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How plasmodium secures nutrients: New targets for drugs?: Reply
24
How Plasmodium Secures Nutrients: New Targets for Drugs?: Reply The membrane trafficking of ions and nutrients in intraetythrocytic malana parasites has been of general Interest for many years. With the recent advent of new methods to study transport, the understanding of plasmodial nutrient acquisition has progressed at a quickened pace, as dlscussed by Olliaro (this issue). We used the patch-clamp method to study the movement of nutrients across the parasitophorous vacuole membrane (PVM), the outer of the two membranes surrounding the intraetythrocytic parasite. This relatively new method overcame several limitations inherent to the previous radioisotope flux measurements in etythrocyte-free plasmodia. Fiti, while flux studies must use imperfect methods to produce large populations of etythrocytefree parasites, we were able selectively to disrupt the plasma membrane of a single parasitized red blood cell, verify that the parasite was liberated but not damaged, and record current fluxes across a small patch of
Immunizing Against Toxic Malarial Antigens: Reply We agree with the overall direction of Ian Clark and Kirk Rockett’s Comment on ‘Immunizing Against Toxic Malarial Antigens’ (this issue), but a few points need clarification. First, the phospholipids coupled to keyhole limpet haemocyanin (KLH) were commercially available and were not parasite extracts as stated. We agree that tumour necrosis factor-a (TNF-a) is not the only mediator that may mediate the pathology of malaria. It seems likely that a cascade of pathogenic cytokines is secreted In response to malarial toxic antigens so that the use of any one cytokine inhibitor is unlikely to protect against clinical disease. This is precisely why we are attempting to Inhibit the toxins which induce pathological cytoklne productIon. We have focused on TNF-a because it is both a convenient marker of toxin inhibItIon and a proximal part of the cytokine cascade. This approach is vindicated by our results which show that mice immunized with KLH-phospholipid conjugates are protected against hypoglycaemia induced by malarial toxic antigens. Although the pathogenesis of hypoglycaemia in this model appears to be multifactorial, involving a number of cytokines and possibly the direct actions of insulinomimetic malarial antigens’,2, our
Parasjtoiogy Today, vol.
IO, no. I, I 994
the exposed PVM. Second, nonspecIfic leaks in the PVM induced by liberation from the red blood cell would produce an attifactually elevated uptake rate with radioisotope studies, but would not alter patch-clamp data. Such leaks could produce a lower resistance seal on the patch pipette, but could not produce the unambiguous channel events we reported. Finally, by studying the activity of a single ion channel rather than net fluxes in a population of cells, transport properties could be examined at a molecular level; the Identified nutrient-permeable channel has a slope conductance of I40 pS, a high open probability, three subconductance states and a unique voltage dependence. Despite these advances, the relative physiologic significance of the various proposed pathways of nutrient acquisition is far from fully understood. Olliaro raises an interesting problem: if the nutrientpermeable channel and the parasitophorous duct are active simultaneously, nutrients flowing from the etythrocyte cytosol through the channel into the parasitophorous space might diffuse down the ducts and leak into the extracellular space. This concern is not unique to our channel. The direct transport of nutrients
from the red blood cell cytosol into the parasite has long been studied with radlolsotope fluxes and is generally accepted as important. Indeed, previous studies suggest that there are transporters for purines, glucose and amino acids on the PVM. With each of these, the concern of extracellular leakage via the parasitophorous ducts arises. The channel we have reported may provide a solution to this dilemma: the extracellular leakage of erythrocyte nutrients that might occur with channels or other transporters that span only the PVM would not occur with a nutrient-permeable channel that spans both the PVM and the parasite plasma membrane, an alternative configuration similar to that of gap junction channels. This two-membrane-spanning configuration has been seen only with channels and can not be readily invoked with simple carrier-type transporter;. Further studies will obviously be needed before this and other issues can be resolved.
results show that we have managed to target the critical antigens. Clark and Rockett raise the possibiltty of a multi-parasite anti-disease vaccine since malaria toxic antigens from different Plasmodium species show serological cross-reactivity3 suggesting that a conserved epitope is responsible for TNF induction in malaria. However, we have no evidence that other parasites produce serologically cross-reactive TNF-inducing components. A vaccine which induces antibodies with broad cross-react&y might prove problematical so it is worth pointing out that the antitoxic IgM antibodies we have described show some specificity in that they bind to phosphatldylinositol (PI) but not other phospholipid liposomes4. Furthermore, inhibitory IgG specifically bound to liposomes containing either PI or phosphatidylsetine (PS). These observations suggest that the epitope recognized by inhibitory antibodies is not the common
phosphate ester group, as stated in the Comment article, but a more specific PI-like structure.
Sanjay Desai Department of Medicine Box 31215 Duke University Medical Center Durham, NC 277 IO, USA
References I Taylor, K. et al. ( 1992) Clin. Exp. immunol. 90. I-5 2 Schofield, L. and Hackett, F. (1993) J. Exp. Med. 177. 145-152 3 Bate, C.A.W. et oi. (1992) Infect. immun. 60, 1241-1243 4 Bate, C.A.W. et a/. (1992) immunology 76, 35-41 Clive Bate Dominic P. Kwiatkowski Department of Paediattics Institute of Molecular Medicine John Radcliffe Hospital Oxford, UK OX3 9DU Janice Taverne John H.L. Playfair Department of Immunology UCL Medical School London, UK WI P 9PG
Letters to the Editor Parasitology Today welcomes letters to the Editor on any subject of interest to parasitologists. If stimulated by any article published in this journal, the original author will be given the opportunity to reply. Please mark clearly whether or not your letter is intended for publication, and address to: Dr Theresa Saklatvala, Parasitology Today, Elsevier Trends Journals, 68 Hills Road, Cambridge, UK CB2 I LA.
How Plasmodium Secures Nutrients: New Targets for Drugs?: Reply The membrane trafficking of ions and nutrients in intraetythrocytic malana parasites has been of general Interest for many years. With the recent advent of new methods to study transport, the understanding of plasmodial nutrient acquisition has progressed at a quickened pace, as dlscussed by Olliaro (this issue). We used the patch-clamp method to study the movement of nutrients across the parasitophorous vacuole membrane (PVM), the outer of the two membranes surrounding the intraetythrocytic parasite. This relatively new method overcame several limitations inherent to the previous radioisotope flux measurements in etythrocyte-free plasmodia. Fiti, while flux studies must use imperfect methods to produce large populations of etythrocytefree parasites, we were able selectively to disrupt the plasma membrane of a single parasitized red blood cell, verify that the parasite was liberated but not damaged, and record current fluxes across a small patch of
Immunizing Against Toxic Malarial Antigens: Reply We agree with the overall direction of Ian Clark and Kirk Rockett’s Comment on ‘Immunizing Against Toxic Malarial Antigens’ (this issue), but a few points need clarification. First, the phospholipids coupled to keyhole limpet haemocyanin (KLH) were commercially available and were not parasite extracts as stated. We agree that tumour necrosis factor-a (TNF-a) is not the only mediator that may mediate the pathology of malaria. It seems likely that a cascade of pathogenic cytokines is secreted In response to malarial toxic antigens so that the use of any one cytokine inhibitor is unlikely to protect against clinical disease. This is precisely why we are attempting to Inhibit the toxins which induce pathological cytoklne productIon. We have focused on TNF-a because it is both a convenient marker of toxin inhibItIon and a proximal part of the cytokine cascade. This approach is vindicated by our results which show that mice immunized with KLH-phospholipid conjugates are protected against hypoglycaemia induced by malarial toxic antigens. Although the pathogenesis of hypoglycaemia in this model appears to be multifactorial, involving a number of cytokines and possibly the direct actions of insulinomimetic malarial antigens’,2, our
Parasjtoiogy Today, vol.
IO, no. I, I 994
the exposed PVM. Second, nonspecIfic leaks in the PVM induced by liberation from the red blood cell would produce an attifactually elevated uptake rate with radioisotope studies, but would not alter patch-clamp data. Such leaks could produce a lower resistance seal on the patch pipette, but could not produce the unambiguous channel events we reported. Finally, by studying the activity of a single ion channel rather than net fluxes in a population of cells, transport properties could be examined at a molecular level; the Identified nutrient-permeable channel has a slope conductance of I40 pS, a high open probability, three subconductance states and a unique voltage dependence. Despite these advances, the relative physiologic significance of the various proposed pathways of nutrient acquisition is far from fully understood. Olliaro raises an interesting problem: if the nutrientpermeable channel and the parasitophorous duct are active simultaneously, nutrients flowing from the etythrocyte cytosol through the channel into the parasitophorous space might diffuse down the ducts and leak into the extracellular space. This concern is not unique to our channel. The direct transport of nutrients
from the red blood cell cytosol into the parasite has long been studied with radlolsotope fluxes and is generally accepted as important. Indeed, previous studies suggest that there are transporters for purines, glucose and amino acids on the PVM. With each of these, the concern of extracellular leakage via the parasitophorous ducts arises. The channel we have reported may provide a solution to this dilemma: the extracellular leakage of erythrocyte nutrients that might occur with channels or other transporters that span only the PVM would not occur with a nutrient-permeable channel that spans both the PVM and the parasite plasma membrane, an alternative configuration similar to that of gap junction channels. This two-membrane-spanning configuration has been seen only with channels and can not be readily invoked with simple carrier-type transporter;. Further studies will obviously be needed before this and other issues can be resolved.
results show that we have managed to target the critical antigens. Clark and Rockett raise the possibiltty of a multi-parasite anti-disease vaccine since malaria toxic antigens from different Plasmodium species show serological cross-reactivity3 suggesting that a conserved epitope is responsible for TNF induction in malaria. However, we have no evidence that other parasites produce serologically cross-reactive TNF-inducing components. A vaccine which induces antibodies with broad cross-react&y might prove problematical so it is worth pointing out that the antitoxic IgM antibodies we have described show some specificity in that they bind to phosphatldylinositol (PI) but not other phospholipid liposomes4. Furthermore, inhibitory IgG specifically bound to liposomes containing either PI or phosphatidylsetine (PS). These observations suggest that the epitope recognized by inhibitory antibodies is not the common
phosphate ester group, as stated in the Comment article, but a more specific PI-like structure.
Sanjay Desai Department of Medicine Box 31215 Duke University Medical Center Durham, NC 277 IO, USA
References I Taylor, K. et al. ( 1992) Clin. Exp. immunol. 90. I-5 2 Schofield, L. and Hackett, F. (1993) J. Exp. Med. 177. 145-152 3 Bate, C.A.W. et oi. (1992) Infect. immun. 60, 1241-1243 4 Bate, C.A.W. et a/. (1992) immunology 76, 35-41 Clive Bate Dominic P. Kwiatkowski Department of Paediattics Institute of Molecular Medicine John Radcliffe Hospital Oxford, UK OX3 9DU Janice Taverne John H.L. Playfair Department of Immunology UCL Medical School London, UK WI P 9PG
Letters to the Editor Parasitology Today welcomes letters to the Editor on any subject of interest to parasitologists. If stimulated by any article published in this journal, the original author will be given the opportunity to reply. Please mark clearly whether or not your letter is intended for publication, and address to: Dr Theresa Saklatvala, Parasitology Today, Elsevier Trends Journals, 68 Hills Road, Cambridge, UK CB2 I LA.