Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein

Investigative systems for purified membrane transporters are almost exclusively reliant on the use of phospholipid vesicles or liposomes. Liposomes provide an environment to support protein function; however, they also have numerous drawbacks and should not be considered as a “one-size fits all” system. The use of artificial vesicles comprising block co-polymers (polymersomes) offers considerable advantages in terms of structural stability; provision of sufficient lateral pressure; and low passive permeability, which is a particular issue for transport assays using hydrophobic compounds. The present investigation demonstrates strategies to reconstitute ATP binding cassette (ABC) transporters into hybrid vesicles combining phospholipids and the block co-polymer poly (butadiene)-poly (ethylene oxide). Two efflux pumps were chosen; namely the Novosphingobium aromaticivorans Atm1 protein and human P-glycoprotein (Pgp). Polymersomes were generated with one of two lipid partners, either purified palmitoyl-oleoyl-phosphatidylcholine, or a mixture of crude E. coli lipid extract and cholesterol. Hybrid polymersomes were characterised for size, structural homogeneity, stability to detergents, and permeability. Two transporters, NaAtm1 and P-gp, were successfully reconstituted into pre-formed and surfactant-destabilised hybrid polymersomes using a detergent adsorption strategy. Reconstitution of both proteins was confirmed by density gradient centrifugation and the hybrid polymersomes supported substrate dependent ATPase activity of both transporters. The hybrid polymersomes also displayed low passive permeability to a fluorescent probe (calcein acetomethoxyl-ester (C-AM)) and offer the potential for quantitative measurements of transport activity for hydrophobic compounds

Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles

Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito.Neta Regev-Rudzki, Danny W. Wilson, Teresa G. Carvalho, Xavier Sisquella, Bradley M. Coleman, Melanie Rug, Dejan Bursac, Fiona Angrisano, Michelle Gee, Andrew F. Hill, Jake Baum, Alan F. Cowma

Seasonal change in the daily timing of behaviour of the common vole, Microtus arvalis

1. Seasonal effects on daily activity patterns in the common vole were established by periodic trapping in the field and continuous year round recording of running wheel and freeding activity in cages exposed to natural meteorological conditions. 2. Trapping revealed decreased nocturnality in winter as compared to summer. This was paralelled by a winter reduction in both nocturnal wheel running and feeding time in cages. 3. Frequent trap checks revealed a 2 h rhythm in daytime catches in winter, not in summer. Cage feeding activity in daytime was always organized in c. 2 h intervals, but day-to-day variations in phase blurred the rhythm in summer in a summation of individual daily records. Thus both seasonal and short-term temporal patterns are consistent between field trappings and cage feeding records. 4. Variables associated with the seasonal change in daily pattern were: reproductive state (sexually active voles more nocturnal), age (juveniles more nocturnal), temperature (cold days: less nocturnal), food (indicated by feeding experiments), habitat structure (more nocturnal in habitat with underground tunnels). 5. Minor discrepancies between field trappings and cage feeding activity can be explained by assuming increased trappability of voles in winter. Cage wheel running is not predictive of field trapping patterns and is thought to reflect behavioral motivations not associated with feeding but with other activities (e.g., exploratory, escape, interactive behaviour) undetected by current methods, including radiotelemetry and passage-counting. 6. Winter decrease in nocturnality appears to involve a reduction in nocturnal non-feeding and feeding behaviour and is interpreted primarily as an adaptation to reduce energy expenditure in adverse but socially stable winter conditions.

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts

Protein export and host membrane remodeling are crucial for multiple Plasmodium species to establish a niche in infected hosts. To better understand the contribution of these processes to successful parasite infection in vivo, we sought to find and characterize protein components of the intraerythrocytic Plasmodium berghei-induced membrane structures (IBIS) that form in the cytoplasm of infected erythrocytes. We identified proteins that immunoprecipitate with IBIS1, a signature member of the IBIS in P. berghei-infected erythrocytes. In parallel, we also report our data describing proteins that co-precipitate with the PTEX (Plasmodium translocon of exported proteins) component EXP2. To validate our findings, we examined the location of three candidate IBIS1-interactors that are conserved across multiple Plasmodium species, and we found they localized to IBIS in infected red blood cells and two further colocalized with IBIS1 in the liver-stage parasitophorous vacuole membrane. Successful gene deletion revealed that these two tryptophan-rich domain-containing proteins, termed here IPIS2 and IPIS3 (for intraerythrocytic Plasmodium-induced membrane structures), are required for efficient blood-stage growth. Erythrocytes infected with IPIS2-deficient schizonts in particular fail to bind CD36 as efficiently as wild-type P. berghei-infected cells and therefore fail to effectively sequester out of the circulating blood. Our findings support the idea that intra-erythrocytic membrane compartments are required across species for alterations of the host erythrocyte that facilitate interactions of infected cells with host tissues

The Plasmodium Export Element Revisited

We performed a bioinformatical analysis of protein export elements (PEXEL) in the putative proteome of the malaria parasite Plasmodium falciparum. A protein family-specific conservation of physicochemical residue profiles was found for PEXEL-flanking sequence regions. We demonstrate that the family members can be clustered based on the flanking regions only and display characteristic hydrophobicity patterns. This raises the possibility that the flanking regions may contain additional information for a family-specific role of PEXEL. We further show that signal peptide cleavage results in a positional alignment of PEXEL from both proteins with, and without, a signal peptide

Dissection of the Role of PfEMP1 and ICAM-1 in the Sensing of Plasmodium falciparum-Infected Erythrocytes by Natural Killer Cells

BACKGROUND: Host innate immunity contributes to malaria clinical outcome by providing protective inflammatory cytokines such as interferon-γ, and by shaping the adaptive immune response. Plasmodium falciparum (Pf) is the etiologic agent of the most severe forms of human malaria. Natural Killer (NK) cells are lymphocytes of the innate immune system that are the first effectors to produce interferon-γ in response to Pf. However, the molecular bases of Pf-NK cell recognition events are unknown. Our study focuses on the role of Pf erythrocyte membrane protein 1 (PfEMP1), a major Pf virulence factor. PfEMP1 is expressed on parasitized-erythrocytes and participates to vascular obstruction through the binding to several host receptors. PfEMP1 is also a pivotal target for host antibody response to Pf infection. METHODOLOGY/PRINCIPAL FINDINGS: Using genetically-engineered parasite mutant strains, a human genetic deficiency, and blocking antibodies, we identified two receptor-ligand pairs involved in two uncoupled events occurring during the sensing of Pf infection by NK cells. First, PfEMP1 interaction with one of its host receptor, chondroitin sulfate A, mediates the cytoadhesion of Pf-infected erythrocytes to human NK cell lines, but is not required for primary NK cell activation. Second, intercellular adhesion molecule-1 (ICAM-1), another host receptor for PfEMP1, is mandatory for NK cell interferon-γ response. In this case, ICAM-1 acts via its engagement with its host ligand, LFA-1, and not with PfEMP1, consistent with the obligatory cross-talk of NK cells with macrophages for their production of interferon-γ. CONCLUSION/SIGNIFICANCE: PfEMP1-independent but ICAM-1/LFA-1-dependent events occurring during NK cell activation by Pf highlight the fundamental role of cellular cooperation during innate immune response to malaria

The kinetics of antibody binding to Plasmodium falciparum VAR2CSA PfEMP1 antigen and modelling of PfEMP1 antigen packing on the membrane knobs

<p>Abstract</p> <p>Background</p> <p>Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by <it>Plasmodium falciparum </it>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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 <it>P. falciparum</it>-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.</p> <p>Conclusions</p> <p>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.</p

Investigating the Host Binding Signature on the Plasmodium falciparum PfEMP1 Protein Family

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family plays a central role in antigenic variation and cytoadhesion of P. falciparum infected erythrocytes. PfEMP1 proteins/var genes are classified into three main subfamilies (UpsA, UpsB, and UpsC) that are hypothesized to have different roles in binding and disease. To investigate whether these subfamilies have diverged in binding specificity and test if binding could be predicted by adhesion domain classification, we generated a panel of 19 parasite lines that primarily expressed a single dominant var transcript and assayed binding against 12 known host receptors. By limited dilution cloning, only UpsB and UpsC var genes were isolated, indicating that UpsA var gene expression is rare under in vitro culture conditions. Consequently, three UpsA variants were obtained by rosette purification and selection with specific monoclonal antibodies to create a more representative panel. Binding assays showed that CD36 was the most common adhesion partner of the parasite panel, followed by ICAM-1 and TSP-1, and that CD36 and ICAM-1 binding variants were highly predicted by adhesion domain sequence classification. Binding to other host receptors, including CSA, VCAM-1, HABP1, CD31/PECAM, E-selectin, Endoglin, CHO receptor “X”, and Fractalkine, was rare or absent. Our findings identify a category of larger PfEMP1 proteins that are under dual selection for ICAM-1 and CD36 binding. They also support that the UpsA group, in contrast to UpsB and UpsC var genes, has diverged from binding to the major microvasculature receptor CD36 and likely uses other mechanisms to sequester in the microvasculature. These results demonstrate that CD36 and ICAM-1 have left strong signatures of selection on the PfEMP1 family that can be detected by adhesion domain sequence classification and have implications for how this family of proteins is specializing to exploit hosts with varying levels of anti-malaria immunity

clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion

BACKGROUND: The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1. METHODOLOGY/PRINCIPAL FINDINGS: Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36. CONCLUSIONS/SIGNIFICANCE: Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications