ORCID Profile
0000-0002-5073-1405
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Animal Developmental and Reproductive Biology | Pollution and contamination | Animal protection (incl. pests and pathogens) | Biochemistry and Cell Biology | Crop and Pasture Biochemistry and Physiology | Veterinary bacteriology | Pollution and contamination not elsewhere classified | Biochemistry and Cell Biology not elsewhere classified |
Expanding Knowledge in the Agricultural and Veterinary Sciences | Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences
Publisher: Elsevier BV
Date: 03-2020
Publisher: Public Library of Science (PLoS)
Date: 15-11-2011
Publisher: Oxford University Press (OUP)
Date: 17-08-2019
Abstract: Complement-fixing antibodies targeting Plasmodium vivax merozoite surface protein 3α are prevalent in both children and adults with infection, with both immunoglobulin G and M mediating complement fixation. Magnitudes of complement-fixing antibodies are influenced by antigenic region.
Publisher: eLife Sciences Publications, Ltd
Date: 23-05-2023
DOI: 10.7554/ELIFE.88088
Abstract: Apicomplexan parasites exhibit tremendous ersity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the s le ∼4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the microtubule organizing center (MTOC) and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still iding, are concurrently segregated and maintain an association to the MTOC until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an MTOC association during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.
Publisher: Elsevier BV
Date: 03-2021
Publisher: Public Library of Science (PLoS)
Date: 26-12-2012
Publisher: Elsevier BV
Date: 09-2017
DOI: 10.1016/J.IJPARA.2017.05.004
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 h ered 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 in iduals. The number of merozoite antigens that elicit an immune response, and their structural and functional ersity, 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.
Publisher: Cold Spring Harbor Laboratory
Date: 31-01-2021
DOI: 10.1101/2021.01.26.21250284
Abstract: Microscopic examination of blood smears remains the gold standard for diagnosis and laboratory studies with malaria. Inspection of smears is, however, a tedious manual process dependent on trained microscopists with results varying in accuracy between in iduals, given the heterogeneity of parasite cell form and disagreement on nomenclature. To address this, we sought to develop an automated image analysis method that improves accuracy and standardisation of cytological smear inspection but retains the capacity for expert confirmation and archiving of images. Here we present a machine-learning method that achieves red blood cell (RBC) detection, differentiation between infected and uninfected RBCs and parasite life stage categorisation from raw, unprocessed heterogeneous images of thin blood films. The method uses a pre-trained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection that performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. A residual neural network (ResNet)-50 model applied to detect infection in segmented RBCs also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Lastly, using a regression model our method successfully recapitulates intra-erythrocytic developmental cycle (IDC) stages with accurate categorisation (ring, trophozoite, schizont), as well as differentiating asexual stages from gametocytes. To accelerate our method’s utility, we have developed a mobile-friendly web-based interface, PlasmoCount, which is capable of automated detection and staging of malaria parasites from uploaded heterogeneous input images of Giemsa-stained thin blood smears. Results gained using either laboratory or phone-based images permit rapid navigation through and review of results for quality assurance. By standardising the assessment of parasite development from microscopic blood smears, PlasmoCount markedly improves user consistency and reproducibility and thereby presents a realistic route to automating the gold standard of field-based malaria diagnosis. Microscopy inspection of Giemsa-stained thin blood smears on glass slides has been used in the diagnosis of malaria and monitoring of malaria cultures in laboratory settings for years. Manual evaluation is, however, time-consuming, error-prone and subjective with no currently available tool that permits reliable automated counting and archiving of Giemsa-stained images. Here, we present a machine learning method for automated detection and staging of parasite infected red cells from heterogeneous smears. Our method calculates parasitaemia and frequency data on the malaria parasite intraerythrocytic development cycle directly from raw images, standardizing smear assessment and providing reproducible and archivable results. Developed into a web tool, PlasmoCount, this method provides improved standardisation of smear inspection for malaria research and potentially field diagnosis.
Publisher: American Society for Microbiology
Date: 05-2005
DOI: 10.1128/IAI.73.5.2848-2856.2005
Abstract: Malaria in pregnancy is associated with placental accumulation of Plasmodium falciparum -infected erythrocytes (IE) that adhere to chondroitin sulfate A (CSA). Adhesion is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1), a variant parasite protein expressed on the surface of IE and encoded by var genes. Rabbit antiserum was generated against the CSA-adherent P. falciparum line CS2, in which the dominant var transcribed is var2csa , a relatively conserved var gene that has been associated with CSA adhesion. Anti-CS2 recognized genetically distinct CSA-adherent P. falciparum lines but not CD36-adherent parent lines. Reactivity with anti-CS2 correlated with the level of adhesion to CSA. Fluorescence-activated cell sorting according to binding of anti-CS2 showed reactivity was associated with CSA adhesion and transcription of var2csa . These data are consistent with the hypothesis that var2csa encodes a PfEMP1 expressed on the surface of IE, which mediates adhesion to CSA and is relatively conserved between genetically distinct strains of P. falciparum .
Publisher: American Society for Microbiology
Date: 08-2016
DOI: 10.1128/IAI.00145-16
Abstract: It is unclear whether naturally acquired immunity to Plasmodium falciparum results from the acquisition of antibodies to multiple, erse antigens or to fewer, highly conserved antigens. Moreover, the specific antibody functions required for malaria immunity are unknown, and hence informative immunological assays are urgently needed to address these knowledge gaps and guide vaccine development. In this study, we investigated whether merozoite-opsonizing antibodies are associated with protection from malaria in a strain-specific or strain-transcending manner by using a novel field isolate and an immune plasma-matched cohort from Papua New Guinea with our validated assay of merozoite phagocytosis. Highly correlated opsonization responses were observed across the 15 parasite strains tested, as were strong associations with protection (composite phagocytosis score across all strains in children uninfected at baseline: hazard ratio of 0.15, 95% confidence interval of 0.04 to 0.63). Opsonizing antibodies had a strong strain-transcending component, and the opsonization of transgenic parasites deficient for MSP3, MSP6, MSPDBL1, or P. falciparum MSP1-19 (PfMSP1-19) was similar to that of wild-type parasites. We have provided the first evidence that merozoite opsonization is predominantly strain transcending, and the highly consistent associations with protection against erse parasite strains strongly supports the use of merozoite opsonization as a correlate of immunity for field studies and vaccine trials. These results demonstrate that conserved domains within merozoite antigens targeted by opsonization generate strain-transcending immune responses and represent promising vaccine candidates.
Publisher: Wiley
Date: 15-03-2005
DOI: 10.1111/J.1365-2958.2005.04577.X
Abstract: Transcription of the majority of the members of the Plasmodium falciparum var multigene family were analysed in two isolates by a quantitative approach. Both of these isolates had been repeatedly selected for adhesion to chondroitin sulphate A (CSA) and one had also been selected for adhesion to hyaluronic acid (HA). These adhesion phenotypes are expressed by many parasites isolated from placentae and are associated with malaria disease in pregnancy. Increased transcription of the var gene var2csa, or its homologue IT4 var4, was associated with the CSA and HA adhesion phenotypes in all parasites suggesting that it was the dominant, if not the only, var gene that encoded adhesion to CSA in these allogeneic isolates. Some var genes were consistently transcribed at higher levels than others regardless of expressed adhesion phenotypes suggesting a transcriptional hierarchy. Unspliced or partial transcripts were detected for most var genes tested. These atypical var gene transcripts may have implications for the regulation of var gene transcription.
Publisher: Cambridge University Press (CUP)
Date: 2021
DOI: 10.1017/S2633903X21000015
Abstract: Microscopic examination of blood smears remains the gold standard for laboratory inspection and diagnosis of malaria. Smear inspection is, however, time-consuming and dependent on trained microscopists with results varying in accuracy. We sought to develop an automated image analysis method to improve accuracy and standardization of smear inspection that retains capacity for expert confirmation and image archiving. Here, we present a machine learning method that achieves red blood cell (RBC) detection, differentiation between infected/uninfected cells, and parasite life stage categorization from unprocessed, heterogeneous smear images. Based on a pretrained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection, our model performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. Application of a residual neural network-50 model to infected cells also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Finally, combining our method with a regression model successfully recapitulates intraerythrocytic developmental cycle with accurate lifecycle stage categorization. Combined with a mobile-friendly web-based interface, called PlasmoCount, our method permits rapid navigation through and review of results for quality assurance. By standardizing assessment of Giemsa smears, our method markedly improves inspection reproducibility and presents a realistic route to both routine lab and future field-based automated malaria diagnosis.
Publisher: Public Library of Science (PLoS)
Date: 20-09-2012
Publisher: Elsevier BV
Date: 08-2002
Publisher: American Society for Microbiology
Date: 27-08-2019
Abstract: Malaria claims about half a million lives each year. Plasmodium falciparum , the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for in idual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.
Publisher: Public Library of Science (PLoS)
Date: 05-12-2012
Publisher: Springer Science and Business Media LLC
Date: 05-02-2019
DOI: 10.1038/S41467-019-08528-Z
Abstract: Antibodies against P . falciparum merozoites fix complement to inhibit blood-stage replication in naturally-acquired and vaccine-induced immunity however, specific targets of these functional antibodies and their importance in protective immunity are unknown. Among malaria-exposed in iduals, we show that complement-fixing antibodies to merozoites are more strongly correlated with protective immunity than antibodies that inhibit growth quantified using the current reference assay for merozoite vaccine evaluation. We identify merozoite targets of complement-fixing antibodies and identify antigen-specific complement-fixing antibodies that are strongly associated with protection from malaria in a longitudinal study of children. Using statistical modelling, combining three different antigens targeted by complement-fixing antibodies could increase the potential protective effect to over 95%, and we identify antigens that were common in the most protective combinations. Our findings support antibody-complement interactions against merozoite antigens as important anti-malaria immune mechanisms, and identify specific merozoite antigens for further evaluation as vaccine candidates.
Publisher: Proceedings of the National Academy of Sciences
Date: 20-09-2010
Abstract: Plasmodium falciparum is responsible for the most severe form of malaria disease in humans, causing more than 1 million deaths each year. As an obligate intracellular parasite, P. falciparum ’s ability to invade erythrocytes is essential for its survival within the human host. P. falciparum invades erythrocytes using multiple host receptor–parasite ligand interactions known as invasion pathways. Here we show that CR1 is the host erythrocyte receptor for PfRh4, a major P. falciparum ligand essential for sialic acid–independent invasion. PfRh4 and CR1 interact directly, with a K d of 2.9 μM. PfRh4 binding is strongly correlated with the CR1 level on the erythrocyte surface. Parasite invasion via sialic acid–independent pathways is reduced in low-CR1 erythrocytes due to limited availability of this receptor on the surface. Furthermore, soluble CR1 can competitively block binding of PfRh4 to the erythrocyte surface and specifically inhibit sialic acid–independent parasite invasion. These results demonstrate that CR1 is an erythrocyte receptor used by the parasite ligand PfRh4 for P. falciparum invasion.
Publisher: American Society for Microbiology
Date: 11-2011
DOI: 10.1128/IAI.05412-11
Abstract: One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum . Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands athways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.
Publisher: Cold Spring Harbor Laboratory
Date: 24-12-2021
DOI: 10.1101/2021.12.21.473770
Abstract: Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum , an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures – the food vacuole, the apicoplast, and the parasite plasma membrane – and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1, PF3D7_1135300), a unique Plasmodium -specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium . Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have erse intracellular localizations and putative functions. Plasmodium falciparum -infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes do not only facilitate movement of nutrients, metabolites and other molecules between these compartments, but are common therapeutic targets and can also confer antimalarial drug resistance. Orphan membrane transporter in P. falciparum without sequence homology to transporters in other evolutionary lineages and ergent to host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed into their importance during asexual parasite growth using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.BMC.2018.12.022
Abstract: The 26S proteasome and calpain are linked to a number of important human diseases. Here, we report a series of analogues of the prototypical tripeptide aldehyde inhibitor MG132 that show a unique combination of high activity and selectivity for calpains over proteasome. Tripeptide aldehydes (1-3) with an aromatic P3 substituent show enhanced activity and selectivity against ovine calpain 2 relative to chymotrypsin-like activity of proteasome. Docking studies reveal the key contacts between inhibitors and calpain to confirm the importance of the S3 pocket with respect to selectivity between calpains 1 and 2 and the proteasome.
Publisher: Springer Science and Business Media LLC
Date: 03-06-2010
Abstract: Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development. The D10 P. falciparum line was transfected to express green fluorescent protein (GFP). In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum s les, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry. Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of s les when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody s les was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 s le dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts. Flow cytometry based assays using GFP-fluorescent parasites proved sensitive and highly reproducible for quantifying the growth-inhibitory activity of antibodies and anti-malarials, with superior reproducibility to light microscopy, and are suitable for high-throughput applications.
Publisher: Public Library of Science (PLoS)
Date: 27-07-2012
Publisher: Springer Science and Business Media LLC
Date: 09-02-2022
DOI: 10.1038/S42003-022-03020-9
Abstract: Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum . Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1 . We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade and was associated with elongation of the rhoptry organelle during merozoite development and inhibition of rhoptry antigen processing. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.
Publisher: Springer Science and Business Media LLC
Date: 05-07-2018
DOI: 10.1038/S41598-018-28457-Z
Abstract: Plasmodium knowlesi , a zoonotic parasite causing severe-to-lethal malaria disease in humans, has only recently been adapted to continuous culture with human red blood cells (RBCs). In comparison with the most virulent human malaria, Plasmodium falciparum , there are, however, few cellular tools available to study its biology, in particular direct investigation of RBC invasion by blood-stage P . knowlesi merozoites. This leaves our current understanding of biological differences across pathogenic Plasmodium spp. incomplete. Here, we report a robust method for isolating viable and invasive P . knowlesi merozoites to high purity and yield. Using this approach, we present detailed comparative dissection of merozoite invasion (using a variety of microscopy platforms) and direct assessment of kinetic differences between knowlesi and falciparum merozoites. We go on to assess the inhibitory potential of molecules targeting discrete steps of invasion in either species via a quantitative invasion inhibition assay, identifying a class of polysulfonate polymer able to efficiently inhibit invasion in both, providing a foundation for pan- Plasmodium merozoite inhibitor development. Given the close evolutionary relationship between P . knowlesi and P . vivax , the second leading cause of malaria-related morbidity, this study paves the way for inter-specific dissection of invasion by all three major pathogenic malaria species.
Publisher: Cold Spring Harbor Laboratory
Date: 24-03-2023
DOI: 10.1101/2023.03.22.533773
Abstract: Apicomplexan parasites exhibit tremendous ersity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the s le ∼4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the microtubule organizing center (MTOC) and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still iding, are concurrently segregated and maintain an association to the MTOC until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an MTOC association during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.
Publisher: Oxford University Press (OUP)
Date: 11-02-2019
Abstract: Plasmodium spp. parasites that cause malaria disease remain a significant global-health burden. With the spread of parasites resistant to artemisinin combination therapies in Southeast Asia, there is a growing need to develop new antimalarials with novel targets. Invasion of the red blood cell by Plasmodium merozoites is essential for parasite survival and proliferation, thus representing an attractive target for therapeutic development. Red blood cell invasion requires a co-ordinated series of protein rotein interactions, protease cleavage events, intracellular signals, organelle release and engagement of an actin-myosin motor, which provide many potential targets for drug development. As these steps occur in the bloodstream, they are directly susceptible and exposed to drugs. A number of invasion inhibitors against a erse range of parasite proteins involved in these different processes of invasion have been identified, with several showing potential to be optimised for improved drug-like properties. In this review, we discuss red blood cell invasion as a drug target and highlight a number of approaches for developing antimalarials with invasion inhibitory activity to use in future combination therapies.
Publisher: Springer Science and Business Media LLC
Date: 18-07-2015
Publisher: American Society for Microbiology
Date: 05-2005
DOI: 10.1128/AAC.49.5.2102-2105.2005
Abstract: The pfcrt allelic type and adjacent microsatellite marker type were determined for 82 Plasmodium falciparum isolates from the Philippines. Mutant pfcrt allelic types P1a and P2a/P2b were dominant in different locations. Microsatellite analysis revealed that P2a/P2b evolved independently in the Philippines, while P1a shared common ancestry with Papua New Guinea chloroquine-resistant parasites.
Publisher: Public Library of Science (PLoS)
Date: 25-05-2010
Publisher: Springer Science and Business Media LLC
Date: 23-09-2016
Publisher: Public Library of Science (PLoS)
Date: 29-10-2008
Publisher: Springer Science and Business Media LLC
Date: 29-09-2020
DOI: 10.1186/S12915-020-00859-4
Abstract: Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium spp. malaria parasites is urgently needed. Azithromycin is a clinically used macrolide antibiotic proposed as a partner drug for combination therapy in malaria, which has also been tested as monotherapy. However, its slow-killing ‘delayed-death’ activity against the parasite’s apicoplast organelle and suboptimal activity as monotherapy limit its application as a potential malaria treatment. Here, we explore a panel of azithromycin analogues and demonstrate that chemical modifications can be used to greatly improve the speed and potency of antimalarial action. Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin with less than 48 hrs treatment in vitro. Analogues were effective against zoonotic Plasmodium knowlesi malaria parasites and against both multi-drug and artemisinin-resistant Plasmodium falciparum lines. Metabolomic profiles of azithromycin analogue-treated parasites suggested activity in the parasite food vacuole and mitochondria were disrupted. Moreover, unlike the food vacuole-targeting drug chloroquine, azithromycin and analogues were active across blood-stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on ‘quick-killing’ activity. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. We show that azithromycin and analogues can rapidly kill malaria parasite asexual blood stages via a fast action mechanism. Development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed-death mechanism of action in a single, multifactorial chemotype.
Publisher: Elsevier BV
Date: 12-2001
Publisher: eLife Sciences Publications, Ltd
Date: 23-05-2023
Abstract: Apicomplexan parasites exhibit tremendous ersity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the s le ∼4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the microtubule organizing center (MTOC) and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still iding, are concurrently segregated and maintain an association to the MTOC until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an MTOC association during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.
Publisher: Elsevier BV
Date: 02-2020
DOI: 10.1016/J.JMB.2019.12.024
Abstract: Apicomplexan parasites contain rhoptries, which are specialized secretory organelles that coordinate host cell invasion. During the process of invasion, rhoptries secrete their contents to facilitate interaction with, and entry into, the host cell. Here, we report the crystal structure of the rhoptry protein Armadillo Repeats-Only (ARO) from the human malaria parasite, Plasmodium falciparum (PfARO). The structure of PfARO comprises five tandem Armadillo-like (ARM) repeats, with adjacent ARM repeats stacked in a head-to-tail orientation resulting in PfARO adopting an elongated curved shape. Interestingly, the concave face of PfARO contains two distinct patches of highly conserved residues that appear to play an important role in protein-protein interaction. We functionally characterized the P. falciparum homolog of ARO interacting protein (PfAIP) and demonstrate that it localizes to the rhoptries. We show that conditional mislocalization of PfAIP leads to deficient red blood cell invasion. Guided by the structure, we identified mutations of PfARO that lead to mislocalization of PfAIP. Using proximity-based biotinylation we probe into PfAIP interacting proteins.
Publisher: Springer Science and Business Media LLC
Date: 11-08-2020
DOI: 10.1038/S41467-020-17829-7
Abstract: Intracellular pathogens mobilize host signaling pathways of their host cell to promote their own survival. Evidence is emerging that signal transduction elements are activated in a-nucleated erythrocytes in response to infection with malaria parasites, but the extent of this phenomenon remains unknown. Here, we fill this knowledge gap through a comprehensive and dynamic assessment of host erythrocyte signaling during infection with Plasmodium falciparum . We used arrays of 878 antibodies directed against human signaling proteins to interrogate the activation status of host erythrocyte phospho-signaling pathways at three blood stages of parasite asexual development. This analysis reveals a dynamic modulation of many host signalling proteins across parasite development. Here we focus on the hepatocyte growth factor receptor (c-MET) and the MAP kinase pathway component B-Raf, providing a proof of concept that human signaling kinases identified as activated by malaria infection represent attractive targets for antimalarial intervention.
Publisher: American Society for Microbiology
Date: 03-2013
DOI: 10.1128/AAC.01881-12
Abstract: Most current antimalarials for treatment of clinical Plasmodium falciparum malaria fall into two broad drug families and target the food vacuole of the trophozoite stage. No antimalarials have been shown to target the brief extracellular merozoite form of blood-stage malaria. We studied a panel of 12 drugs, 10 of which have been used extensively clinically, for their invasion, schizont rupture, and growth-inhibitory activity using high-throughput flow cytometry and new approaches for the study of merozoite invasion and early intraerythrocytic development. Not surprisingly, given reported mechanisms of action, none of the drugs inhibited merozoite invasion in vitro . Pretreatment of erythrocytes with drugs suggested that halofantrine, lumefantrine, piperaquine, amodiaquine, and mefloquine diffuse into and remain within the erythrocyte and inhibit downstream growth of parasites. Studying the inhibitory activity of the drugs on intraerythrocytic development, schizont rupture, and reinvasion enabled several different inhibitory phenotypes to be defined. All drugs inhibited parasite replication when added at ring stages, but only artesunate, artemisinin, cycloheximide, and trichostatin A appeared to have substantial activity against ring stages, whereas the other drugs acted later during intraerythrocytic development. When drugs were added to late schizonts, only artemisinin, cycloheximide, and trichostatin A were able to inhibit rupture and subsequent replication. Flow cytometry proved valuable for in vitro assays of antimalarial activity, with the free merozoite population acting as a clear marker for parasite growth inhibition. These studies have important implications for further understanding the mechanisms of action of antimalarials, studying and evaluating drug resistance, and developing new antimalarials.
Publisher: Elsevier BV
Date: 12-2003
DOI: 10.1046/J.1523-1747.2003.12621.X
Abstract: Parasitic infestation of the skin by the mite Sarcoptes scabiei is a significant problem worldwide, particularly in socially disadvantaged communities. A multigene family of at least 24 homologs of a serine protease allergen have been identified in S. scabiei. Surprisingly, the products of all but one of these genes are predicted to be catalytically inactive, due to mutations at a critical triad of amino acids at the active site. We discuss the possibility that these genes for inactivated proteases have been conserved because they mediate a novel host defense evasion strategy that the mite has evolved as an adaptation to parasitism of the epidermis. The identification of this family, and elucidation of its value to the parasite, may present an unanticipated approach to protective vaccination.
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.IJPARA.2012.11.002
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.
Publisher: Wiley
Date: 23-08-2012
DOI: 10.1111/J.1600-0854.2012.01402.X
Abstract: The establishment of parasite infection within the human erythrocyte is an essential stage in the development of malaria disease. As such, significant interest has focused on the mechanics that underpin invasion and on characterization of parasite molecules involved. Previous evidence has implicated a presenilin-like signal peptide peptidase (SPP) from the most virulent human malaria parasite, Plasmodium falciparum, in the process of invasion where it has been proposed to function in the cleavage of the erythrocyte cytoskeletal protein Band 3. The role of a traditionally endoplasmic reticulum (ER) protease in the process of red blood cell invasion is unexpected. Here, using a combination of molecular, cellular and chemical approaches we provide evidence that PfSPP is, instead, a bona fide ER-resident peptidase that remains intracellular throughout the invasion process. Furthermore, SPP-specific drug inhibition has no effect on erythrocyte invasion whilst having low micromolar potency against intra-erythrocytic development. Contrary to previous reports, these results show that PfSPP plays no role in erythrocyte invasion. Nonetheless, PfSPP clearly represents a potential chemotherapeutic target to block parasite growth, supporting ongoing efforts to develop antimalarial-targeting protein maturation and trafficking during intra-erythrocytic development.
Publisher: American Society for Microbiology
Date: 06-2009
DOI: 10.1128/IAI.00048-09
Abstract: Plasmodium falciparum invasion into human erythrocytes relies on the interaction between multiple parasite ligands and their respective erythrocyte receptors. The sialic acid-independent invasion pathway is dependent on the expression of P. falciparum reticulocyte binding protein-like homologue 4 (PfRh4), as disruption of the gene abolishes the ability of parasites to switch to this pathway. We show that PfRh4 is present as an invasion ligand in culture supernatants as a 160-kDa proteolytic fragment. We confirm that PfRh4 binds to the surfaces of erythrocytes through recognition of an erythrocyte receptor that is neuraminidase resistant but trypsin and chymotrypsin sensitive. Serum antibodies from malaria-exposed in iduals show reactivity against the binding domain of PfRh4. Purified immunoglobulin G raised in rabbits against the binding domain of PfRh4 blocked the binding of native PfRh4 to the surfaces of erythrocytes and inhibited erythrocyte invasion of parasites using sialic acid-independent invasion pathways and grown in neuraminidase-treated erythrocytes. Our results suggest PfRh4 is a potential vaccine candidate.
Publisher: The American Association of Immunologists
Date: 15-07-2013
Abstract: Abs that inhibit Plasmodium falciparum invasion of erythrocytes form an important component of human immunity against malaria, but key target Ags are largely unknown. Phenotypic variation by P. falciparum mediates the evasion of inhibitory Abs, contributing to the capacity of P. falciparum to cause repeat and chronic infections. However, Ags involved in mediating immune evasion have not been defined, and studies of the function of human Abs are limited. In this study, we used novel approaches to determine the importance of P. falciparum erythrocyte-binding Ags (EBAs), which are important invasion ligands, as targets of human invasion-inhibitory Abs and define their role in contributing to immune evasion through variation in function. We evaluated the invasion-inhibitory activity of acquired Abs from malaria-exposed children and adults from Kenya, using P. falciparum with disruption of genes encoding EBA140, EBA175, and EBA181, either in idually or combined as EBA140/EBA175 or EBA175/EBA181 double knockouts. Our findings provide important new evidence that variation in the expression and function of the EBAs plays an important role in evasion of acquired Abs and that a substantial amount of phenotypic ersity results from variation in expression of different EBAs that contributes to immune evasion by P. falciparum. All three EBAs were identified as important targets of naturally acquired inhibitory Abs demonstrated by differential inhibition of parental parasites greater than EBA knockout lines. This knowledge will help to advance malaria vaccine development and suggests that multiple invasion ligands need to be targeted to overcome the capacity of P. falciparum for immune evasion.
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1016/J.EJMECH.2017.10.010
Abstract: In this study, a structure-activity relationship (SAR) compound series based on the NDH-2 inhibitor diphenyleneiodonium (DPI) was synthesised. Compounds were evaluated primarily for in vitro efficacy against Gram-positive and Gram-negative bacteria, commonly responsible for nosocomial and community acquired infections. In addition, we also assessed the activity of these compounds against Mycobacterium tuberculosis (Tuberculosis) and Plasmodium spp. (Malaria). This led to the discovery of highly potent compounds active against bacterial pathogens and malaria parasites in the low nanomolar range, several of which were significantly less toxic to mammalian cells.
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.CHOM.2016.06.004
Abstract: Plasmodium falciparum parasites in the merozoite stage invade human erythrocytes and cause malaria. Invasion requires multiple interactions between merozoite ligands and erythrocyte receptors. P. falciparum reticulocyte binding homolog 5 (PfRh5) forms a complex with the PfRh5-interacting protein (PfRipr) and Cysteine-rich protective antigen (CyRPA) and binds erythrocytes via the host receptor basigin. However, the specific role that PfRipr and CyRPA play during invasion is unclear. Using P. falciparum lines conditionally expressing PfRipr and CyRPA, we show that loss of PfRipr or CyRPA function blocks growth due to the inability of merozoites to invade erythrocytes. Super-resolution microscopy revealed that PfRipr, CyRPA, and PfRh5 colocalize at the junction between merozoites and erythrocytes during invasion. PfRipr, CyRPA, and PfRipr/CyRPA/PfRh5-basigin complex is required for triggering the Ca(2+) release and establishing the tight junction. Together, these results establish that the PfRh5/PfRipr/CyRPA complex is essential in the sequential molecular events leading to parasite invasion of human erythrocytes.
Publisher: Elsevier BV
Date: 07-2011
DOI: 10.1016/J.IMBIO.2010.12.010
Abstract: This study aimed to evaluate whether intermittent preventive treatment in infants with sulfadoxine-pyrimethamine (IPTi-SP) had an effect on the acquisition of IgG against Plasmodium falciparum variant surface antigens (VSA) and growth-inhibitory antibodies in Manhiça, Mozambique. In addition, we assessed factors affecting the magnitude of these responses and the association between antibody levels and protection against malaria. IgG to VSA expressed by MOZ2, R29 and E8B parasite isolates were measured in plasma s les collected at 5, 9, 12 and 24 months of age by flow cytometry. Growth-inhibitory antibodies in dialyzed plasmas using GFP-D10 parasites were measured by flow cytometry at 12 and 24 months. IPTi-SP did not significantly modify the levels of IgG against VSA nor the growth-inhibitory capacity of antibodies up to 2 years of age. Age but not previous episodes of malaria influenced the magnitude of these responses. In addition, anti-VSA IgG levels were 7% higher in children with current P. falciparum infection and were associated with neighborhood of residence. Children aged 24 months had 10% less parasite growth than those aged 12 months (95% CI 0.88-0.93, P<0.0001). Growth-inhibitory antibodies correlated with levels of IgG against AMA-1, when evaluating the 10% (R(2)=0.444, P=0.049) and 20% (R(2)=0.230, P=0.037) highest inhibitory s les. None of the responses were associated with subsequent risk of malaria. In conclusion, IPTi-SP does not negatively affect the development of antibody responses thought to be major contributors to the acquisition of immunity to malaria in infancy.
Publisher: American Society for Microbiology
Date: 03-2011
DOI: 10.1128/IAI.01021-10
Abstract: Plasmodium falciparum causes the most severe form of malaria in humans and invades erythrocytes using multiple ligand-receptor interactions. Two important protein families involved in erythrocyte binding are the erythrocyte binding-like (EBL) and the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) proteins. We constructed P. falciparum lines lacking expression of EBL proteins by creating single and double knockouts of the corresponding genes for eba-175 , eba-181 , and eba-140 and show that the EBL and PfRh proteins function cooperatively, consistent with them playing a similar role in merozoite invasion. We provide evidence that PfRh and EBL proteins functionally interact, as loss of function of EBA-181 ablates the ability of PfRh2a/b protein antibodies to inhibit merozoite invasion. Additionally, loss of function of some ebl genes results in selection for increased transcription of the PfRh family. This provides a rational basis for considering PfRh and EBL proteins for use as a combination vaccine against P. falciparum . We immunized rabbits with combinations of PfRh and EBL proteins to test the ability of antibodies to block merozoite invasion in growth inhibition assays. A combination of EBA-175, PfRh2a/b, and PfRh4 recombinant proteins induced antibodies that potently blocked merozoite invasion. This validates the use of a combination of these ligands as a potential vaccine that would have broad activity against P. falciparum .
Publisher: Cold Spring Harbor Laboratory
Date: 27-11-2020
DOI: 10.1101/2020.11.26.400549
Abstract: Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum . Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1 , a related cytosolically exposed rhoptry bulb protein. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to an inhibition of merozoite invasion after tight junction formation. PfCERLI2 knockdown was associated with inhibition of rhoptry antigen processing and a significant elongation of the rhoptries, suggesting that the inability of merozoites to invade is caused by aberrant rhoptry function due to PfCERLI2 deficiency. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.
Publisher: Cold Spring Harbor Laboratory
Date: 24-05-2019
DOI: 10.1101/646299
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 C ytosolically E xposed R hoptry L eaflet I nteracting 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.
Publisher: Elsevier BV
Date: 05-2013
DOI: 10.1016/J.CELL.2013.04.029
Abstract: 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.
Publisher: Springer Science and Business Media LLC
Date: 16-03-2020
DOI: 10.1038/S41467-020-15127-W
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.
No related organisations have been discovered for Danny Wilson.
Start Date: 07-2023
End Date: 07-2028
Amount: $4,508,426.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2021
End Date: 06-2022
Amount: $909,079.00
Funder: Australian Research Council
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