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Legionella pneumophila is a Gram-negative bacterium found in natural and man-made water systems where it replicates within amoebas and ciliates. In humans, once inside the lungs, L. pneumophila replicates in alveolar macrophages and causes Legionnaires’ disease, a severe pneumonia. The Icm/Dot type IVb secretion system is a major virulence factor required for intracellular multiplication. The Icm/Dot system allows the secretion of effectors into the cytoplasm of the host cell. These effectors modify host cell vesicular trafficking and prevent maturation of the phagosome. The innate immune response is crucial in restricting L. pneumophila proliferation. TNF-α is one of the major cytokines involved in this process as it renders macrophages more resistant to L. pneumophila infection and induces apoptosis of L. pneumophila-infected macrophages. Tail-specific proteases (Tsp) are involved in tolerating thermal stress and in virulence. We have previously characterized the Tsp encoded by L. pneumophila, showing that it is important for surviving thermal stress and for infection of amoeba when a temperature change occurs during infection. Here, we demonstrated that Tsp is required for intracellular multiplication in macrophages. Absence of tsp is associated with higher production of TNF-α by macrophages in response to L. pneumophila infection. This effect is independent of the Icm/Dot secretion system.

Malaria is a complex parasitic disease, caused by Plasmodium spp. More than a century after the discovery of malaria parasites, this disease continues to pose a global public health problem and the pathogenesis of the severe forms of malaria remains incompletely understood. Extracellular vesicles (EVs), including exosomes and microvesicles, have been increasingly researched in the field of malaria in a bid to fill these knowledge gaps. EVs released from Plasmodium-infected red blood cells and other host cells during malaria infection are now believed to play key roles in disease pathogenesis and are suggested as vital components of the biology of Plasmodium spp. Malaria-derived EVs have been identified as potential disease biomarkers and therapeutic tools. In this review, key findings of malaria EV studies over the last 20 years are summarized and critically analysed. Outstanding areas of research into EV biology are identified. Unexplored EV research foci for the future that will contribute to consolidating the potential for EVs as agents in malaria prevention and control are proposed.

The Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0–8.0), although PfM1AAP also possesses some activity (

Background Malaria kills one child every 30 seconds reaching up to 3000 children a day. The mosquito borne malarial parasite invades the blood stream and hijacks red blood cells (RBCs). One of the medical successes of the 20th century was development of malaria diagnostic tests. However, poor specificity and sensitivity along with the inability of these assays to distinguish active malarial infections has put the management scheme in jeopardy. Aim To develop an in-vitro functional assay to predict active malarial infections. Methods Plasmodium falciparum (3D7) parasites were incubated with delta-aminolevulinic acid (ALA) for 7 h and imaged using a confocal microscope for protoporphyrin IX (PpIX) fluorescence. Similarly, PpIX was extracted, and fluorescence was estimated by fluorimetry. Results Imaging showed that the falciparum-infected RBCs fluoresced while the non-infected cells did not. Moreover, fluorimetry showed fluorescent peaks only in actively infected RBCs. Conclusions ALA was only taken up by infected RBCs. When the parasites were loaded with ALA, they fluoresced. These proof-of-concept results demonstrate the first functional assay to detect/diagnose active malaria.

Background Malaria continues to be endemic in the coast and Amazon regions of Ecuador. Clarifying current Plasmodium falciparum resistance in the country will support malaria elimination efforts. In this study, Ecuadorian P. falciparum parasites were analysed to determine their drug resistance genotypes and phenotypes. Methods Molecular analyses were performed to search for mutations in known resistance markers (Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, k13). Pfmdr1 copy number was determined by qPCR. PFMDR1 transporter activity was characterized in live parasites using live cell imaging in combination with the Fluo-4 transport assay. Chloroquine, quinine, lumefantrine, mefloquine, dihydroartemisinin, and artemether sensitivities were measured by in vitro assays. Results The majority of samples from this study presented the CVMNT genotype for Pfcrt (72–26), NEDF SDFD mutations in Pfmdr1 and wild type genotypes for Pfdhfr, Pfdhps and k13. The Ecuadorian P. falciparum strain ESM-2013 showed in vitro resistance to chloroquine, but sensitivity to quinine, lumefantrine, mefloquine, dihydroartemisinin and artemether. In addition, transport of the fluorochrome Fluo-4 from the cytosol into the digestive vacuole (DV) of the ESM-2013 strain was minimally detected in the DV. All analysed samples revealed one copy of Pfmdr1. Conclusion This study indicates that Ecuadorian parasites presented the genotype and phenotype for chloroquine resistance and were found to be sensitive to SP, artemether-lumefantrine, quinine, mefloquine, and dihydroartemisinin. The results suggest that the current malaria treatment employed in the country remains effective. This study clarifies the status of anti-malarial resistance in Ecuador and informs the P. falciparum elimination campaigns in the country.

Chloroquine (CQ) treatment failure in Plasmodium falciparum parasites has been documented for decades, but the pharmacological explanation of this phenotype is not fully understood. Current concepts attribute CQ resistance to reduced accumulation of the drug at a given external CQ concentration ([CQ]ex) in resistant compared to sensitive parasites. The implication of this explanation is that the mechanisms of CQ-induced toxicity in resistant and sensitive strains are similar once lethal internal concentrations have been reached. To test this hypothesis, we investigated the mechanism of CQ-induced toxicity in CQ-sensitive (CQS) versus CQ-resistant (CQR) parasites by analyzing the time-course of cellular responses in these strains after exposure to varying [CQ]ex as determined in 72 h toxicity assays. Parasite killing was delayed in CQR parasites for up to 10 h compared to CQS parasites when exposed to equipotent [CQ]ex. In striking contrast, brief exposure (1 h) to lethal [CQ]ex in CQS but not CQR parasites caused the appearance of hitherto undescribed hemozoin (Hz)-containing compartments in the parasite cytosol. Hz-containing compartments were very rarely observed in CQR parasites even after CQ exposures sufficient to cause irreversible cell death. These findings challenge current concepts that CQ killing of malaria parasites is solely concentration-dependent, and instead suggest that CQS and CQR strains fundamentally differ in the consequences of CQ exposure.

Malaria is a major disease in the tropics where chemotherapy remains the main mode of treatment and as such the rise and spread of drug-resistant malaria can lead to human tragedy. Two membrane transport proteins, PfMDR1 (Plasmodium falciparum multidrug resistance protein 1) and PfCRT (P. falciparum chloroquine resistance transporter), have been shown to cause resistance to several antimalarials. Both PfMDR1 and PfCRT are localized to the digestive vacuolar membrane and appear to regulate the transport of drugs and physiological metabolites. In this study we have used MK571, a 2-amino quinoline, to explore its interaction with PfMDR1 and PfCRT in chloroquine-sensitive and -resistant strains of P. falciparum. Our results show that chloroquine-resistant strains (e.g., K1, Dd2, and 7G8) are consistently more sensitive to MK571 than chloroquine-sensitive strains (e.g., 3D7, 106/1 and D10). This association, however, was not maintained with the chloroquine-resistant strain FCB which IC50 value was similar to chloroquine-sensitive strains. Moreover, the susceptibility of chloroquine-sensitive and -resistant strains to MK571 does not correlate with mutated PfCRT, nor is it reversible with verapamil; but correlates with mutations in PfMDR1. Furthermore, MK571 appears to target the parasite's digestive vacuole (DV), as demonstrated by the ability of MK571 to: (1) block the accumulation of the fluorescent dye Fluo-4 AM, a PfMDR1 substrate, into the digestive vacuole; (2) reduce the transvacuolar pH gradient; and (3) inhibit the formation of β-hematin in vitro. Moreover, the presence of non-toxic concentrations of MK571 sensitized both chloroquine-sensitive and -resistant parasites to mefloquine and halofantrine, likely by competing against PfMDR1-mediated sequestering of the drugs into the DV compartment and away from the drugs' cytosolic targets. Our data, nevertheless, found only a minimal decrease in MK571 IC50 value in FCB parasite which second pfmdr1 copy was inactivated via gene disruption. Taken together, the findings of this study suggest that MK571 interacts with native and mutant PfMDR1 and modulates the import of drugs or solutes into the parasite's DV and, as such, MK571 may be a useful tool in the characterization of PfMDR1 drug interactions and substrate specificity.

Mutations in the multidrug resistance transporter of Plasmodium falciparum PfMDR1 have been implicated to play a significant role in the emergence of worldwide drug resistance, yet the molecular and biochemical mechanisms of this transporter are not well understood. Although it is generally accepted that drug resistance in P. falciparum is partly associated with PfMDR1 transport activity situated in the membrane of the digestive vacuole, direct estimates of the pump rate of this transport process in the natural environment of the intact host-parasite system have never been analysed. The fluorochrome Fluo-4 is a well-documented surrogate substrate of PfMDR1 and has been found to accumulate by actively being transported into the digestive vacuole of several parasitic strains. In the present study, we designed an approach to use Fluo-4 fluorescence uptake as a measure of compartmental Fluo-4 concentration accumulation in the different compartments of the host-parasite system. We performed a 'reverse Fluo-4 imaging' approach to relate fluorescence intensity to changes in dye concentration rather than Ca(2+) fluctuations and were able to calculate the overall rate of transport for PfMDR1 in Dd2 parasites. With this assay, we provide a powerful method to selectively measure the effect of PfMDR1 mutations on substrate transport kinetics. This will be of high significance for future compound screening to test for new drugs in resistant P. falciparum strains.

We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards. © 2012.

During the development of malaria parasites within human erythrocytes, the fusion of digestive vesicles gives rise to a large digestive vacuole (DV). This organelle, which is maintained at low pH, processes 60-80 percent of the erythrocyte hemoglobin to provide a pool of amino acids that is crucial for parasite growth and development. During proteolysis, heme is released from hemoglobin as a toxic byproduct and is detoxified by biocrystallization to hemozoin. Proteases that contribute to hemoglobin breakdown, as well as other DV-associated proteins, arrive at this site via several different transport pathways. Antimalarial quinoline drugs, such as chloroquine, act by binding to heme and thus prevent its sequestration into hemozoin. Other drugs, such as artemisinin, may cause oxidative damage of DV macromolecules and membranes. The membrane of the DV contains ion pumps and transporters that maintain its low pH but are also pivotal in the development of parasite resistance to several antimalarial drugs. Methods for the isolation of the DV organelle have been developed to study the biogenesis and function of this important organelle.

The human malaria parasite Plasmodium falciparum is capable of adapting to vastly different extracellular Ca(2+) environments while maintaining tight control of its intracellular Ca(2+) concentration. The mechanisms underpinning Ca(2+) homeostasis in this important pathogen are only partly understood. Here we have functionally expressed the putative Ca(2+)/H(+) antiporter PfCHA in Xenopus laevis oocytes. Our data suggest that PfCHA mediates H(+)-coupled Ca(2+) and Mn(2+) exchange. The apparent dissociation constant K(M) for Ca(2+) of 2.2 +/- 0.7 mM and the maximal velocity V(max) of 0.6 +/- 0.1 nmol per oocyte per hour are consistent with PfCHA being a low-affinity high-capacity Ca(2+) carrier. In the parasite, PfCHA was found to localize to the mitochondrion. Physiological studies conducted with live parasitized erythrocytes, and using Fluo-4 and Rhod-2 to monitor cytoplasmic and mitochondrial Ca(2+) dynamics, suggest that the mitochondrion serves as a dynamic Ca(2+) store and that PfCHA functions as a Ca(2+) efflux system expelling excess Ca(2+) from the mitochondrion. PfCHA lacks appreciable homologies to the human mitochondrial Ca(2+)/H(+) exchanger and might represent an evolutionary divergent class of mitochondrial cation antiporter, which, in turn, might provide novel opportunities for intervention.

Resistance to chloroquine has been linked to polymorphisms within the pfcrt gene of the human malarial parasite Plasmodium falciparum. Here, we have investigated the prevalence of the pfcrt allele associated with chloroquine resistance in the peripheral blood and the placenta of pregnant women diagnosed with a P. falciparum infection. Our molecular epidemiological data show an unequal distribution with a significant under-representation of parasites carrying the mutated pfcrt allele in the placenta, as compared to the peripheral blood. In comparison, no differences were seen with regard to pfmdr1 polymorphisms of these parasites. Our data suggest a selective disadvantage of the polymorphic and a selective advantage of the wild-type pfcrt haplotype in the placenta, supporting the model that the human host provides various microenvironments that favor genetically distinct P. falciparum populations.

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasite's food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans-stimulation efflux protocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine-sensitive and -resistant parasites, however, with different transport modalities.

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of action of several antimalarial drugs, such as chloroquine, which accumulate in this organelle due to their properties as amphiphilic weak bases that inhibit haem detoxification. It has been suggested that changes in the pH of the digestive vacuole, affecting either drug partitioning or haem solubility and/or biomineralization rates, would correlate with reduced intracellular chloroquine accumulation and, hence, would determine the chloroquine-resistance phenotype. The techniques previously used to quantify digestive vacuolar pH mainly relied on lysed or isolated parasites, with unpredictable consequences on internal pH homeostasis. In this study, we have investigated the baseline steady-state pH of the cytoplasm and digestive vacuole of a chloroquine-sensitive (HB3) and a chloroquine-resistant (Dd2) parasite using a pH-sensitive green fluorescent protein, termed pHluorin. This non-invasive technique allows for in vivo pH measurements in intact P. falciparum-infected erythrocytes under physiological conditions. The data suggest that the pH of the cytoplasm is approximately 7.15 +/- 0.07 and that of the digestive vacuole approximately 5.18 +/- 0.05. No significant differences in baseline pH values were recorded for the chloroquine-sensitive and chloroquine-resistant parasites.

The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several anti-malarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of approximately 90, approximately 350, and approximately 400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.

Resistance to the antimalarial drug chloroquine has been linked with polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum, yet the mechanism by which this gene confers the reduced drug accumulation phenotype associated with resistance is largely unknown. To investigate the role of pfcrt in mediating chloroquine resistance, we challenged P. falciparum clones differing only in their pfcrt allelic form with the "varying-trans" procedure. In this procedure, movement of labeled substrate across a membrane is measured when unlabeled substrate is present on the trans side of the membrane. If a transporter is mediating the substrate flow, a stimulation of cis-to-trans movement may be observed with increasing concentrations of trans substrate. We present evidence for an association of those pfcrt alleles found in chloroquine-resistant P. falciparum strains with the phenomenon of stimulated chloroquine accumulation under varying-trans conditions. Such an association is not seen with polymorphisms within pfmdr1, which encodes a homologue of the human multidrug resistance efflux pump. Our data are interpreted in terms of a model in which pfcrt is directly or indirectly involved in carrier-mediated chloroquine efflux from resistant cells.

The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized—particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in P. falciparum.

In den vergangenen Jahren stieg sowohl das wissenschaftliche als auch das kommerzielle Interesse an rekombinanten Antikorpern vor allem aufgrund des einfachen Weges zur Gewinnung komplett humaner IgGs. Innovative Selektionsmethoden wie das “phage display” ermoglichen heute sogar die Isolierung von humanen Antikorpern gegen giftige oder stark pathogene Antigene, wie sie unser eigenes Immunsystem nicht hervorzubringen vermag. Der Einsatz der Gentechnik ermoglicht die Herstellung kleiner Immunglobulinfragmente, welche die notwendige Information eines Antikorpers zur Antigenbindung auf ein Minimum reduzieren. Diese Fragmente konnen dann zu multivalenten, hochaffinen Molekulen mit optimierten pharmakokinetischen Eigenschaften aufgebaut werden, was ihre Anwendungsmoglichkeiten in Forschung, Diagnostik und Therapie gegenuber herkommlichen Immunglobulinen stark erweitert.

Erythrocytes infected with the human malarial para-site Plasmodium falciparum acquire adhesive propertiesand sequester in the microvasculature. This mechanism,referred to as cytoadhesion, involves the transport of par-asite encoded immunovariant adhesions, collectively calledP. falciparum erythrocyte membrane protein 1 (PfEMP1),to the erythrocyte’s surface, where they act as ligands forcytoadhesive events [1].In cells of higher eukaryotes, intracellular protein trans-port between the various organelles of the endomembranesystem and the plasma membrane is facilitated by vesi-cles. Many, if not all of the vesicles possess a protein coatat their cytosolic surface. The first coat described was theclathrin coat, which is characteristic of endosomal vesicu-lar transport from the plasma membrane to endosomes [2].Distinct from clathrin-coated vesicles are COPI (coat pro-tein I)-coated vesicles[3] that mediate transport steps withinthe Golgi stack and back to the endoplasmic reticulum (ER)[4,5]. The COPI coat was shown to consist of the regulatoryGTPase ARF and seven different protein subunits termed al-pha, beta, beta , gamma, delta, epsilon and zeta [6]. Antero-grade protein traffic from the ER to the Golgi is executed bythe COPII-coated vesicles[7,8]. COPII coats also require ac-tivity of a small GTPase, referred to here as Sar1-activatingcomplex. In addition, two combined complexes consistingof Sec13/31 and Sec23/24 have been described in COPIIprotein-coated vesicles in yeast[9,10], and the structure of

Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasitePlasmodium falciparum, including pH homeostasis, Ca2+ signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasite's acidic food vacuole, brought about by lipid peroxidation initiated by light-induced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.

We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1–5×10 7 independent clones giving a final combined library of 4×10 9 members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.

Light microscopy plays an important role in cell biology and has been instrumental in the description of the living world. Fluorescent probes and new advances in microscopic techniques have revolutionized the application, enabling real time investigation of biological processes in 3D living cells using physiological conditions. Current applications of live cell imaging may include quantification of ion concentrations in cellular compartments, monitoring ion fluxes and signaling events, protein sorting and trafficking processes, as well as protein-protein interactions. Because of its potential, live cell imaging has been widely applied to study the biology and pathophysiology of the human malaria parasite Plasmodium falciparum. Apart from its clinical importance, P. falciparum has fascinated the cell biologist because of its ability to exist and develop within an inert cell: the human erythrocyte. Our understanding of intracellular ion regulation in malaria parasites has been greatly enhanced since the introduction of fluorescence microscopy and live cell imaging. This chapter provides an overview of the various fluorescent molecules available to monitor ion homeostasis in this parasite and outline the techniques used for ion quantification.

Light microscopy plays an important role in cell biology and has been instrumental in the description of the living world. Fluorescent probes and new advances in microscopic techniques have revolutionized the application, enabling real time investigation of biological processes in 3D living cells using physiological conditions. Current applications of live cell imaging may include quantification of ion concentrations in cellular compartments, monitoring ion fluxes and signaling events, protein sorting and trafficking processes, as well as protein-protein interactions. Because of its potential, live cell imaging has been widely applied to study the biology and pathophysiology of the human malaria parasite Plasmodium falciparum. Apart from its clinical importance, P. falciparum has fascinated the cell biologist because of its ability to exist and develop within an inert cell: the human erythrocyte. Our understanding of intracellular ion regulation in malaria parasites has been greatly enhanced since the introduction of fluorescence microscopy and live cell imaging. This chapter provides an overview of the various fluorescent molecules available to monitor ion homeostasis in this parasite and outline the techniques used for ion quantification.

The human malaria parasite Plasmodium falciparum is capable of adapting to vastly different extracellular Ca(2+) environments while maintaining tight control of its intracellular Ca(2+) concentration. The mechanisms underpinning Ca(2+) homeostasis in this important pathogen are only partly understood. Here we have functionally expressed the putative Ca(2+)/H(+) antiporter PfCHA in Xenopus laevis oocytes. Our data suggest that PfCHA mediates H(+)-coupled Ca(2+) and Mn(2+) exchange. The apparent dissociation constant K(M) for Ca(2+) of 2.2 +/- 0.7 mM and the maximal velocity V(max) of 0.6 +/- 0.1 nmol per oocyte per hour are consistent with PfCHA being a low-affinity high-capacity Ca(2+) carrier. In the parasite, PfCHA was found to localize to the mitochondrion. Physiological studies conducted with live parasitized erythrocytes, and using Fluo-4 and Rhod-2 to monitor cytoplasmic and mitochondrial Ca(2+) dynamics, suggest that the mitochondrion serves as a dynamic Ca(2+) store and that PfCHA functions as a Ca(2+) efflux system expelling excess Ca(2+) from the mitochondrion. PfCHA lacks appreciable homologies to the human mitochondrial Ca(2+)/H(+) exchanger and might represent an evolutionary divergent class of mitochondrial cation antiporter, which, in turn, might provide novel opportunities for intervention.

Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer's clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.

The steady-state regulation of intracellular levels of essential ions and ionic gradients is critical for almost all functions within a cell. Thus, it is not surprising to find that ions have been shown to play an important role in numerous parasitic processes, such as invasion, development and possibly drug resistance mechanisms. Live cell imaging has become a widespread technique to visualize and quantify several of these processes, including pH and Ca(2+) homeostasis, in an effort to better understand the biology and physiology of cells. This is now also the case for many human pathogens. The aim of this review is to emphasize the importance of this technique and provide an overview of what we have learned so far, using the malaria parasite Plasmodium falciparum as a paradigm.

The spread of parasitic resistance has necessitated the development of new drugs and drug targets for the treatment of malaria. Microtubules, which have gained outstanding importance as target molecules for the development of anticancer drugs, are likely to be potent antimalarial targets. The clinical implementation of microtubule inhibitors has given rise to a detailed mechanistic understanding of their interaction with tubulin on the molecular level and their effects on the cellular level. By comparison, our knowledge on Plasmodium falciparum, the causative agent of the most severe form of malaria, is rather poor. This article gives an overview on the microtubule inhibitors that have been explored in the parasite, reviews their effects on parasite growth and assesses their potential as novel antimalarials.

The P-glycoprotein homolog of the human malaria parasite Plasmodium falciparum (Pgh-1) has been implicated in decreased susceptibility to several antimalarial drugs, including quinine, mefloquine and artemisinin. Pgh-1 mainly resides within the parasite's food vacuolar membrane. Here, we describe a surrogate assay for Pgh-1 function based on the subcellular distribution of Fluo-4 acetoxymethylester and its free fluorochrome. We identified two distinct Fluo-4 staining phenotypes: preferential staining of the food vacuole versus a more diffuse staining of the entire parasite. Genetic, positional cloning and pharmacological data causatively link the food vacuolar Fluo-4 phenotype to those Pgh-1 variants that are associated with altered drug responses. On the basis of our data, we propose that Pgh-1 imports solutes, including certain antimalarial drugs, into the parasite's food vacuole. The implications of our findings for drug resistance mechanisms and testing are discussed.

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasite Plasmodium falciparum, including pH homeostasis, Ca(2+) signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasite's acidic food vacuole, brought about by lipid peroxidation initiated by light-induced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.

Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4×107) of single chain antibodies (scFv). The affinities of both the anti-estradiol and anti-progesterone scFv were approximately 108 M−1. Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response.

Background The Plasmodium falciparum multidrug resistance 1 transporter, PfMDR1, contains five amino acid polymorphisms that are suggested to be involved in altered drug transport from the parasite’s cytosol into the digestive vacuole (DV). Transport of a substrate into another intracellular compartment influences drug availability at its site of action, therefore making the parasite more susceptible or resistant to a drug. Fluo-4 is a known fluorescent substrate that can be used as a molecular tool to investigate transport dynamics of PfMDR1 in many parasite strains. Methods Six P. falciparum strains with varying PfMDR1 mutations were loaded with Fluo-4 AM. Accumulation of the fluorophore in the DV was measured using confocal microscopy. The role of a key amino acid mutation was verified using selected parasite clones with point mutations at PfMDR1 amino acid position 1042. Equal expression of PfMDR1 was confirmed by Western blot. Results Fluo-4 was transported by PfMDR1 into the DV of most drug-sensitive and -resistant parasites. Asparagine at PfMDR1 amino acid position 1042 was crucial for Fluo-4 transport, while the N1042D substitution abolished Fluo-4 transport. Competition studies of Fluo-4 with chloroquine, quinine and mefloquine were performed on parasites harbouring asparagine at position 1042. A distinct Fluo-4 transport inhibition pattern for each tested anti-malarial drug was observed in parasite strains of different genetic background. Conclusion This study demonstrates that Fluo-4 can be used to investigate PfMDR1 transport dynamics in both drug-sensitive and -resistant parasites. Furthermore, direct evidence of altered Fluo-4 transport in PfMDR1 is linked to a single amino acid mutation in the substrate binding pocket. This system offers a great tool to investigate the role of substrate transport by PfMDR1 and the mutations necessary to support transport, which would lead to new insights for the development of novel anti-malarial drugs. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0791-3) contains supplementary material, which is available to authorized users.