Divergent roles for the RH5 complex components, CyRPA and RIPR in human-infective malaria parasites.

Malaria is caused by Plasmodium parasites, which invade and replicate in erythrocytes. For Plasmodium falciparum, the major cause of severe malaria in humans, a heterotrimeric complex comprised of the secreted parasite proteins, PfCyRPA, PfRIPR and PfRH5 is essential for erythrocyte invasion, mediated by the interaction between PfRH5 and erythrocyte receptor basigin (BSG). However, whilst CyRPA and RIPR are present in most Plasmodium species, RH5 is found only in the small Laverania subgenus. Existence of a complex analogous to PfRH5-PfCyRPA-PfRIPR targeting BSG, and involvement of CyRPA and RIPR in invasion, however, has not been addressed in non-Laverania parasites. Here, we establish that unlike P. falciparum, P. knowlesi and P. vivax do not universally require BSG as a host cell invasion receptor. Although we show that both PkCyRPA and PkRIPR are essential for successful invasion of erythrocytes by P. knowlesi parasites in vitro, neither protein forms a complex with each other or with an RH5-like molecule. Instead, PkRIPR is part of a different trimeric protein complex whereas PkCyRPA appears to function without other parasite binding partners. It therefore appears that in the absence of RH5, outside of the Laverania subgenus, RIPR and CyRPA have different, independent functions crucial for parasite survival.

Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi.

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

Real-time imaging of the intracellular glutathione redox potential in the malaria parasite Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (EGSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects EGSH changes induced by oxidative and nitrosative stress. The cytosolic basal EGSH of 3D7 and Dd2 were estimated to be -314.2±3.1 mV and -313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in EGSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' EGSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular EGSH in P. falciparum. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in Plasmodium and might also stimulate redox research in other pathogens.

Structural basis for DARC binding in reticulocyte invasion by Plasmodium vivax.

The symptoms of malaria occur during the blood stage of infection, when the parasite replicates within human red blood cells. The human malaria parasite, Plasmodium vivax, selectively invades reticulocytes in a process which requires an interaction between the ectodomain of the human DARC receptor and the Plasmodium vivax Duffy-binding protein, PvDBP. Previous studies have revealed that a small helical peptide from DARC binds to region II of PvDBP (PvDBP-RII). However, it is also known that sulphation of tyrosine residues on DARC affects its binding to PvDBP and these residues were not observed in previous structures. We therefore present the structure of PvDBP-RII bound to sulphated DARC peptide, showing that a sulphate on tyrosine 41 binds to a charged pocket on PvDBP-RII. We use molecular dynamics simulations, affinity measurements and growth-inhibition experiments in parasites to confirm the importance of this interaction. We also reveal the epitope for vaccine-elicited growth-inhibitory antibody DB1. This provides a complete understanding of the binding of PvDBP-RII to DARC and will guide the design of vaccines and therapeutics to target this essential interaction.

Sequential roles for red blood cell binding proteins enable phased commitment to invasion for malaria parasites.

Invasion of red blood cells (RBCs) by Plasmodium merozoites is critical to their continued survival within the host. Two major protein families, the Duffy binding-like proteins (DBPs/EBAs) and the reticulocyte binding like proteins (RBLs/RHs) have been studied extensively in P. falciparum and are hypothesized to have overlapping, but critical roles just prior to host cell entry. The zoonotic malaria parasite, P. knowlesi, has larger invasive merozoites and contains a smaller, less redundant, DBP and RBL repertoire than P. falciparum. One DBP (DBPα) and one RBL, normocyte binding protein Xa (NBPXa) are essential for invasion of human RBCs. Taking advantage of the unique biological features of P. knowlesi and iterative CRISPR-Cas9 genome editing, we determine the precise order of key invasion milestones and demonstrate distinct roles for each family. These distinct roles support a mechanism for phased commitment to invasion and can be targeted synergistically with invasion inhibitory antibodies.

Merozoite surface protein 1 paralog is involved in the human erythrocyte invasion of a zoonotic malaria, Plasmodium knowlesi.

The zoonotic malaria parasite Plasmodium knowlesi is an important public health concern in Southeast Asia. Invasion of host erythrocytes is essential for parasite growth, and thus, understanding the repertoire of parasite proteins that enable this process is vital for identifying vaccine candidates and how some species are able to cause zoonotic infection. Merozoite surface protein 1 (MSP1) is found in all malaria parasite species and is perhaps the most well-studied as a potential vaccine candidate. While MSP1 is encoded by a single gene in P. falciparum, all other human infective species (P. vivax, P. knowlesi, P. ovale, and P. malariae) additionally encode a divergent paralogue known as MSP1P, and little is known about its role or potential functional redundancy with MSP1. We, therefore, studied the function of P. knowlesi merozoite surface protein 1 paralog (PkMSP1P), using both recombinant protein and CRISPR-Cas9 genome editing. The recombinant 19-kDa C-terminus of PkMSP1P (PkMSP1P-19) was shown to bind specifically to human reticulocytes. However, immunoblotting data suggested that PkMSP1P-19-induced antibodies can recognize PkMSP1-19 and vice versa, confounding our ability to separate the properties of these two proteins. Targeted disruption of the pkmsp1p gene profoundly impacts parasite growth, demonstrating for the first time that PkMSP1P is important in in vitro growth of P. knowlesi and likely plays a distinct role from PkMSP1. Importantly, the MSP1P KO also enabled functional characterization of the PkMSP1P-19 antibodies, revealing clear immune cross-reactivity between the two paralogues, highlighting the vital importance of genetic studies in contextualizing recombinant protein studies.

Vaccination with Plasmodium vivax Duffy-binding protein inhibits parasite growth during controlled human malaria infection.

There are no licensed vaccines against Plasmodium vivax. We conducted two phase 1/2a clinical trials to assess two vaccines targeting P. vivax Duffy-binding protein region II (PvDBPII). Recombinant viral vaccines using chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) vectors as well as a protein and adjuvant formulation (PvDBPII/Matrix-M) were tested in both a standard and a delayed dosing regimen. Volunteers underwent controlled human malaria infection (CHMI) after their last vaccination, alongside unvaccinated controls. Efficacy was assessed by comparisons of parasite multiplication rates in the blood. PvDBPII/Matrix-M, given in a delayed dosing regimen, elicited the highest antibody responses and reduced the mean parasite multiplication rate after CHMI by 51% (n = 6) compared with unvaccinated controls (n = 13), whereas no other vaccine or regimen affected parasite growth. Both viral-vectored and protein vaccines were well tolerated and elicited expected, short-lived adverse events. Together, these results support further clinical evaluation of the PvDBPII/Matrix-M P. vivax vaccine.

Cation ATPase (ATP4) Orthologue Replacement in the Malaria Parasite Plasmodium knowlesi Reveals Species-Specific Responses to ATP4-Targeting Drugs.

Several unrelated classes of antimalarial compounds developed against Plasmodium falciparum target a parasite-specific P-type ATP-dependent Na+ pump, PfATP4. We have previously shown that other malaria parasite species infecting humans are less susceptible to these compounds. Here, we generated a series of transgenic Plasmodium knowlesi orthologue replacement (OR) lines in which the endogenous pkatp4 locus was replaced by a recodonized P. knowlesi atp4 (pkatp4) coding region or the orthologous coding region from P. falciparum, Plasmodium malariae, Plasmodium ovale subsp. curtisi, or Plasmodium vivax. Each OR transgenic line displayed a similar growth pattern to the parental P. knowlesi line. We found significant orthologue-specific differences in parasite susceptibility to three chemically unrelated ATP4 inhibitors, but not to comparator drugs, among the P. knowlesi OR lines. The PfATP4OR transgenic line of P. knowlesi was significantly more susceptible than our control PkATP4OR line to three ATP4 inhibitors: cipargamin, PA21A092, and SJ733. The PvATP4OR and PmATP4OR lines were similarly susceptible to the control PkATP4OR line, but the PocATP4OR line was significantly less susceptible to all ATP4 inhibitors than the PkATP4OR line. Cipargamin-induced inhibition of Na+ efflux was also significantly greater with the P. falciparum orthologue of ATP4. This confirms that species-specific susceptibility differences previously observed in ex vivo studies of human isolates are partly or wholly enshrined in the primary amino acid sequences of the respective ATP4 orthologues and highlights the need to monitor efficacy of investigational malaria drugs against multiple species. P. knowlesi is now established as an important in vitro model for studying drug susceptibility in non-falciparum malaria parasites. IMPORTANCE Effective drugs are vital to minimize the illness and death caused by malaria. Development of new drugs becomes ever more urgent as drug resistance emerges. Among promising compounds now being developed to treat malaria are several unrelated molecules that each inhibit the same protein in the malaria parasite-ATP4. Here, we exploited the genetic tractability of P. knowlesi to replace its own ATP4 genes with orthologues from five human-infective species to understand the drug susceptibility differences among these parasites. We previously estimated the susceptibility to ATP4-targeting drugs of each species using clinical samples from malaria patients. These estimates closely matched those of the corresponding "hybrid" P. knowlesi parasites carrying introduced ATP4 genes. Thus, species-specific ATP4 inhibitor efficacy is directly determined by the sequence of the gene. Our novel approach to understanding cross-species susceptibility/resistance can strongly support the effort to develop antimalarials that effectively target all human malaria parasite species.

Impact of a blood-stage vaccine on Plasmodium vivax malaria.

Background: There are no licensed vaccines against Plasmodium vivax , the most common cause of malaria outside of Africa. Methods: We conducted two Phase I/IIa clinical trials to assess the safety, immunogenicity and efficacy of two vaccines targeting region II of P. vivax Duffy-binding protein (PvDBPII). Recombinant viral vaccines (using ChAd63 and MVA vectors) were administered at 0, 2 months or in a delayed dosing regimen (0, 17, 19 months), whilst a protein/adjuvant formulation (PvDBPII/Matrix-M™) was administered monthly (0, 1, 2 months) or in a delayed dosing regimen (0, 1, 14 months). Delayed regimens were due to trial halts during the COVID-19 pandemic. Volunteers underwent heterologous controlled human malaria infection (CHMI) with blood-stage P. vivax parasites at 2-4 weeks following their last vaccination, alongside unvaccinated controls. Efficacy was assessed by comparison of parasite multiplication rate (PMR) in blood post-CHMI, modelled from parasitemia measured by quantitative polymerase-chain-reaction (qPCR). Results: Thirty-two volunteers were enrolled and vaccinated (n=16 for each vaccine). No safety concerns were identified. PvDBPII/Matrix-M™, given in the delayed dosing regimen, elicited the highest antibody responses and reduced the mean PMR following CHMI by 51% (range 36-66%; n=6) compared to unvaccinated controls (n=13). No other vaccine or regimen impacted parasite growth. In vivo growth inhibition of blood-stage P. vivax correlated with functional antibody readouts of vaccine immunogenicity. Conclusions: Vaccination of malaria-naïve adults with a delayed booster regimen of PvDBPII/ Matrix-M™ significantly reduces the growth of blood-stage P. vivax . Funded by the European Commission and Wellcome Trust; VAC069, VAC071 and VAC079 ClinicalTrials.gov numbers NCT03797989 , NCT04009096 and NCT04201431 .

CRISPR-Cas9 Genome Editing of Plasmodium knowlesi.

Plasmodium knowlesi is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After P. falciparum, P. knowlesi is the only other malaria parasite capable of being maintained in long term in vitro culture with human red blood cells (RBCs). Its closer ancestry to other non-falciparum human malaria parasites, more balanced AT-content, larger merozoites and higher transfection efficiencies, gives P. knowlesi some key advantages over P. falciparum for the study of malaria parasite cell/molecular biology. Here, we describe the generation of marker-free CRISPR gene-edited P. knowlesi parasites, the fast and scalable production of transfection constructs and analysis of transfection efficiencies. Our protocol allows rapid, reliable and unlimited rounds of genome editing in P. knowlesi requiring only a single recyclable selection marker.

Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi.

Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition assays against these two species. This protocol gives detailed step-by-step procedures to carry out flow cytometry-based multiplication assays and growth inhibition activity assays to test neutralizing antibodies based on the activity of the parasite enzyme lactate dehydrogenase of Plasmodium knowlesi adapted to human red blood cell culture. Whilst similar assays are well established for P. falciparum, P. knowlesi is more closely related to all other human infective species ( Pacheco et al., 2018 ) and so can be used as a surrogate for testing drugs and vaccines for other malaria species such as P. vivax, which is the most widespread cause of malaria outside of Africa, but cannot yet be cultured under laboratory conditions.

The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin µ Subunit is Clathrin Independent and Essential for Schizont Maturation.

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the µ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2µ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2µ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2µ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2µ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the µ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research.

Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody.

The most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells through a process requiring interaction of the P. vivax Duffy binding protein (PvDBP) with its human receptor, the Duffy antigen receptor for chemokines. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to P. vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunized in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to the Duffy antigen receptor for chemokines, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of P. vivax. This identified a broadly neutralizing human monoclonal antibody that inhibited invasion of all tested strains of P. vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.

Stable Integration and Comparison of hGrx1-roGFP2 and sfroGFP2 Redox Probes in the Malaria Parasite Plasmodium falciparum.

Studying redox metabolism in malaria parasites is of great interest for understanding parasite biology, parasite-host interactions, and mechanisms of drug action. Genetically encoded fluorescent redox sensors have recently been described as powerful tools for determining the glutathione-dependent redox potential in living parasites. In the present study, we genomically integrated and expressed the ratiometric redox sensors hGrx1-roGFP2 (human glutaredoxin 1 fused to reduction-oxidation sensitive green fluorescent protein) and sfroGFP2 (superfolder roGFP2) in the cytosol of NF54- attB blood-stage Plasmodium falciparum parasites. Both sensors were evaluated in vitro and in cell culture with regard to their fluorescence properties and reactivity. As genomic integration allows for the stable expression of redox sensors in parasites, we systematically compared single live-cell imaging with plate reader detection. For these comparisons, short-term effects of redox-active compounds were analyzed along with mid- and long-term effects of selected antimalarial agents. Of note, the single components of the redox probes themselves did not influence the redox balance of the parasites. Our analyses revealed comparable results for both the hGrx1-roGFP2 and sfroGFP2 probes, with sfroGFP2 exhibiting a more pronounced fluorescence intensity in cellulo. Accordingly, the sfroGFP2 probe was employed to monitor the fluorescence signals throughout the parasites' asexual life cycle. Through the use of stable genomic integration, we demonstrate a means of overcoming the limitations of transient transfection, allowing more detailed in-cell studies as well as high-throughput analyses using plate reader-based approaches.

Comparative Heterochromatin Profiling Reveals Conserved and Unique Epigenome Signatures Linked to Adaptation and Development of Malaria Parasites.

Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium.

Cellular dissection of malaria parasite invasion of human erythrocytes using viable Plasmodium knowlesi merozoites.

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.

A dual colour fluorescence in situ hybridization (FISH) assay for identifying the zoonotic malaria parasite Plasmodium knowlesi with a potential application for the specific diagnosis of knowlesi malaria in peripheral-level laboratories of Southeast Asia.

BACKGROUND: Plasmodium knowlesi is primarily responsible for zoonotic malaria in several Southeast Asian countries. Precise identification of the parasite in the blood of patients presently relies on an expensive and elaborate PCR procedure because microscopic examination of blood and other available field identification techniques lack adequate specificity. Therefore, the use of a simple and inexpensive dual-colour fluorescence in situ hybridization (FISH) assay, analogous to FISH assays recently described for Plasmodium falciparum and Plasmodium vivax, was investigated as a potential tool for identifying P. knowlesi. RESULTS: A P. knowlesi 18S rDNA sequence-based DNA probe was used to test thin blood smears of P. knowlesi by FISH, and fluorescence viewed in a light microscope fitted with a light emitting diode light source and appropriate emission and barrier filters. The limit of detection in the P. knowlesi FISH assay was 84 parasites per µl in infected monkey blood and 61 parasites per µl for P. knowlesi cultured in human blood. The P. knowlesi-specific FISH probe detected only P. knowlesi and not P. falciparum, Plasmodium malariae, Plasmodium ovale, P. vivax or a panel of other human blood-borne pathogens. A previously described Plasmodium genus-specific probe used simultaneously in the P. knowlesi FISH assay reacted with all tested Plasmodium species. CONCLUSIONS: To our knowledge, this is the first description of a FISH assay for P. knowlesi that is potentially useful for diagnosing infections in remote laboratories in endemic countries.

Determination of glutathione redox potential and pH value in subcellular compartments of malaria parasites.

The malaria parasite Plasmodium falciparum is exposed to multiple sources of oxidative challenge during its complex life cycle in the Anopheles vector and its human host. In order to further elucidate redox-based parasite host cell interactions and mechanisms of drug action, we targeted the genetically encoded glutathione redox sensor roGFP2 coupled to human glutaredoxin 1 (roGFP2-hGrx1) as well as the ratiometric pH sensor pHluorin to the apicoplast and the mitochondrion of P. falciparum. Using live cell imaging, this allowed for the first time the determination of the pH values of the apicoplast (7.12±0.40) and mitochondrion (7.37±0.09) in the intraerythrocytic asexual stages of the parasite. Based on the roGFP2-hGrx1 signals, glutathione-dependent redox potentials of -267mV and -328mV, respectively, were obtained. Employing these novel tools, initial studies on the effects of redox-active agents and clinically employed antimalarial drugs were carried out on both organelles.

Comparison of methods probing the intracellular redox milieu in Plasmodium falciparum.

Glutathione plays a crucial role in the redox regulation of the malaria parasite Plasmodium falciparum and is linked to drug resistance mechanisms, especially in resistance against the antimalarial drug chloroquine (CQ). The determination of the glutathione-dependent redox potential was recently established in living parasites using a cytosolically expressed biosensor comprising redox-sensitive green fluorescent protein coupled to human glutaredoxin 1 (hGrx1-roGFP2). In order to further elucidate redox changes induced by antimalarial drugs and to consolidate the application spectrum of the ratiometric biosensor we systematically compared it to other methods probing thiol and redox metabolism. Among these methods were cell disruptive and non-disruptive approaches including spectrophotometric assays with Ellman's reagent and naphthalene dicarboxyaldehyde as well as molecular probes such as ThiolTracker™ Violet and the dichlorofluorescein-based probe CM-H2DCFDA. To directly compare the methods, blood stages of the CQ-sensitive P. falciparum 3D7 strain were challenged with the oxidative agent diamide and the antimalarial drugs artemisinin and CQ for 1h, 4h, and 24h. For all conditions, dose-dependent changes in the different redox parameters could be monitored which are compared and discussed. We furthermore detected slight differences in thiol status of parasites transiently transfected with hGrx1-roGFP2 in comparison with control 3D7 cells. In conclusion, ThiolTracker™ Violet and, even more so, the hGrx1-roGFP2 probe reacted reliably and sensitively to drug induced changes in intracellular redox metabolism. These results were substantiated by classical cell disruptive methods.