Dataset Information

Dynamic association of PfEMP1 and KAHRP in knobs mediates cytoadherence during Plasmodium invasion.

ABSTRACT: Plasmodium falciparum infected erythrocytes display membrane knobs that are essential for their adherence to vascular endothelia and for prevention of clearance by the spleen. The knob associated histidine rich protein (KAHRP) is indispensable to knob formation and has been implicated in the recruitment and tethering of P. falciparum erythrocyte membrane protein-1 (PfEMP1) by binding to its cytoplasmic domain termed VARC. However, the precise mechanism of interaction between KAHRP and VARC is not very well understood. Here we report that both the proteins co-localize to membrane knobs of P. falciparum infected erythrocytes and have identified four positively charged linear sequence motifs of high intrinsic mobility on KAHRP that interact electrostatically with VARC in solution to form a fuzzy complex. The current study provides molecular insight into interaction between KAHRP and VARC in solution that takes place at membrane knobs.

SUBMITTER: Ganguly AK

PROVIDER: S-EPMC4345318 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Dynamic association of PfEMP1 and KAHRP in knobs mediates cytoadherence during Plasmodium invasion.

Ganguly Akshay Kumar AK Ranjan Priyatosh P Kumar Ashutosh A Bhavesh Neel Sarovar NS

Scientific reports 20150302

Plasmodium falciparum infected erythrocytes display membrane knobs that are essential for their adherence to vascular endothelia and for prevention of clearance by the spleen. The knob associated histidine rich protein (KAHRP) is indispensable to knob formation and has been implicated in the recruitment and tethering of P. falciparum erythrocyte membrane protein-1 (PfEMP1) by binding to its cytoplasmic domain termed VARC. However, the precise mechanism of interaction between KAHRP and VARC is no ...[more]

PMID: 25726759

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Project description: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 antibody-receptor 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. falciparum-infected 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.

Project description:BackgroundThe ability of mature forms of Plasmodium falciparum infected erythrocytes to bind to a range of host receptors including those displayed on endothelial cells has been associated with the pathology of this infection. Investigations into this adhesive phenomenon have used protein and cell-based adhesion assays to quantify the ability of infected red blood cells to bind. These adhesion assays tend to have relatively high inherent variability and so require multiple experiments in order to provide good quantitation. This means that investigators doing these experiments must count many fields of adherent parasites, a task that is time-consuming and laborious. To address this issue and to facilitate cytoadherence research, developed automated protocols were developed for counting parasite adhesion.MethodsParasite adhesion assays were mainly carried out under static conditions using purified receptors, which is the simplest form of these assays and is translatable to the field. Two different software platforms were used, one commercial (Image Pro-Plus (Media Cybernetics)) and one available in the public domain (ImageSXM) based on the freely available NIH Image software. The adhesion assays were performed and parasite binding quantified using standard manual techniques. Images were also captured using video microscopy and analysed using the two automated systems. The results generated by each system were compared using the Bland and Altman method for assessing the agreement between two methods.ResultsBoth automated counting programs showed concordance compared to the 'gold standard' manual counting within the normal range of adhesion seen with these assays, although the ImageSXM technique had some systematic bias. There was some fall-off in accuracy at very high parasite densities, but this can be resolved through good design of the experiments. Cell based assays were also used as inputs to one of the automated systems (ImageSXM) and produced variable, but encouraging, results.ConclusionsThe automated counting programs are an accurate and practical way of quantifying static parasite binding assays to purified proteins. They are less accurate when applied to cell based systems, but can still provide a reasonable level of accuracy to give a semi-quantitative readout.

Dataset Information

Dynamic association of PfEMP1 and KAHRP in knobs mediates cytoadherence during Plasmodium invasion.

Publications

Dynamic association of PfEMP1 and KAHRP in knobs mediates cytoadherence during Plasmodium invasion.

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