Imaging liver‐stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High‐speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver‐stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.     

The MTIP-myosin A complex in blood stage malaria parasites

Parasites of the Apicomplexa phylum use an actomyosin motor to drive invasion of host cells. The motor complex is located at the parasite's periphery between the plasma membrane and an inner membrane complex. A crucial component of this complex is myosin tail domain interacting protein (MTIP) identified in the murine malaria parasite Plasmodium yoelii. Here, we show that MTIP is expressed in Plasmodium falciparum merozoites, localises to the periphery of the cell and is present in a complex with myosin A. The MTIP-myosin A tail interaction has a Kd of 235 nM and calcium ions do not play a role in modulating the binding affinity of the two molecules, despite reports of a predicted EF-hand in MTIP. Antibodies to MTIP were used to immobilise the MTIP-myosin A complex, allowing actin binding and motility to be examined. Measurement of actin filament velocities powered by myosin A revealed a velocity of 3.51 microm s(-1), a speed comparable to fast muscle myosins. A short peptide derived from the tail of myosin A (C-MyoA) bound to MTIP and was able to disrupt the association of MTIP and myosin A in parasite lysates. C-MyoA peptidomimetic compounds that disrupt the MTIP-myosin A interaction are predicted to inhibit parasite motility and host cell invasion, which may be targets for new therapeutic approaches.     

Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood‐ and mosquito‐stage development

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species.     

Thermal biology of mosquito‐borne disease

Mosquito‐borne diseases cause a major burden of disease worldwide. The vital rates of these ectothermic vectors and parasites respond strongly and nonlinearly to temperature and therefore to climate change. Here, we review how trait‐based approaches can synthesise and mechanistically predict the temperature dependence of transmission across vectors, pathogens, and environments. We present 11 pathogens transmitted by 15 different mosquito species – including globally important diseases like malaria, dengue, and Zika – synthesised from previously published studies. Transmission varied strongly and unimodally with temperature, peaking at 23–29ºC and declining to zero below 9–23ºC and above 32–38ºC. Different traits restricted transmission at low versus high temperatures, and temperature effects on transmission varied by both mosquito and parasite species. Temperate pathogens exhibit broader thermal ranges and cooler thermal minima and optima than tropical pathogens. Among tropical pathogens, malaria and Ross River virus had lower thermal optima (25–26ºC) while dengue and Zika viruses had the highest (29ºC) thermal optima. We expect warming to increase transmission below thermal optima but decrease transmission above optima. Key directions for future work include linking mechanistic models to field transmission, combining temperature effects with control measures, incorporating trait variation and temperature variation, and investigating climate adaptation and migration.     

Diversity, loss, and gain of malaria parasites in a globally invasive bird

Invasive species can displace natives, and thus identifying the traits that make aliens successful is crucial for predicting and preventing biodiversity loss. Pathogens may play an important role in the invasive process, facilitating colonization of their hosts in new continents and islands. According to the Novel Weapon Hypothesis, colonizers may out-compete local native species by bringing with them novel pathogens to which native species are not adapted. In contrast, the Enemy Release Hypothesis suggests that flourishing colonizers are successful because they have left their pathogens behind. To assess the role of avian malaria and related haemosporidian parasites in the global spread of a common invasive bird, we examined the prevalence and genetic diversity of haemosporidian parasites (order Haemosporida, genera Plasmodium and Haemoproteus) infecting house sparrows (Passer domesticus). We sampled house sparrows (N = 1820) from 58 locations on 6 continents. All the samples were tested using PCR-based methods; blood films from the PCR-positive birds were examined microscopically to identify parasite species. The results show that haemosporidian parasites in the house sparrows'' native range are replaced by species from local host-generalist parasite fauna in the alien environments of North and South America. Furthermore, sparrows in colonized regions displayed a lower diversity and prevalence of parasite infections. Because the house sparrow lost its native parasites when colonizing the American continents, the release from these natural enemies may have facilitated its invasion in the last two centuries. Our findings therefore reject the Novel Weapon Hypothesis and are concordant with the Enemy Release Hypothesis.     

Transmission stage investment of malaria parasites in response to in-host competition

Conspecific competition occurs in a multitude of organisms, particularly in parasites, where several clones are commonly sharing limited resources inside their host. In theory, increased or decreased transmission investment might maximize parasite fitness in the face of competition, but, to our knowledge, this has not been tested experimentally. We developed and used a clone-specific, stage-specific, quantitative PCR protocol to quantify Plasmodium chabaudi replication and transmission stage densities in mixed-clone infections. We co-infected mice from two strains with an avirulent and virulent parasite clone and found competitive suppression of in-host (blood-stage) parasite densities and generally corresponding reductions in transmission stage production, with the virulent clone obtaining overall competitive superiority. In response to competitive suppression, there was little evidence of any alteration in transmission stage investment, apart from a small reduction by one of the two clones in one of the two host strains. This alteration did not result in a competitive advantage, although it might have reduced the disadvantage. This study supports much of the current literature, which predicts that conspecific in-host competition will result in a competitive advantage and positive selection for virulent clones and thus the evolution of higher virulence.     

Imaging ion flux and ion homeostasis in blood stage malaria parasites

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 Ca²⁺ 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.     

Directing traffic: Chaperone‐mediated protein transport in malaria parasites

The ability of eukaryotic parasites from the phylum Apicomplexa to cause devastating diseases is predicated upon their ability to maintain faithful and precise protein trafficking mechanisms. Their parasitic life cycle depends on the trafficking of effector proteins to the infected host cell, transport of proteins to several critical organelles required for survival, as well as transport of parasite and host proteins to the digestive organelles to generate the building blocks for parasite growth. Several recent studies have shed light on the molecular mechanisms parasites utilise to transform the infected host cells, transport proteins to essential metabolic organelles and for biogenesis of organelles required for continuation of their life cycle. Here, we review key pathways of protein transport originating and branching from the endoplasmic reticulum, focusing on the essential roles of chaperones in these processes. Further, we highlight key gaps in our knowledge that prevents us from building a holistic view of protein trafficking in these deadly human pathogens.     

Identification of plant-like galactolipids in Chromera velia, a photosynthetic relative of malaria parasites

Apicomplexa are protist parasites that include Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, responsible for toxoplasmosis. Most Apicomplexa possess a relict plastid, the apicoplast, which was acquired by secondary endosymbiosis of a red alga. Despite being nonphotosynthetic, the apicoplast is otherwise metabolically similar to algal and plant plastids and is essential for parasite survival. Previous studies of Toxoplasma gondii identified membrane lipids with some structural features of plastid galactolipids, the major plastid lipid class. However, direct evidence for the plant-like enzymes responsible for galactolipid synthesis in Apicomplexan parasites has not been obtained. Chromera velia is an Apicomplexan relative recently discovered in Australian corals. C. velia retains a photosynthetic plastid, providing a unique model to study the evolution of the apicoplast. Here, we report the unambiguous presence of plant-like monogalactosyldiacylglycerol and digalactosyldiacylglycerol in C. velia and localize digalactosyldiacylglycerol to the plastid. We also provide evidence for a plant-like biosynthesis pathway and identify candidate galactosyltranferases responsible for galactolipid synthesis. Our study provides new insights in the evolution of these important enzymes in plastid-containing eukaryotes and will help reconstruct the evolution of glycerolipid metabolism in important parasites such as Plasmodium and Toxoplasma.     

Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes

The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. We report that, just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte.     

Subpatent infection with nucleoside transporter 1‐deficient Plasmodium blood stage parasites confers sterile protection against lethal malaria in mice

Repeated immunizations with whole Plasmodium blood stage parasites and concomitant drug cure of infection confer protective immunity against parasite challenge in mice, monkeys and humans. Moreover, it was recently shown that infections with genetically modified rodent malaria blood stage parasites conferred sterile protection against lethal blood stage challenge. However, in these models vaccination resulted in high parasitemias and, in consequence, carries risk of vaccine‐induced pathology and death. Herein, we generated a novel, completely blood stage‐attenuated P. yoelii rodent malaria strain by targeted deletion of parasite nucleoside transporter 1 (NT1). Immunization of inbred and outbred mouse strains with a single low dose of Pynt1^‐ blood stages did not induce any patent infections and conferred complete sterile protection against lethal heterologous blood stage and sporozoite challenges. Partial protection was observed against lethal challenges with another parasite species, P. berghei. Importantly, subcutaneous immunization with Pynt1^‐ conferred sterile protection against lethal blood stage challenges. We show that cellular and humoral immune responses are both essential for sterile protection. The study demonstrates that genetic manipulation provides a platform for the designed, complete attenuation of malaria parasite blood stages and suggests testing the safety and efficacy of P. falciparum NT1 knockout strains in humans.