calcium facilitates parasite invasion presumably causing activation of the intracellular calpain in the host erythrocytes [7]. In
* Corresponding author. Tel.: /1-617-789-2677; fax: /1-617-7893111 E-mail address: [email protected] (M. Hanspal).
general, the calpain activity in mammalian cells is contributed by two isoforms of calpain. The mu-calpain is activated at micromolar concentrations of calcium whereas the activation of m-calpain requires millimolar calcium concentrations [8]. Early evidence suggested that the mu-calpain is the dominant source of calpain activity in the mammalian erythrocytes [9]. Despite the importance and extensive interest in defining the physiological role of calpain activity in erythrocytes, a major limitation of these studies continues to be their reliance on the use of synthetic inhibitors of calpain activity. Since these inhibitors cannot distinguish between mu- and m-calpains, and more importantly between host and parasite calpains, the specific role of erythrocyte calpain activity remains to be established in the malaria parasite life cycle. Recently, we generated mu-calpain deficient mice by genetic inactivation of the mu-calpain catalytic subunit gene (Capn 1 ) using homologous recombination in embryonic stem cells [10]. The expression of m-calpain in Capn 1 null mice was not affected. Casein zymography measurements demonstrated that the mu-calpain (///) erythrocytes are completely deficient of all calpain activity [10]. Furthermore, a monoclonal antibody that reacts to both mu- and m-isoforms of mouse calpain failed to detect any calpain in the whole cell lysate of mu-calpain null erythrocytes by Western blotting indicating that mu-calpain is the only isoform present in mouse erythrocytes and that mu-calpain deficiency does not induce or amplify m-calpain in the mutant erythrocytes [10]. The availability of mu-calpain deficient erythrocytes, which are completely devoid of any cysteine protease activity, provided a unique opportunity to test the role of host erythrocyte calpain in the malaria parasite life cycle both in vivo and in vitro.
0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 1 0 4 - 4
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M. Hanspal et al. / Molecular & Biochemical Parasitology 122 (2002) 227 /229
To investigate the role of erythrocyte calpain in the invasion and growth of malaria parasites in vivo, we infected the mu-calpain null mice with P. yoelii. The P. yoelii 17 XL strain was revived into C57 BL/6J mice and blood was collected after the parasitemia reached / 50%. Infected erythrocytes (/105 cells) in 0.2 ml volume were injected into wild-type and mu-calpain null mice. Each experimental group contained five to six animals. Blood smears were made after every 24 h for up to 8 days post-infection, and stained with Giemsa. A total of 1200/1600 erythrocytes were counted from each slide to calculate % parasitemia. As shown in Fig. 1, there was no significant difference in the % parasitemia between wild-type and mu-calpain null mice. These results demonstrate that host mu-calpain does not participate in the P. yoelii invasion of erythrocytes, its intraerythrocytic growth, and the release of merozoites from erythrocytes in vivo. Next, we examined the invasion potential of P. falciparum (3D7 strain) into mu-calpain null mouse erythrocytes in vitro. Human malaria P. falciparum invades wild-type mouse RBCs at a reduced rate and sustain its growth for a short period in culture [11,12]. Blood was drawn by cardiac puncture from wild-type (///), mu-calpain heterozygous (///), and homozygous (///) mice. Erythrocytes were washed three times with RPMI and leukocytes were removed as previously described [13]. Washed erythrocytes were resuspended in RPMI to attain /50% hematocrit. The trophozoite stage of P. falciparum was isolated using 70% Percoll gradient to achieve a parasitemia of /95%. Human serum was pre-adsorbed with wild-type erythrocytes at 37 8C for 1 h. The following components were mixed to initiate the invasion assay, 3 ml of human erythrocytes containing the trophozoite stage P. falciparum at /95%
Fig. 1. Role of mu-calpain in the invasion and growth of P. falciparum . Wild-type and mu-calpain null mice were infected with /50% parasitemia of P. yoelii 17 XL strain. Blood smears were made after every 24 h for 8 days post-infection, and stained with Giemsa. Parasitemias were determined by counting 1200 /1600 erythrocytes from each slide. Data were plotted as mean % parasitemia9/standard error.
parasitemia; 50 ml of 50% hematocrit of defined erythrocytes and 1.2 ml of RPMI containing 10% preadsorbed human serum. The culture was incubated for 22 /24 h at 37 8C in a gas mixture containing 5% CO2, 1% O2, and 94% N2 [14]. Thin smears were made the next day and stained with Giemsa. Newly invaded rings were counted from a total of /4000 erythrocytes for each sample to determine the extent of parasitemia. The experiment was repeated twice. As shown in Fig. 2, the invasion efficiency of P. falciparum in mu-calpain null erythrocytes is indistinguishable from that of wild-type and mu-calpain heterozygous erythrocytes. Furthermore, the number of ring-infected erythrocytes in the calpain (///) erythrocyte sample (mean % parasitemia9/S.D. /0.99%9/0.14) was significantly greater (P B/0.05) than the number of uninfected human erythrocytes originally introduced at the start of the invasion assay (maximum possible % parasitemia, if all human erythrocytes in the culture were infected / 0.56%). These results demonstrate that P. falciparum did indeed invade mouse erythrocytes, albeit at a reduced rate relative to human erythrocytes, and that the mouse erythrocyte calpain is not required for P. falciparum invasion and growth in vitro. Taken together, our results suggest that the endogenous calcium-dependent cysteine protease, mu-calpain, of mouse erythrocytes is not required for the blood-stage malaria parasite development in vivo and in vitro at least in P. falciparum and P. yoelii malaria. In contrast, compelling evidence suggests that the parasite-derived cysteine proteases are obligatory at all stages of parasite development in the host erythrocytes [1] and appear to be good targets for the development of specific inhibitors [15,16]. The availability of mu-calpain null mice will
Fig. 2. In vitro invasion of P. falciparum (3D7 strain) into mu-calpain null mouse erythrocytes. Normal human RBCs and RBCs from wild type (///), mu-calpain heterozygous (///), and homozygous (///) mice were incubated with purified trophozoite/schizont stage of P. falciparum at /95% parasitemia for 22 /24 h at 37 8C in a gas mixture containing 5% CO2, 1% O2, and 94% N2. Parasitemia was determined by counting the rings */ /4000 erythrocytes from Giemsastained smears. Data were plotted as mean invasion rate (9/standard error) relative to the human erythrocyte control sample (100% invasion).
M. Hanspal et al. / Molecular & Biochemical Parasitology 122 (2002) 227 /229
allow future testing of such inhibitors against parasite cysteine proteases in an environment where the host calpain activity is absent.
Acknowledgements We thank Dr M. Azam of our laboratory for sharing his stock of mu-calpain null mice for this study. This work was supported in part by the National Institutes of Health grants, HL 60152 (to MH) and HL 51445 (to AHC), and Tufts University Earl P. Charlton grant (to SSO).
References [1] Rosenthal P.J.. Proteases of malaria parasites: New targets for chemotherapy. Emerging Infect Dis 1998;4:49 /57. [2] Raphael P., Takakuwa Y., Manno S., Liu S.-C., Chishti A.H., Hanspal M.. A cysteine protease activity from Plasmodium falciparum cleaves human erythrocyte ankyrin. Mol Biochem Parasitol 2000;110:259 /72. [3] Dua M., Raphael P., Sijwali P.S., Rosenthal P.J., Hanspal M.. Recombinant falcipain-2 cleaves erythrocyte membrane ankyrin and protein 4.1. Mol Biochem Parasitol 2001;116:95 /9. [4] McKerrow J.H., Sun E., Rosenthal P.J., Bouvier J.. The proteases and pathogenicity of parasitic protozoa. Annu Rev Microbiol 1993;47:821 /53.
229
[5] Salmon B.L., Oksman A., Goldberg D.E.. Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis. Proc Natl Acad Sci USA 2001;98:271 /6. [6] Olaya P., Wasserman M.. Effect of calpain inhibitors on the invasion of human erythrocytes by the parasite Plasmodium falciparum . Biochim Biophys Acta 1991;1096:217 /21. [7] Wasserman M., Alarcon C., Mendoza P.M.. Effects of Ca2 depletion on the asexual cell cycle of Plasmodium falciparum . Am J Trop Med Hyg 1982;31:711 /7. [8] Suzuki K., Sorimachi H., Yoshizawa T., Kinbara K., Ishiura S.. Calpain: novel family members, activation, and physiologic function. Biol Chem Hoppe-Seyler 1995;376:523 /9. [9] Kawasaki H., Kawashima S.. Regulation of the calpain /calpastatin system by membranes. Mol Membr Biol 1996;13:217 /24. [10] Azam M., Andrabi S.S., Sahr K.E., Kamath L., Kuliopulos A., Chishti A.H.. Disruption of the mouse mu-calpain gene reveals an essential role in platelet function. Mol Cell Biol 2001;21:2213 /20. [11] Klotz F.W., Chulay J.D., Daniel W., Miller L.H.. Invasion of mouse erythrocytes by the human malaria parasite, Plasmodium falciparum . J Exp Med 1987;165:1713 /8. [12] Sam-Yellowe T.Y., Perkins M.E.. Binding of Plasmodium falciparum rhoptry proteins to mouse erythrocytes and their possible role in invasion. Mol Biochem Parasitol 1990;39:91 /100. [13] Bennett V., Stenbuck P.J.. Human erythrocyte ankyrin. J Biol Chem 1980;255:2540 /8. [14] Trager W., Jensen J.B.. Human malaria parasites in continuous culture. Science 1976;193:673 /5. [15] McKerrow J.H., Engel J.C., Caffrey C.R.. Cysteine protease inhibitors as chemotherapy for parasitic infections. Bioorg Med Chem 1999;7:639 /44. [16] Sabnis Y., Rosenthal P.J., Desai P., Avery M.A.. Homology modeling of falcipain-2: validation, de novo ligand design and synthesis of novel inhibitors. J Biomol Struct Dyn 2002;19:765 / 74.
Short communication
Erythrocyte calpain is dispensable for malaria parasite invasion and growth Manjit Hanspal *, Vikas K. Goel, Steven S. Oh, Athar H. Chishti Departments of Medicine, Anatomy, and Cell Biology, St. Elizabeth’s Medical Center, Tufts University School of Medicine, Biomedical Research, ACH 406, Boston, MA 02135, USA Received 20 February 2002; received in revised form 23 April 2002; accepted 3 May 2002 Keywords: Plasmodium falciparum ; Malaria; mu-Calpain; Cysteine proteases
Plasmodium falciparum causes the most severe form of human malaria and is responsible for nearly all malaria-related mortality in the holoendemic areas. Clinical disease results from asexual replication of the blood-stage malaria parasite in circulating erythrocytes of the human host. The erythrocytic life cycle of the parasite begins when free merozoites invade erythrocytes in the peripheral circulation. The intraerythrocytic parasites mature from ring forms to trophozoites and then to the schizonts. Mature segmented schizonts rupture the host erythrocytes and release merozoites, which rapidly invade other erythrocytes to reinitiate the infection cycle. Mounting evidence now suggests a pivotal role of parasite-derived proteases at all stages of the parasite development within erythrocytes, including parasite invasion, hemoglobin degradation, host cytoskeleton remodeling, and the rupture of the erythrocyte membrane [1 /3]. Cysteine proteases have been implicated during the parasite invasion and release from the red blood cells [4,5]. Specifically, the erythrocyte calpain, a calciumdependent cysteine protease, has been reported to play an important role in the parasite invasion of the human red blood cells [6]. In this previous report, two peptidebased inhibitors of cysteine proteases were used to block P. falciparum invasion into red blood cells prompting the authors to suggest that host calpain plays a role in the invasion process [6]. This view was further supported by the demonstration that the influx of calcium facilitates parasite invasion presumably causing activation of the intracellular calpain in the host erythrocytes [7]. In
* Corresponding author. Tel.: /1-617-789-2677; fax: /1-617-7893111 E-mail address: [email protected] (M. Hanspal).
general, the calpain activity in mammalian cells is contributed by two isoforms of calpain. The mu-calpain is activated at micromolar concentrations of calcium whereas the activation of m-calpain requires millimolar calcium concentrations [8]. Early evidence suggested that the mu-calpain is the dominant source of calpain activity in the mammalian erythrocytes [9]. Despite the importance and extensive interest in defining the physiological role of calpain activity in erythrocytes, a major limitation of these studies continues to be their reliance on the use of synthetic inhibitors of calpain activity. Since these inhibitors cannot distinguish between mu- and m-calpains, and more importantly between host and parasite calpains, the specific role of erythrocyte calpain activity remains to be established in the malaria parasite life cycle. Recently, we generated mu-calpain deficient mice by genetic inactivation of the mu-calpain catalytic subunit gene (Capn 1 ) using homologous recombination in embryonic stem cells [10]. The expression of m-calpain in Capn 1 null mice was not affected. Casein zymography measurements demonstrated that the mu-calpain (///) erythrocytes are completely deficient of all calpain activity [10]. Furthermore, a monoclonal antibody that reacts to both mu- and m-isoforms of mouse calpain failed to detect any calpain in the whole cell lysate of mu-calpain null erythrocytes by Western blotting indicating that mu-calpain is the only isoform present in mouse erythrocytes and that mu-calpain deficiency does not induce or amplify m-calpain in the mutant erythrocytes [10]. The availability of mu-calpain deficient erythrocytes, which are completely devoid of any cysteine protease activity, provided a unique opportunity to test the role of host erythrocyte calpain in the malaria parasite life cycle both in vivo and in vitro.
0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 1 0 4 - 4
228
M. Hanspal et al. / Molecular & Biochemical Parasitology 122 (2002) 227 /229
To investigate the role of erythrocyte calpain in the invasion and growth of malaria parasites in vivo, we infected the mu-calpain null mice with P. yoelii. The P. yoelii 17 XL strain was revived into C57 BL/6J mice and blood was collected after the parasitemia reached / 50%. Infected erythrocytes (/105 cells) in 0.2 ml volume were injected into wild-type and mu-calpain null mice. Each experimental group contained five to six animals. Blood smears were made after every 24 h for up to 8 days post-infection, and stained with Giemsa. A total of 1200/1600 erythrocytes were counted from each slide to calculate % parasitemia. As shown in Fig. 1, there was no significant difference in the % parasitemia between wild-type and mu-calpain null mice. These results demonstrate that host mu-calpain does not participate in the P. yoelii invasion of erythrocytes, its intraerythrocytic growth, and the release of merozoites from erythrocytes in vivo. Next, we examined the invasion potential of P. falciparum (3D7 strain) into mu-calpain null mouse erythrocytes in vitro. Human malaria P. falciparum invades wild-type mouse RBCs at a reduced rate and sustain its growth for a short period in culture [11,12]. Blood was drawn by cardiac puncture from wild-type (///), mu-calpain heterozygous (///), and homozygous (///) mice. Erythrocytes were washed three times with RPMI and leukocytes were removed as previously described [13]. Washed erythrocytes were resuspended in RPMI to attain /50% hematocrit. The trophozoite stage of P. falciparum was isolated using 70% Percoll gradient to achieve a parasitemia of /95%. Human serum was pre-adsorbed with wild-type erythrocytes at 37 8C for 1 h. The following components were mixed to initiate the invasion assay, 3 ml of human erythrocytes containing the trophozoite stage P. falciparum at /95%
Fig. 1. Role of mu-calpain in the invasion and growth of P. falciparum . Wild-type and mu-calpain null mice were infected with /50% parasitemia of P. yoelii 17 XL strain. Blood smears were made after every 24 h for 8 days post-infection, and stained with Giemsa. Parasitemias were determined by counting 1200 /1600 erythrocytes from each slide. Data were plotted as mean % parasitemia9/standard error.
parasitemia; 50 ml of 50% hematocrit of defined erythrocytes and 1.2 ml of RPMI containing 10% preadsorbed human serum. The culture was incubated for 22 /24 h at 37 8C in a gas mixture containing 5% CO2, 1% O2, and 94% N2 [14]. Thin smears were made the next day and stained with Giemsa. Newly invaded rings were counted from a total of /4000 erythrocytes for each sample to determine the extent of parasitemia. The experiment was repeated twice. As shown in Fig. 2, the invasion efficiency of P. falciparum in mu-calpain null erythrocytes is indistinguishable from that of wild-type and mu-calpain heterozygous erythrocytes. Furthermore, the number of ring-infected erythrocytes in the calpain (///) erythrocyte sample (mean % parasitemia9/S.D. /0.99%9/0.14) was significantly greater (P B/0.05) than the number of uninfected human erythrocytes originally introduced at the start of the invasion assay (maximum possible % parasitemia, if all human erythrocytes in the culture were infected / 0.56%). These results demonstrate that P. falciparum did indeed invade mouse erythrocytes, albeit at a reduced rate relative to human erythrocytes, and that the mouse erythrocyte calpain is not required for P. falciparum invasion and growth in vitro. Taken together, our results suggest that the endogenous calcium-dependent cysteine protease, mu-calpain, of mouse erythrocytes is not required for the blood-stage malaria parasite development in vivo and in vitro at least in P. falciparum and P. yoelii malaria. In contrast, compelling evidence suggests that the parasite-derived cysteine proteases are obligatory at all stages of parasite development in the host erythrocytes [1] and appear to be good targets for the development of specific inhibitors [15,16]. The availability of mu-calpain null mice will
Fig. 2. In vitro invasion of P. falciparum (3D7 strain) into mu-calpain null mouse erythrocytes. Normal human RBCs and RBCs from wild type (///), mu-calpain heterozygous (///), and homozygous (///) mice were incubated with purified trophozoite/schizont stage of P. falciparum at /95% parasitemia for 22 /24 h at 37 8C in a gas mixture containing 5% CO2, 1% O2, and 94% N2. Parasitemia was determined by counting the rings */ /4000 erythrocytes from Giemsastained smears. Data were plotted as mean invasion rate (9/standard error) relative to the human erythrocyte control sample (100% invasion).
M. Hanspal et al. / Molecular & Biochemical Parasitology 122 (2002) 227 /229
allow future testing of such inhibitors against parasite cysteine proteases in an environment where the host calpain activity is absent.
Acknowledgements We thank Dr M. Azam of our laboratory for sharing his stock of mu-calpain null mice for this study. This work was supported in part by the National Institutes of Health grants, HL 60152 (to MH) and HL 51445 (to AHC), and Tufts University Earl P. Charlton grant (to SSO).
References [1] Rosenthal P.J.. Proteases of malaria parasites: New targets for chemotherapy. Emerging Infect Dis 1998;4:49 /57. [2] Raphael P., Takakuwa Y., Manno S., Liu S.-C., Chishti A.H., Hanspal M.. A cysteine protease activity from Plasmodium falciparum cleaves human erythrocyte ankyrin. Mol Biochem Parasitol 2000;110:259 /72. [3] Dua M., Raphael P., Sijwali P.S., Rosenthal P.J., Hanspal M.. Recombinant falcipain-2 cleaves erythrocyte membrane ankyrin and protein 4.1. Mol Biochem Parasitol 2001;116:95 /9. [4] McKerrow J.H., Sun E., Rosenthal P.J., Bouvier J.. The proteases and pathogenicity of parasitic protozoa. Annu Rev Microbiol 1993;47:821 /53.
229
[5] Salmon B.L., Oksman A., Goldberg D.E.. Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis. Proc Natl Acad Sci USA 2001;98:271 /6. [6] Olaya P., Wasserman M.. Effect of calpain inhibitors on the invasion of human erythrocytes by the parasite Plasmodium falciparum . Biochim Biophys Acta 1991;1096:217 /21. [7] Wasserman M., Alarcon C., Mendoza P.M.. Effects of Ca2 depletion on the asexual cell cycle of Plasmodium falciparum . Am J Trop Med Hyg 1982;31:711 /7. [8] Suzuki K., Sorimachi H., Yoshizawa T., Kinbara K., Ishiura S.. Calpain: novel family members, activation, and physiologic function. Biol Chem Hoppe-Seyler 1995;376:523 /9. [9] Kawasaki H., Kawashima S.. Regulation of the calpain /calpastatin system by membranes. Mol Membr Biol 1996;13:217 /24. [10] Azam M., Andrabi S.S., Sahr K.E., Kamath L., Kuliopulos A., Chishti A.H.. Disruption of the mouse mu-calpain gene reveals an essential role in platelet function. Mol Cell Biol 2001;21:2213 /20. [11] Klotz F.W., Chulay J.D., Daniel W., Miller L.H.. Invasion of mouse erythrocytes by the human malaria parasite, Plasmodium falciparum . J Exp Med 1987;165:1713 /8. [12] Sam-Yellowe T.Y., Perkins M.E.. Binding of Plasmodium falciparum rhoptry proteins to mouse erythrocytes and their possible role in invasion. Mol Biochem Parasitol 1990;39:91 /100. [13] Bennett V., Stenbuck P.J.. Human erythrocyte ankyrin. J Biol Chem 1980;255:2540 /8. [14] Trager W., Jensen J.B.. Human malaria parasites in continuous culture. Science 1976;193:673 /5. [15] McKerrow J.H., Engel J.C., Caffrey C.R.. Cysteine protease inhibitors as chemotherapy for parasitic infections. Bioorg Med Chem 1999;7:639 /44. [16] Sabnis Y., Rosenthal P.J., Desai P., Avery M.A.. Homology modeling of falcipain-2: validation, de novo ligand design and synthesis of novel inhibitors. J Biomol Struct Dyn 2002;19:765 / 74.