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Ioannis Vakonakis - One of the best experts on this subject based on the ideXlab platform.
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a spiral scaffold underlies cytoadherent Knobs in plasmodium falciparum infected erythrocytes
Blood, 2016Co-Authors: Jean M. Watermeyer, Victoria L. Hale, Fiona Hackett, Daniel K. Clare, Erin E. Cutts, Ioannis Vakonakis, Roland A. Fleck, Michael J. Blackman, Helen R. SaibilAbstract:Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as Knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of Knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the Knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte Knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of Knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.
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A spiral scaffold underlies cytoadherent Knobs in Plasmodium falciparum–infected erythrocytes
Blood, 2015Co-Authors: Jean M. Watermeyer, Victoria L. Hale, Fiona Hackett, Daniel K. Clare, Erin E. Cutts, Ioannis Vakonakis, Roland A. Fleck, Michael J. Blackman, Helen R. SaibilAbstract:Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as Knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of Knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the Knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte Knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of Knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.
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a plasmodium falciparum phist protein binds the virulence factor pfemp1 and comigrates to Knobs on the host cell surface
The FASEB Journal, 2014Co-Authors: Alexander Oberli, Erin E. Cutts, Leanne M Slater, Francoise Brand, Esther Mundwilerpachlatko, Sebastian Rusch, Martin F G Masik, Michele C Erat, Hanspeter Beck, Ioannis VakonakisAbstract:Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as Knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in Knobs. PHIST variants that do not locate in Knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in Knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.
Erin E. Cutts - One of the best experts on this subject based on the ideXlab platform.
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a spiral scaffold underlies cytoadherent Knobs in plasmodium falciparum infected erythrocytes
Blood, 2016Co-Authors: Jean M. Watermeyer, Victoria L. Hale, Fiona Hackett, Daniel K. Clare, Erin E. Cutts, Ioannis Vakonakis, Roland A. Fleck, Michael J. Blackman, Helen R. SaibilAbstract:Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as Knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of Knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the Knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte Knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of Knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.
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A spiral scaffold underlies cytoadherent Knobs in Plasmodium falciparum–infected erythrocytes
Blood, 2015Co-Authors: Jean M. Watermeyer, Victoria L. Hale, Fiona Hackett, Daniel K. Clare, Erin E. Cutts, Ioannis Vakonakis, Roland A. Fleck, Michael J. Blackman, Helen R. SaibilAbstract:Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as Knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of Knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the Knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte Knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of Knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.
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a plasmodium falciparum phist protein binds the virulence factor pfemp1 and comigrates to Knobs on the host cell surface
The FASEB Journal, 2014Co-Authors: Alexander Oberli, Erin E. Cutts, Leanne M Slater, Francoise Brand, Esther Mundwilerpachlatko, Sebastian Rusch, Martin F G Masik, Michele C Erat, Hanspeter Beck, Ioannis VakonakisAbstract:Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as Knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in Knobs. PHIST variants that do not locate in Knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in Knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.
Helen R. Saibil - One of the best experts on this subject based on the ideXlab platform.
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a spiral scaffold underlies cytoadherent Knobs in plasmodium falciparum infected erythrocytes
Blood, 2016Co-Authors: Jean M. Watermeyer, Victoria L. Hale, Fiona Hackett, Daniel K. Clare, Erin E. Cutts, Ioannis Vakonakis, Roland A. Fleck, Michael J. Blackman, Helen R. SaibilAbstract:Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as Knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of Knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the Knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte Knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of Knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.
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A spiral scaffold underlies cytoadherent Knobs in Plasmodium falciparum–infected erythrocytes
Blood, 2015Co-Authors: Jean M. Watermeyer, Victoria L. Hale, Fiona Hackett, Daniel K. Clare, Erin E. Cutts, Ioannis Vakonakis, Roland A. Fleck, Michael J. Blackman, Helen R. SaibilAbstract:Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as Knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of Knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the Knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte Knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of Knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.
Lea Barfod - One of the best experts on this subject based on the ideXlab platform.
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Plasmodium falciparum-Infected Erythrocyte Knob Density Is Linked to the PfEMP1 Variant Expressed
mBio, 2015Co-Authors: Ramesh Subramani, Tue Hassenkam, Lars Hviid, Katharina A. Quadt, Anine Jeppesen, Casper Hempel, Jens E. V. Petersen, Lea BarfodAbstract:Members of the clonally variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediate adhesion of infected erythrocytes (IEs) to vascular receptors. PfEMP1 expression is normally confined to nanoscale knob protrusions on the IE surface membrane. To investigate the relationship between the densities of these IE surface Knobs and the PfEMP1 variant expressed, we used specific antibody panning to generate three sublines of the P. falciparum clone IT4, which expresses the PfEMP1 variants IT4VAR04, IT4VAR32b, and IT4VAR60. The knob density in each subline was then determined by atomic force microscopy (AFM) and scanning electron microscopy (SEM) and compared to PfEMP1 and knob-associated histidine-rich protein (KAHRP) expression. Selection for uniform expression of IT4VAR04 produced little change in knob density, compared to unselected IEs. In contrast, selection for IT4VAR32b expression increased knob density approximately 3-fold, whereas IEs selected for IT4VAR60 expression were essentially knobless. When IT4VAR60(+) IEs were subsequently selected to express IT4VAR04 or IT4VAR32b, they again displayed low and high knob densities, respectively. All sublines expressed KAHRP regardless of the PfEMP1 expressed. Our study documents for the first time that knob density is related to the PfEMP1 variant expressed. This may reflect topological requirements to ensure optimal adhesive properties of the IEs. Infections with Plasmodium falciparum malaria parasites are still responsible for many deaths, especially among children and pregnant women. New interventions are needed to reduce severe illness and deaths caused by this malaria parasite. Thus, a better understanding of the mechanisms behind the pathogenesis is essential. A main reason why Plasmodium falciparum malaria is more severe than disease caused by other malaria species is its ability to express variant antigens on the infected erythrocyte surface. These antigens are presented on membrane protrusions known as Knobs. This study set out to investigate the interplay between different variant antigens on the surface of P. falciparum-infected erythrocytes and the density of the Knobs on which the antigens are expressed. Such a direct analysis of this relationship has not been reported before but adds to the important understanding of the complexity of malaria antigen presentation. Copyright © 2015 Subramani et al.
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the density of Knobs on plasmodium falciparum infected erythrocytes depends on developmental age and varies among isolates
PLOS ONE, 2012Co-Authors: Katharina A. Quadt, Lea Barfod, Tue Hassenkam, Daniel Andersen, Jonas Bruun, Ben Gyan, Michael F Ofori, Lars HviidAbstract:Background The virulence of Plasmodium falciparum malaria is related to the parasite’s ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on dome-shaped protrusions called Knobs on the IE surface is central to both. Differences in receptor specificity and affinity of expressed PfEMP1 are important for IE adhesiveness, but it is not known whether differences in the number and size of the Knobs on which the PfEMP1 proteins are expressed also play a role. Therefore, the aim of this study was to provide detailed information on isolate- and time-dependent differences in knob size and density.
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the kinetics of antibody binding to plasmodium falciparum var2csa pfemp1 antigen and modelling of pfemp1 antigen packing on the membrane Knobs
Malaria Journal, 2010Co-Authors: Lars M Joergensen, Ali Salanti, Tina Dobrilovic, Lea Barfod, Tue Hassenkam, Thor G Theander, Lars Hviid, David E ArnotAbstract:Background: Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibodyreceptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target. Methods: A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno-fluorescence microscopy was used to visualize antibody-mediated adhesion between the surfaces of live infected erythrocytes and atomic force microscopy was used to obtain higher resolution images of the membrane Knobs on the infected erythrocyte to estimate knob surface areas and model VAR2CSA packing density on the knob. Results: Kinetic analysis indicates that antibody dissociation from the VAR2CSA PfEMP1 antigen is extremely slow when there is a high avidity interaction. High avidity binding to PfEMP1 antigens on the surface of P. falciparuminfected erythrocytes in turn requires bivalent cross-linking of epitopes positioned within the distance that can be bridged by antibody. Calculations of the surface area of the Knobs and the possible densities of PfEMP1 packing on the Knobs indicate that high-avidity cross-linking antibody reactions are constrained by the architecture of the Knobs and the large size of PfEMP1 molecules. Conclusions: High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18 nm sweep radius of an antibody. The large size of PfEMP1 will also constrain intra-knob cross-linking interactions. This analysis indicates that effective vaccines targeting the parasite's vulnerable adhesion receptors should primarily induce strongly adhering, high avidity antibodies whose association rate constant is less important than their dissociation rate constant.
Michael J. Blackman - One of the best experts on this subject based on the ideXlab platform.
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a spiral scaffold underlies cytoadherent Knobs in plasmodium falciparum infected erythrocytes
Blood, 2016Co-Authors: Jean M. Watermeyer, Victoria L. Hale, Fiona Hackett, Daniel K. Clare, Erin E. Cutts, Ioannis Vakonakis, Roland A. Fleck, Michael J. Blackman, Helen R. SaibilAbstract:Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as Knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of Knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the Knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte Knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of Knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.
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A spiral scaffold underlies cytoadherent Knobs in Plasmodium falciparum–infected erythrocytes
Blood, 2015Co-Authors: Jean M. Watermeyer, Victoria L. Hale, Fiona Hackett, Daniel K. Clare, Erin E. Cutts, Ioannis Vakonakis, Roland A. Fleck, Michael J. Blackman, Helen R. SaibilAbstract:Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as Knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of Knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the Knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte Knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of Knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.