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.     

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.     

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.     

Enzymes involved in plastid‐targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver‐stage development

Malaria parasites scavenge nutrients from their host but also harbour enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast‐targeted type II fatty acid synthesis, which is essential for late liver‐stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast‐targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three‐step reaction utilizing three enzymes: glycerol 3‐phosphate dehydrogenase, glycerol 3‐phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbour genes for both apicoplast‐ and cytosol/endoplasmic reticulum‐targeted phosphatidic acid synthesis. Our research shows that apicoplast‐targeted Plasmodium yoelii glycerol 3‐phosphate dehydrogenase and glycerol 3‐phosphate acyltransferase are expressed only during liver‐stage development and deletion of the encoding genes resulted in late liver‐stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast‐targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite life cycle. Our results suggest that P. yoelii has an incomplete apicoplast‐targeted phosphatidic acid synthesis pathway that is essential for liver‐stage maturation.     

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.     

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.     

Protective immunity against malaria liver stage after vaccination with live parasites

Despite nearly 100 years of research and control efforts, malaria remains one of the most important infectious diseases. An efficient vaccine would be a powerful to tool to reduce mortality and morbidity. Experimentally, induction of sterile immunity in humans after vaccination with attenuated sporozoites has been obtained. This observation has spurred the search for subunit vaccines that aim to reproduce this protection. As yet none of the current candidate subunit vaccines achieved complete protection reproducibly. This failure coupled to the recent advent of genetically modified Plasmodium parasites has led to a renewed interest in the use of live parasites for vaccination against malaria pre-erythrocytic stages. In this article, we review and discuss the recent developments in this field.     

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.     

An apicoplast‐resident folate transporter is essential for sporogony of malaria parasites

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co‐factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2‐deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single‐membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2‐deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.     

Avian malaria parasites (

There is concern that avian malaria may be partly responsible for fluctuations in yellow-eyed penguin (Megadyptes antipodes) populations in New Zealand. Recent findings, however, have provided no evidence of avian malaria parasites infecting yellow-eyed penguins on the Otago Peninsula, raising questions as to whether this area is currently free of such parasites. To test this possibility we collected blood samples from 109 individuals of five non-native bird species known to carry malarial parasites elsewhere in New Zealand. Molecular screening by polymerase chain reaction revealed 6% of the sampled birds were positive for malarial parasites, indicating that a local reservoir of infection is present. Sequence data revealed a generalist strain of Plasmodium is present, one that infects a number of native and non-native bird species elsewhere in the country. The absence of this generalist strain in yellow-eyed penguins, some of which were sampled during the same period as the current study, may be due to low levels of mosquito vectors of disease during the study period, low densities of non-native birds around yellow-eyed penguin colonies, or infected penguins dying before they could be sampled. Continued monitoring of mosquito populations and the factors that affect their densities should be included in the future management of native birds in this area.     

Chromosomes of malaria parasites

The advent of pulsed field gradient electrophoresis has proved remarkably useful for studying chromosomes of the genetically intractable malaria parasite Plasmodium falciparum. Advances include determination of the karyotype, a linkage map and restriction maps of individual chromosomes that enable the ordering of genes. The structural basis underlying a frequently occurring form of chromosome size polymorphism is now understood and other polymorphisms are providing tantalizing clues to the mechanisms underlying drug resistance.     

Measurably recombining malaria parasites

Genomic epidemiology has guided research and policy for various viral pathogens and there has been a parallel effort towards using genomic epidemiology to combat diseases that are caused by eukaryotic pathogens, such as the malaria parasite. However, the central concept of viral genomic epidemiology, namely that of measurably mutating pathogens, does not apply easily to sexually recombining parasites. Here we introduce the related but different concept of measurably recombining malaria parasites to promote convergence around a unifying theoretical framework for malaria genomic epidemiology. Akin to viral phylodynamics, we anticipate that an inferential framework developed around recombination will help guide practical research and thus realize the full public health potential of genomic epidemiology for malaria parasites and other sexually recombining pathogens.     

Phylogeography of Caribbean lizard malaria: tracing the history of vector‐borne parasites

The Anolis lizards of the eastern Caribbean islands are parasitized by several species of malaria parasites (Plasmodium). Here I focus on two species of Plasmodium, using molecular data (mitochondrial cytochrome b sequences) to recover the phylogeography of the parasites throughout the Lesser Antilles and Puerto Rico. The two parasites were originally described as a single species, P. azurophilum, which infects both red and white blood cells. Here the two species are termed P. azurophilum Red and P. azurophilum White based on their host cell type. Six haplotypes were found in 100 infections sequenced of P. azurophilum Red and six in 45 infections of P. azurophilum White. Nested clade analysis revealed a significant association of geographical location and clades as well as a pattern of past fragmentation of parasite populations. This is consistent with the hypothesis that vector‐borne parasites such as malaria may be subject to frequent local extinctions and recolonizations. Comparison of the phylogeography of the lizard and parasites shows only weak concordance; that is, the parasites colonized the lizards in the islands, but dispersal events between islands via vectors or failed lizard colonizations were present. The two parasites had different histories, P. azurophilum Red colonized the islands from both the north and south, and P. azurophilum White originated in the central Lesser Antilles, probably from P. azurophilum Red, then moved to both north and south. This is the first study to examine the biogeography of a pair of sibling species of vector‐borne parasites within an island archipelago system.