The Experts below are selected from a list of 6621 Experts worldwide ranked by ideXlab platform

Michael J Blackman - One of the best experts on this subject based on the ideXlab platform.

  • the malaria parasite sheddase sub2 governs host red blood cell membrane sealing at invasion
    bioRxiv, 2020
    Co-Authors: Christine R Collins, Michael J Blackman, Fiona Hackett, Ambrosius P. Snijders, Matthew Rg Russell, Steven Howell, Lucy M Collinson
    Abstract:

    Abstract Red blood cell (RBC) invasion by malaria Merozoites involves formation of a parasitophorous vacuole into which the parasite moves. The vacuole membrane seals and pinches off behind the parasite through an unknown mechanism, enclosing the parasite within the RBC. During invasion, several parasite surface proteins are shed by a membrane-bound protease called SUB2. Here we show that genetic depletion of SUB2 abolishes shedding of a range of parasite proteins, identifying previously unrecognized SUB2 substrates. Interaction of SUB2-null Merozoites with RBCs leads to either abortive invasion with rapid RBC lysis, or successful entry but developmental arrest. Selective failure to shed the most abundant SUB2 substrate, MSP1, reduces intracellular replication, whilst conditional ablation of the substrate AMA1 produces host RBC lysis. We conclude that SUB2 activity is critical for host RBC membrane sealing following parasite internalisation and for correct functioning of merozoite surface proteins. Key highlights Many malaria parasite surface proteins are shed by SUB2 during RBC invasion SUB2-null Merozoites either induce rapid host RBC lysis, or invade then die Merozoite surface protein shedding is crucial for host RBC membrane sealing

  • the malarial serine protease sub1 plays an essential role in parasite liver stage development
    PLOS Pathogens, 2013
    Co-Authors: Catherine Suarez, Katrin Volkmann, Ana Rita Gomes, Oliver Billker, Michael J Blackman
    Abstract:

    Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter Merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage Merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic Merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.

  • global identification of multiple substrates for plasmodium falciparum sub1 an essential malarial processing protease
    Infection and Immunity, 2011
    Co-Authors: Natalie Silmon C De Monerri, Fiona Hackett, Chrislaine Withersmartinez, Konstantinos Koussis, Helen R Flynn, Marta G Campos, Mark J Skehel, Michael J Blackman
    Abstract:

    The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious Merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important merozoite surface and PV proteins. These include the essential merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptry-associated protein 1) and the merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target.

  • a multifunctional serine protease primes the malaria parasite for red blood cell invasion
    The EMBO Journal, 2009
    Co-Authors: Konstantinos Koussis, Fiona Hackett, Chrislaine Withersmartinez, Hermann Bujard, Sharon Yeoh, Matthew A Child, Ellen Knuepfer, Luiz Juliano, Ute Woehlbier, Michael J Blackman
    Abstract:

    The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter Merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in ‘priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.

  • pfsub 2 a second subtilisin like protein in plasmodium falciparum Merozoites
    Molecular and Biochemical Parasitology, 1999
    Co-Authors: Fiona Hackett, Chrislaine Withersmartinez, Mohammed Sajid, Muni Grainger, Michael J Blackman
    Abstract:

    Erythrocyte invasion by the malaria merozoite requires the activity of merozoite proteases. We have previously identified a Plasmodium falciparum protein belonging to the superfamily of subtilisin-like serine proteases, which is expressed in a subset of secretory organelles in free Merozoites. Here we describe the identification of a second P. falciparum subtilisin-like merozoite protein. Called PfSUB-2, it is encoded by a single copy gene and is expressed as a large putative type I integral membrane protein which undergoes extensive post-translational processing. The terminal processing product is expressed in an apical location in Merozoites. PfSUB-2 may mediate one or more of the serine protease activities known to be associated with erythrocyte invasion.

Alan F Cowman - One of the best experts on this subject based on the ideXlab platform.

  • recruitment of human c1 esterase inhibitor controls complement activation on blood stage plasmodium falciparum Merozoites
    Journal of Immunology, 2017
    Co-Authors: Alan F Cowman, Alexander T Kennedy, Lakshmi C Wijeyewickrema, Alisee Huglo, Clara S Lin, Robert N Pike
    Abstract:

    The complement system is a front-line defense system that opsonizes and lyses invading pathogens. To survive, microbes exposed to serum must evade the complement response. To achieve this, many pathogens recruit soluble human complement regulators to their surfaces and hijack their regulatory function for protection from complement activation. C1 esterase inhibitor (C1-INH) is a soluble regulator of complement activation that negatively regulates the classical and lectin pathways of complement to protect human tissue from aberrant activation. In this article, we show that Plasmodium falciparum Merozoites, the invasive form of blood stage malaria parasites, actively recruit C1-INH to their surfaces when exposed to human serum. We identified PfMSP3.1, a member of the merozoite surface protein 3 family of merozoite surface proteins, as the direct interaction partner. When bound to the merozoite surface, C1-INH retains its ability to complex with and inhibit C1s, MASP1, and MASP2, the activating proteases of the complement cascade. P. falciparum Merozoites that lack PfMSP3.1 showed a marked reduction in C1-INH recruitment and increased C3b deposition on their surfaces. However, these ΔPfMSP3.1 Merozoites exhibit enhanced invasion of RBCs in the presence of active complement. This study characterizes an immune-evasion strategy used by malaria parasites and highlights the complex relationship between Merozoites and the complement system.

  • the merozoite surface protein 1 complex is a platform for binding to human erythrocytes by plasmodium falciparum
    Journal of Biological Chemistry, 2014
    Co-Authors: Clara S Lin, Alessandro D Uboldi, Danushka S Marapana, Peter E Czabotar, Christian Epp, Hermann Bujard, Nicole L Taylor, Matthew A Perugini, Anthony N Hodder, Alan F Cowman
    Abstract:

    Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of Merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of Merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2–4 × 10−7 m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.

  • electron tomography of plasmodium falciparum Merozoites reveals core cellular events that underpin erythrocyte invasion
    Cellular Microbiology, 2013
    Co-Authors: Eric Hanssen, Alan F Cowman, David T Riglar, Chaitali Dekiwadia, Melanie Rug, Leandro Lemgruber, Marek Cyrklaff
    Abstract:

    Erythrocyte invasion by Merozoites forms of the malaria parasite is a key step in the establishment of human malaria disease. To date, efforts to understand cellular events underpinning entry have been limited to insights from non-human parasites, with no studies at sub-micrometer resolution undertaken using the most virulent human malaria parasite, Plasmodium falciparum. This leaves our understanding of the dynamics of merozoite sub-cellular compartments during infectionincomplete, in particular that of the secretory organelles. Using advances in P. falciparum merozoite isolation and new imaging techniques we present a three-dimensional study of invasion using electron microscopy, cryo-electron tomography and cryo-X-ray tomography. We describe the core architectural features of invasion and identify fusion between rhoptries at the commencement of invasion as a hitherto overlooked event that likely provides a critical step that initiates entry. Given the centrality of merozoite organelle proteins to vaccine development, these insights provide a mechanistic framework to understand therapeutic strategies targeted towards the cellular events of invasion.

  • efficient measurement of opsonising antibodies to plasmodium falciparum Merozoites
    PLOS ONE, 2012
    Co-Authors: Danika L Hill, Danny W. Wilson, Alan F Cowman, Emily M Eriksson, Amandine Carmagnac, Diana S Hansen, Louis Schofield
    Abstract:

    Background: Antibodies targeting Merozoites are important in protection from malaria. Therefore, merozoite surface proteins are attractive vaccine candidates. There is a need for robust functional assays to investigate mechanisms of acquired immunity and vaccine efficacy. To date, the study of merozoite phagocytosis has been confounded by the complexity and variability of in vitro assays. Methodology/Principal findings: We have developed a new flow cytometry-based merozoite phagocytosis assay. An optimized merozoite preparation technique produced high yields of Merozoites separated from haemozoin. Phagocytosis by the undifferentiated THP-1 monocytic cell line was mediated only by Fc Receptors, and was therefore ideal for studying opsonising antibody responses. The assay showed robust phagocytosis with highly diluted immune sera and strong inter-assay correlation. The assay effectively measured differences in opsonisation-dependent phagocytosis among individuals. Conclusions/Significance: This highly reproducible assay has potential applications in assessing the role of opsonic phagocytosis in naturally acquired immunity and vaccine trials.

  • selective inhibition of a two step egress of malaria parasites from the host erythrocyte
    Journal of Biological Chemistry, 2003
    Co-Authors: Mark Wickham, Janetta G Culvenor, Alan F Cowman
    Abstract:

    Abstract Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16–32 daughter Merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which Merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.

Danny W. Wilson - One of the best experts on this subject based on the ideXlab platform.

  • PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes.
    Nature communications, 2020
    Co-Authors: Benjamin Liffner, Sonja Frölich, Gary K. Heinemann, Boyin Liu, Stuart A. Ralph, Matthew W. A. Dixon, Tim-wolf Gilberger, Danny W. Wilson
    Abstract:

    The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by Merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology.

  • PfCERLI1, a conserved rhoptry associated protein essential for invasion by Plasmodium falciparum Merozoites.
    2019
    Co-Authors: Benjamin Liffner, Gary K. Heinemann, Boyin Liu, Matthew W. A. Dixon, Tim-wolf Gilberger, S. Frölich, Danny W. Wilson
    Abstract:

    Abstract The disease-causing blood stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by Merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparumCytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is the first rhoptry associated protein identified to have a direct role in function of this essential malaria invasion organelle which has broader implications for understanding apicomplexan invasion biology.

  • Targets of complement-fixing antibodies in protective immunity against malaria in children
    Nature Publishing Group, 2019
    Co-Authors: Linda Reiling, Danny W. Wilson, Michelle J Boyle, Jack S Richards, Michael T. White, Gaoqian Feng, Rupert Weaver, Herbert D. Opi, Kristina E. M. Persson, Peter M. Siba
    Abstract:

    Antibodies against Plasmodium falciparum Merozoites that fix complement can inhibit blood-stage replication. Here, Reiling et al. show that complement-fixing antibodies strongly correlate with protective immunity in children, identify the merozoite targets, and predict antigen combinations that should result in strong protection

  • igg opsonization of Merozoites multiple immune mechanisms for malaria vaccine development
    International Journal for Parasitology, 2017
    Co-Authors: Danny W. Wilson, Louis Schofield, Danika L Hill
    Abstract:

    Global eradication of the human-infecting malaria parasite Plasmodium falciparum, the major cause of malaria mortality, is unlikely to be achieved without an effective vaccine. However, our limited understanding of how protective immune responses target malaria parasites in humans, and how to best elicit these immune responses through vaccination, has hampered vaccine development. The red blood cell invading stage of the parasite lifecycle (merozoite) displays antigens that are attractive vaccine candidates as they are accessible to antibodies and raise high antibody titres in naturally immune individuals. The number of merozoite antigens that elicit an immune response, and their structural and functional diversity, has led to a large number of lead antigens being pursued as vaccine candidates. Despite being seemingly spoilt for choice in terms of vaccine candidates, there is still a lack of consensus on exactly how merozoite antibodies reduce parasitemia and malaria disease. In this review we describe the various immune mechanisms that can result from IgG opsonization of Merozoites, and highlight recent developments that support a role for these functional antibodies in naturally acquired and vaccine-induced immunity.

  • new approaches to studying plasmodium falciparum merozoite invasion and insights into invasion biology
    International Journal for Parasitology, 2013
    Co-Authors: Michelle J Boyle, Danny W. Wilson, James G Beeson
    Abstract:

    Merozoite invasion of human red blood cells by Plasmodium falciparum is essential for blood stage asexual replication and the development of malaria disease. Despite this, many of the processes involved in invasion are poorly understood. Recent advances have been made in methods to isolate viable Merozoites for studies of invasion. The application of these approaches is providing new insights into the kinetics of invasion and merozoite survival, as well as proteins and interactions involved in invasion, and will facilitate the development and testing of anti-merozoite vaccines and the identification of invasion-inhibitory compounds with potential for drug development. This review discusses these recent advances and considers potential avenues for future research.

Michelle J Boyle - One of the best experts on this subject based on the ideXlab platform.

  • Targets of complement-fixing antibodies in protective immunity against malaria in children
    Nature Publishing Group, 2019
    Co-Authors: Linda Reiling, Danny W. Wilson, Michelle J Boyle, Jack S Richards, Michael T. White, Gaoqian Feng, Rupert Weaver, Herbert D. Opi, Kristina E. M. Persson, Peter M. Siba
    Abstract:

    Antibodies against Plasmodium falciparum Merozoites that fix complement can inhibit blood-stage replication. Here, Reiling et al. show that complement-fixing antibodies strongly correlate with protective immunity in children, identify the merozoite targets, and predict antigen combinations that should result in strong protection

  • opsonic phagocytosis of plasmodium falciparum Merozoites mechanism in human immunity and a correlate of protection against malaria
    BMC Medicine, 2014
    Co-Authors: Michelle J Boyle, Jack S Richards, Christine Langer, Faith Ha Osier, Linda Reiling, Gaoqian Feng, Jingling Zhou, Fiona J Mccallum, Anthony Jaworowski
    Abstract:

    An understanding of the mechanisms mediating protective immunity against malaria in humans is currently lacking, but critically important to advance the development of highly efficacious vaccines. Antibodies play a key role in acquired immunity, but the functional basis for their protective effect remains unclear. Furthermore, there is a strong need for immune correlates of protection against malaria to guide vaccine development. Using a validated assay to measure opsonic phagocytosis of Plasmodium falciparum Merozoites, we investigated the potential role of this functional activity in human immunity against clinical episodes of malaria in two independent cohorts (n = 109 and n = 287) experiencing differing levels of malaria transmission and evaluated its potential as a correlate of protection. Antibodies promoting opsonic phagocytosis of Merozoites were cytophilic immunoglobulins (IgG1 and IgG3), induced monocyte activation and production of pro-inflammatory cytokines, and were directed against major merozoite surface proteins (MSPs). Consistent with protective immunity in humans, opsonizing antibodies were acquired with increasing age and malaria exposure, were boosted on re-infection, and levels were related to malaria transmission intensity. Opsonic phagocytosis was strongly associated with a reduced risk of clinical malaria in longitudinal studies in children with current or recent infections. In contrast, antibodies to the merozoite surface in standard immunoassays, or growth-inhibitory antibodies, were not significantly associated with protection. In multivariate analyses including several antibody responses, opsonic phagocytosis remained significantly associated with protection against malaria, highlighting its potential as a correlate of immunity. Furthermore, we demonstrate that human antibodies against MSP2 and MSP3 that are strongly associated with protection in this population are effective in opsonic phagocytosis of Merozoites, providing a functional link between these antigen-specific responses and protection for the first time. Opsonic phagocytosis of Merozoites appears to be an important mechanism contributing to protective immunity in humans. The opsonic phagocytosis assay appears to be a strong correlate of protection against malaria, a valuable biomarker of immunity, and provides a much-needed new tool for assessing responses to blood-stage malaria vaccines and measuring immunity in populations.

  • sequential processing of merozoite surface proteins during and after erythrocyte invasion by plasmodium falciparum
    Infection and Immunity, 2014
    Co-Authors: Michelle J Boyle, Anthony N Hodder, Robin F Anders, Christine Langer, Joanne Chan, Ross L Coppel, James G Beeson
    Abstract:

    Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when Merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of Merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium Merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.

  • new approaches to studying plasmodium falciparum merozoite invasion and insights into invasion biology
    International Journal for Parasitology, 2013
    Co-Authors: Michelle J Boyle, Danny W. Wilson, James G Beeson
    Abstract:

    Merozoite invasion of human red blood cells by Plasmodium falciparum is essential for blood stage asexual replication and the development of malaria disease. Despite this, many of the processes involved in invasion are poorly understood. Recent advances have been made in methods to isolate viable Merozoites for studies of invasion. The application of these approaches is providing new insights into the kinetics of invasion and merozoite survival, as well as proteins and interactions involved in invasion, and will facilitate the development and testing of anti-merozoite vaccines and the identification of invasion-inhibitory compounds with potential for drug development. This review discusses these recent advances and considers potential avenues for future research.

  • isolation of viable plasmodium falciparum Merozoites to define erythrocyte invasion events and advance vaccine and drug development
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Danny W. Wilson, Michelle J Boyle, Jack S Richards, David T Riglar, Kevin K A Tetteh, David J Conway
    Abstract:

    During blood-stage infection by Plasmodium falciparum, Merozoites invade RBCs. Currently there is limited knowledge of cellular and molecular invasion events, and no established assays are available to readily measure and quantify invasion-inhibitory antibodies or compounds for vaccine and drug studies. We report the isolation of viable Merozoites that retain their invasive capacity, at high purity and yield, purified by filtration of highly synchronous populations of schizonts. We show that the half-life of merozoite invasive capacity after rupture is 5 min at 37 °C, and 15 min at room temperature. Studying the kinetics of invasion revealed that 80% of invasion events occur within 10 min of mixing Merozoites and RBCs. Invasion efficiency was maximum at low merozoite-to-RBC ratios and occurred efficiently in the absence of serum and with high concentrations of dialyzed nonimmune serum. We developed and optimized an invasion assay by using purified Merozoites that enabled invasion-inhibitory activity of antibodies and compounds to be measured separately from other mechanisms of growth inhibition; the assay was more sensitive for detecting inhibitory activity than established growth-inhibition assays. Furthermore, with the use of purified Merozoites it was possible to capture and fix Merozoites at different stages of invasion for visualization by immunofluorescence microscopy and EM. We thereby demonstrate that processing of the major merozoite antigen merozoite surface protein-1 occurs at the time of RBC invasion. These findings have important implications for defining invasion events and molecular interactions, understanding immune interactions, and identifying and evaluating inhibitors to advance vaccine and drug development.

James G Beeson - One of the best experts on this subject based on the ideXlab platform.

  • sequential processing of merozoite surface proteins during and after erythrocyte invasion by plasmodium falciparum
    Infection and Immunity, 2014
    Co-Authors: Michelle J Boyle, Anthony N Hodder, Robin F Anders, Christine Langer, Joanne Chan, Ross L Coppel, James G Beeson
    Abstract:

    Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when Merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of Merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium Merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.

  • new approaches to studying plasmodium falciparum merozoite invasion and insights into invasion biology
    International Journal for Parasitology, 2013
    Co-Authors: Michelle J Boyle, Danny W. Wilson, James G Beeson
    Abstract:

    Merozoite invasion of human red blood cells by Plasmodium falciparum is essential for blood stage asexual replication and the development of malaria disease. Despite this, many of the processes involved in invasion are poorly understood. Recent advances have been made in methods to isolate viable Merozoites for studies of invasion. The application of these approaches is providing new insights into the kinetics of invasion and merozoite survival, as well as proteins and interactions involved in invasion, and will facilitate the development and testing of anti-merozoite vaccines and the identification of invasion-inhibitory compounds with potential for drug development. This review discusses these recent advances and considers potential avenues for future research.

  • isolation of viable plasmodium falciparum Merozoites to define erythrocyte invasion events and advance vaccine and drug development
    American Journal of Tropical Medicine and Hygiene, 2010
    Co-Authors: Michelle J Boyle, Stuart A. Ralph, Danny W. Wilson, Jack S Richards, David T Riglar, Kevin K A Tetteh, David J Conway, Jake Baum, James G Beeson
    Abstract:

    During blood-stage infection by Plasmodium falciparum, Merozoites invade RBCs. Currently there is limited knowledge of cellular and molecular invasion events, and no established assays are available to readily measure and quantify invasion-inhibitory antibodies or compounds for vaccine and drug studies. We report the isolation of viable Merozoites that retain their invasive capacity, at high purity and yield, purified by filtration of highly synchronous populations of schizonts. We show that the half-life of merozoite invasive capacity after rupture is 5 min at 37 degrees C, and 15 min at room temperature. Studying the kinetics of invasion revealed that 80% of invasion events occur within 10 min of mixing Merozoites and RBCs. Invasion efficiency was maximum at low merozoite-to-RBC ratios and occurred efficiently in the absence of serum and with high concentrations of dialyzed nonimmune serum. We developed and optimized an invasion assay by using purified Merozoites that enabled invasion-inhibitory activity of antibodies and compounds to be measured separately from other mechanisms of growth inhibition; the assay was more sensitive for detecting inhibitory activity than established growth-inhibition assays. Furthermore, with the use of purified Merozoites it was possible to capture and fix Merozoites at different stages of invasion for visualization by immunofluorescence microscopy and EM. We thereby demonstrate that processing of the major merozoite antigen merozoite surface protein-1 occurs at the time of RBC invasion. These findings have important implications for defining invasion events and molecular interactions, understanding immune interactions, and identifying and evaluating inhibitors to advance vaccine and drug development.