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Malaria: Endless Fascination with Merozoite Release
Current Biology Vol 15 No 18 R760
Malaria: Endless Fascination with Merozoite Release The erythrocytic asexual reproduction cycle of Plasmodium falciparum, the most lethal malaria parasite in humans, starts with the invasion of a red blood cell by a merozoite and ends with the release of up to 32 new copies of itself in about 48 hours. A new study reveals that merozoite release is an explosive event ensuring the dispersal of these nonmotile parasites for optimal re-invasion of new red cells. Virgilio L. Lew Among the many open questions on the biology of malaria parasites, the merozoite release process remains one of the most intriguing and controversial. Are merozoites released and dispersed by the sudden lytic rupture of a swollen host cell [1–3]? Or are they released in packaged form, surrounded by the parasitophorous vacuolar membrane (PVM), and subsequently freed by delayed proteolysis of the PVM [4]? Is merozoite release accomplished without immediate host cell lysis [5,6]? Is lysis prevented by the fusion of the PVM and the red cell plasma membrane at the point of rupture [5]? Does the release process in vivo always follow an identical ‘normal’ pattern, or is it a polymorphic process with a frequency distribution of alternative normal patterns [7]? The paper by Glushakova, Yin, Li and Zimmerberg in this issue of Current Biology [8] provides solid and elegant evidence in support of the burst-dispersal model as the norm, with the best images and movies ever recorded on the events preceding and immediately following rupture. But before we consider their results and the new challenges emanating from their work, let us analyse the prescient insights from one of the most experienced observers in the field. William Trager, who passed away on the 22nd January 2005 at the age of 94, and to whom we owe — among many other ground-breaking and insightful contributions — the basic methodology of in vitro malaria culture [9], recently recounted the pleasure he had when first observing the release of
Plasmodium lophurae merozoites from infected duck erythrocytes ~50 years ago [2,10]: “R.B. McGhee and I saw the host cell become rounded and present a bright, refractile appearance. Within five minutes or less, the merozoites would be ejected with a kind of seething motion, which seemed to scatter the merozoites. This was seen repeatedly in preparations of late schizonts that were prepared freshly. However, after an hour or more on a warm stage, the merozoites that emerged were not ejected and scattered, but appeared to ‘drop’ out of the red blood cell as a cluster. We felt that this was not normal and was a result of lessfavourable conditions.” Trager also recounted more recent observations on the release of P. falciparum merozoites [2]: ‘In connection with my work on axenic culture of P. falciparum [11], I have checked many wet mounts taken as samples from schizont suspensions that were incubated at 37°C for the preparation of free merozoites, which constituted the inoculum for such experiments. I would see, using phase-contrast microscopy, before rupture, a rounded, bright, highly refractile cell with a central pigment clump. The merozoites would then be released abruptly with a scattering movement. Except for a lack of membrane vesiculation, it was very similar to the motion picture of P. knowlesi.’ The ‘motion picture’ to which Trager was referring was that produced by Dvorak et al. [1] on the release of merozoites from the simian parasite P. knowlesi in a perfused, temperature-controlled chamber, a first milestone recording of such event.
Let us briefly extract the main features of the merozoite release process as recounted by Trager [2], and documented by Dvorak et al. [1]. Firstly, the conditions of temperature, medium, atmosphere and duration of observation influence the pattern of merozoite release. Secondly, in fresh samples, during the last few minutes preceding rupture there is a dramatic morphological change; the infected cell becomes bright, highly refractile with a central pigment clump. Thirdly, the merozoites are released abruptly with a scattering movement. Fourthly, with time on a warm stage, merozoite release changes to a ‘drop-out’ clustered pattern, without scatter. Finally, the merozoite release process appeared to be quite similar in different Plasmodia species. These, and many other novel features of the merozoite release process, have now been documented in great detail by Glushakova et al. [8] using stateof-the-art methodologies and ingenious experimental designs. They combined multi-channel laser scanning confocal microscopy with the use of separate fluorophores for parasite and host cell membranes to investigate the process of merozoite release from P. falciparum-infected red blood cells in conditions resembling normal cultures. Their results showed that merozoite release is preceded for the last few minutes by a state which the authors termed ‘flowers’, a fairly accurate description of its morphological appearance, reminiscent of the highly refractile cell with central pigment clump (the residual, haemozoin-containing body) described by Trager. The flower stage rapidly evolves to a spherical configuration very near the critical haemolytic volume of the host red cell. The associated movies show that release occurs explosively with rapid merozoite dispersal, as if pressure-released, well in agreement with Trager’s ‘ejected with a kind of seething motion, which seemed to scatter the merozoites’ and ‘abruptly with a scattering movement’. These
Dispatch R761
Figure 1. Predicted 1.8 volume changes of a Critical haemolytic volume human red blood cell during the asexual 1.6 reproduction cycle of P. falciparum; effect of a terminal increase in 1.4 monovalent cation permeability. 1.2 The simulations were carried out using the mathematical model of Lew et 1.0 al. [13]. The black curve reports the predicted 0.8 change in the volume of 8 12 16 20 24 28 32 36 40 47 48 the host cell during the Time post-invasion (hours) parasite asexual cycle, Current Biology expressed relative to that immediately after invasion. The red segment illustrates the effect of a sudden, ten-fold increase in the monovalent cation permeability of the host cell membrane, 10 min before the nominal termination of the 48 h asexual cycle. Note that at this stage the membrane permeability of the host cell is largely determined by parasite-induced permeability pathways, which regulate the traffic of nutrients, waste products and inorganic ions [15]. The simulated permeability increase caused rapid swelling towards the critical haemolytic volume in ~10 min indicating that there is an adequate colloidosmotic driving force in the host’s residual haemoglobin concentration to induce rapid swelling, if sufficiently permeabilized. Relative cell volume
images clearly support terminal swelling and rupture hypotheses and rule out packaged merozoite release. Additional evidence also argues against the PVM–erythrocyte plasma membrane fusion scenario. Based on their results and on previous data in the literature, Glushakova et al. [8] propose a sequence of events pre- and post-rupture (see Figure 4 in [8]). Terminal lytic swelling is additionally supported by the opposite effects of inhibition and promotion of merozoite release induced by hypertonic and hypotonic pulses, respectively. Most surprising is the vesiculation pattern of the residual red cell membrane fragments after rupture. Besides settling the original controversies in favour of the burstdispersal model, the work of Glushakova et al. [8] raises a number of new issues and prompts the reformulation of some old ones. What role does the spectrin–actin cytoskeleton play in the generation of the flower-configuration and in post-rupture vesiculation of the residual host cell membrane? The vesiculation pattern is reminiscent of that generated by red cell lysis in low-ionic strength media [12]. Does cytoskeletal disassembly play a similar role in post-release vesiculation? Does release proceed through similar stages in vivo, when most red cells with mature parasites remain sequestered in the microcirculation, adhered to endothelial cells? What causes rupture? Is it acute terminal swelling alone, protease activity, or a coordinated combination of both? Induction of rapid terminal swelling in the few minutes before rupture requires a permeability increase and a driving force for fluid gain. Since the Na+ and K+ gradients across the host cell membrane are largely dissipated at this stage, the main driving force for fluid gain is in the excess colloidosmotic pressure of intracellular proteins (predominantly haemoglobin). A recent mathematical model of the homeostasis of a P. falciparuminfected red cell, incorporating the known time course of both haemoglobin digestion and membrane permeabilization, predicted terminal swelling, but
just below the critical haemolytic volume (Figure 1), a prediction supported by osmotic fragility studies on P. falciparum cultures [13,14]. The question then is whether the colloidosmotic pressure associated with the reduced haemoglobin content of host cells with late-stage parasites would be sufficient to drive rapid terminal swelling and lysis if the cation permeability of the host membrane were suddenly increased further. Figure 1 tests this point and confirms its plausibility; but whether or not terminal permeabilization contributes to merozoite release remains to be established. The results of Glushakova et al. [8] show that burst-dispursal is the reproducible modality of merozoite release in conditions closest to normal, and point out the likely artifactual origin of alternative release patterns. This work opens new areas of enquiry likely to inspire and guide much of the future research in this field.
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References 1. Dvorak, J.A., Miller, L.H., Whitehouse, W.C., and Shiroishi, T. (1975). Invasion of erythrocytes by malaria merozoites. Science 187, 748–750. 2. Trager, W. (2002). On the release of malaria merozoites. Trends Parasitol. 18, 60–61. 3. Wickham, M.E., Culvenor, J.G., and Cowman, A.F. (2003). Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte. J. Biol. Chem. 278, 37658–37663. 4. Salmon, B.L., Oksman, A., and Goldberg,
15.
D.E. (2001). Malaria parasite exit from the host erythrocyte: A two- step process requiring extraerythrocytic proteolysis. Proc. Natl. Acad. Sci. USA 98, 271–276. Winograd, E., Clavijo, C.A., Bustamante, L.Y., and Jaramillo, M. (1999). Release of merozoites from Plasmodium falciparum-infected erythrocytes could be mediated by a non-explosive event. Parasitol. Res. 85, 621–624. Sherman I.W, Eda S, Winograd E. (2004) Erythrocyte aging and malaria. Cell. Mol. Biol. (Noisy -le-grand) 50, 159–169. Lew, V.L. (2001). Packaged merozoite release without immediate host cell lysis. Trends Parasitol. 17, 401–403. Glushakova, S., Yin, D., Li, T., and Zimmerberg, J. (2005). Membrane transformation during malaria parasite release from human red blood cells. Curr. Biol. 15, this issue. Trager, W., and Jensen, J.B. (1976). Human malaria parasites in continuous culture. Science 193, 673–675. Trager, W., and McGhee, R.B. (1956). The intracellular position of malarial parasites. Trans. R. Soc. Trop. Med. Hyg. 50, 419–420. Trager, W., and Williams, J. (1992). Extracellular (axenic) development in vitro of the erythrocytic cycle of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 89, 5351–5355. Lew, V.L., Hockaday, A., Freeman, C.J., and Bookchin, R.M. (1988). Mechanism of spontaneous inside-out vesiculation of red cell membranes. J. Cell Biol. 106, 1893–1901. Lew, V.L., Tiffert, T., and Ginsburg, H. (2003). Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum-infected red blood cells. Blood 101, 4189–4194. Lew, V.L., Macdonald, L., Ginsburg, H., Krugliak, M., and Tiffert, T. (2004). Excess haemoglobin digestion by malaria parasites: a strategy to prevent premature host cell lysis. Blood Cells Mol. Dis. 32, 353–359. Kirk, K. (2001). Membrane transport in the malaria-infected erythrocyte. Physiol. Rev. 81, 495–537.
Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. DOI: 10.1016/j.cub.2005.08.059
Malaria: Endless Fascination with Merozoite Release The erythrocytic asexual reproduction cycle of Plasmodium falciparum, the most lethal malaria parasite in humans, starts with the invasion of a red blood cell by a merozoite and ends with the release of up to 32 new copies of itself in about 48 hours. A new study reveals that merozoite release is an explosive event ensuring the dispersal of these nonmotile parasites for optimal re-invasion of new red cells. Virgilio L. Lew Among the many open questions on the biology of malaria parasites, the merozoite release process remains one of the most intriguing and controversial. Are merozoites released and dispersed by the sudden lytic rupture of a swollen host cell [1–3]? Or are they released in packaged form, surrounded by the parasitophorous vacuolar membrane (PVM), and subsequently freed by delayed proteolysis of the PVM [4]? Is merozoite release accomplished without immediate host cell lysis [5,6]? Is lysis prevented by the fusion of the PVM and the red cell plasma membrane at the point of rupture [5]? Does the release process in vivo always follow an identical ‘normal’ pattern, or is it a polymorphic process with a frequency distribution of alternative normal patterns [7]? The paper by Glushakova, Yin, Li and Zimmerberg in this issue of Current Biology [8] provides solid and elegant evidence in support of the burst-dispersal model as the norm, with the best images and movies ever recorded on the events preceding and immediately following rupture. But before we consider their results and the new challenges emanating from their work, let us analyse the prescient insights from one of the most experienced observers in the field. William Trager, who passed away on the 22nd January 2005 at the age of 94, and to whom we owe — among many other ground-breaking and insightful contributions — the basic methodology of in vitro malaria culture [9], recently recounted the pleasure he had when first observing the release of
Plasmodium lophurae merozoites from infected duck erythrocytes ~50 years ago [2,10]: “R.B. McGhee and I saw the host cell become rounded and present a bright, refractile appearance. Within five minutes or less, the merozoites would be ejected with a kind of seething motion, which seemed to scatter the merozoites. This was seen repeatedly in preparations of late schizonts that were prepared freshly. However, after an hour or more on a warm stage, the merozoites that emerged were not ejected and scattered, but appeared to ‘drop’ out of the red blood cell as a cluster. We felt that this was not normal and was a result of lessfavourable conditions.” Trager also recounted more recent observations on the release of P. falciparum merozoites [2]: ‘In connection with my work on axenic culture of P. falciparum [11], I have checked many wet mounts taken as samples from schizont suspensions that were incubated at 37°C for the preparation of free merozoites, which constituted the inoculum for such experiments. I would see, using phase-contrast microscopy, before rupture, a rounded, bright, highly refractile cell with a central pigment clump. The merozoites would then be released abruptly with a scattering movement. Except for a lack of membrane vesiculation, it was very similar to the motion picture of P. knowlesi.’ The ‘motion picture’ to which Trager was referring was that produced by Dvorak et al. [1] on the release of merozoites from the simian parasite P. knowlesi in a perfused, temperature-controlled chamber, a first milestone recording of such event.
Let us briefly extract the main features of the merozoite release process as recounted by Trager [2], and documented by Dvorak et al. [1]. Firstly, the conditions of temperature, medium, atmosphere and duration of observation influence the pattern of merozoite release. Secondly, in fresh samples, during the last few minutes preceding rupture there is a dramatic morphological change; the infected cell becomes bright, highly refractile with a central pigment clump. Thirdly, the merozoites are released abruptly with a scattering movement. Fourthly, with time on a warm stage, merozoite release changes to a ‘drop-out’ clustered pattern, without scatter. Finally, the merozoite release process appeared to be quite similar in different Plasmodia species. These, and many other novel features of the merozoite release process, have now been documented in great detail by Glushakova et al. [8] using stateof-the-art methodologies and ingenious experimental designs. They combined multi-channel laser scanning confocal microscopy with the use of separate fluorophores for parasite and host cell membranes to investigate the process of merozoite release from P. falciparum-infected red blood cells in conditions resembling normal cultures. Their results showed that merozoite release is preceded for the last few minutes by a state which the authors termed ‘flowers’, a fairly accurate description of its morphological appearance, reminiscent of the highly refractile cell with central pigment clump (the residual, haemozoin-containing body) described by Trager. The flower stage rapidly evolves to a spherical configuration very near the critical haemolytic volume of the host red cell. The associated movies show that release occurs explosively with rapid merozoite dispersal, as if pressure-released, well in agreement with Trager’s ‘ejected with a kind of seething motion, which seemed to scatter the merozoites’ and ‘abruptly with a scattering movement’. These
Dispatch R761
Figure 1. Predicted 1.8 volume changes of a Critical haemolytic volume human red blood cell during the asexual 1.6 reproduction cycle of P. falciparum; effect of a terminal increase in 1.4 monovalent cation permeability. 1.2 The simulations were carried out using the mathematical model of Lew et 1.0 al. [13]. The black curve reports the predicted 0.8 change in the volume of 8 12 16 20 24 28 32 36 40 47 48 the host cell during the Time post-invasion (hours) parasite asexual cycle, Current Biology expressed relative to that immediately after invasion. The red segment illustrates the effect of a sudden, ten-fold increase in the monovalent cation permeability of the host cell membrane, 10 min before the nominal termination of the 48 h asexual cycle. Note that at this stage the membrane permeability of the host cell is largely determined by parasite-induced permeability pathways, which regulate the traffic of nutrients, waste products and inorganic ions [15]. The simulated permeability increase caused rapid swelling towards the critical haemolytic volume in ~10 min indicating that there is an adequate colloidosmotic driving force in the host’s residual haemoglobin concentration to induce rapid swelling, if sufficiently permeabilized. Relative cell volume
images clearly support terminal swelling and rupture hypotheses and rule out packaged merozoite release. Additional evidence also argues against the PVM–erythrocyte plasma membrane fusion scenario. Based on their results and on previous data in the literature, Glushakova et al. [8] propose a sequence of events pre- and post-rupture (see Figure 4 in [8]). Terminal lytic swelling is additionally supported by the opposite effects of inhibition and promotion of merozoite release induced by hypertonic and hypotonic pulses, respectively. Most surprising is the vesiculation pattern of the residual red cell membrane fragments after rupture. Besides settling the original controversies in favour of the burstdispersal model, the work of Glushakova et al. [8] raises a number of new issues and prompts the reformulation of some old ones. What role does the spectrin–actin cytoskeleton play in the generation of the flower-configuration and in post-rupture vesiculation of the residual host cell membrane? The vesiculation pattern is reminiscent of that generated by red cell lysis in low-ionic strength media [12]. Does cytoskeletal disassembly play a similar role in post-release vesiculation? Does release proceed through similar stages in vivo, when most red cells with mature parasites remain sequestered in the microcirculation, adhered to endothelial cells? What causes rupture? Is it acute terminal swelling alone, protease activity, or a coordinated combination of both? Induction of rapid terminal swelling in the few minutes before rupture requires a permeability increase and a driving force for fluid gain. Since the Na+ and K+ gradients across the host cell membrane are largely dissipated at this stage, the main driving force for fluid gain is in the excess colloidosmotic pressure of intracellular proteins (predominantly haemoglobin). A recent mathematical model of the homeostasis of a P. falciparuminfected red cell, incorporating the known time course of both haemoglobin digestion and membrane permeabilization, predicted terminal swelling, but
just below the critical haemolytic volume (Figure 1), a prediction supported by osmotic fragility studies on P. falciparum cultures [13,14]. The question then is whether the colloidosmotic pressure associated with the reduced haemoglobin content of host cells with late-stage parasites would be sufficient to drive rapid terminal swelling and lysis if the cation permeability of the host membrane were suddenly increased further. Figure 1 tests this point and confirms its plausibility; but whether or not terminal permeabilization contributes to merozoite release remains to be established. The results of Glushakova et al. [8] show that burst-dispursal is the reproducible modality of merozoite release in conditions closest to normal, and point out the likely artifactual origin of alternative release patterns. This work opens new areas of enquiry likely to inspire and guide much of the future research in this field.
5.
6. 7. 8.
9. 10.
11.
12.
13.
14.
References 1. Dvorak, J.A., Miller, L.H., Whitehouse, W.C., and Shiroishi, T. (1975). Invasion of erythrocytes by malaria merozoites. Science 187, 748–750. 2. Trager, W. (2002). On the release of malaria merozoites. Trends Parasitol. 18, 60–61. 3. Wickham, M.E., Culvenor, J.G., and Cowman, A.F. (2003). Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte. J. Biol. Chem. 278, 37658–37663. 4. Salmon, B.L., Oksman, A., and Goldberg,
15.
D.E. (2001). Malaria parasite exit from the host erythrocyte: A two- step process requiring extraerythrocytic proteolysis. Proc. Natl. Acad. Sci. USA 98, 271–276. Winograd, E., Clavijo, C.A., Bustamante, L.Y., and Jaramillo, M. (1999). Release of merozoites from Plasmodium falciparum-infected erythrocytes could be mediated by a non-explosive event. Parasitol. Res. 85, 621–624. Sherman I.W, Eda S, Winograd E. (2004) Erythrocyte aging and malaria. Cell. Mol. Biol. (Noisy -le-grand) 50, 159–169. Lew, V.L. (2001). Packaged merozoite release without immediate host cell lysis. Trends Parasitol. 17, 401–403. Glushakova, S., Yin, D., Li, T., and Zimmerberg, J. (2005). Membrane transformation during malaria parasite release from human red blood cells. Curr. Biol. 15, this issue. Trager, W., and Jensen, J.B. (1976). Human malaria parasites in continuous culture. Science 193, 673–675. Trager, W., and McGhee, R.B. (1956). The intracellular position of malarial parasites. Trans. R. Soc. Trop. Med. Hyg. 50, 419–420. Trager, W., and Williams, J. (1992). Extracellular (axenic) development in vitro of the erythrocytic cycle of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 89, 5351–5355. Lew, V.L., Hockaday, A., Freeman, C.J., and Bookchin, R.M. (1988). Mechanism of spontaneous inside-out vesiculation of red cell membranes. J. Cell Biol. 106, 1893–1901. Lew, V.L., Tiffert, T., and Ginsburg, H. (2003). Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum-infected red blood cells. Blood 101, 4189–4194. Lew, V.L., Macdonald, L., Ginsburg, H., Krugliak, M., and Tiffert, T. (2004). Excess haemoglobin digestion by malaria parasites: a strategy to prevent premature host cell lysis. Blood Cells Mol. Dis. 32, 353–359. Kirk, K. (2001). Membrane transport in the malaria-infected erythrocyte. Physiol. Rev. 81, 495–537.
Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. DOI: 10.1016/j.cub.2005.08.059