The role of Plasmodium V-ATPase in vacuolar physiology and antimalarial drug uptake.

To ensure their survival in the human bloodstream, malaria parasites degrade up to 80% of the host erythrocyte hemoglobin in an acidified digestive vacuole. Here, we combine conditional reverse genetics and quantitative imaging approaches to demonstrate that the human malaria pathogen Plasmodium falciparum employs a heteromultimeric V-ATPase complex to acidify the digestive vacuole matrix, which is essential for intravacuolar hemoglobin release, heme detoxification, and parasite survival. We reveal an additional function of the membrane-embedded V-ATPase subunits in regulating morphogenesis of the digestive vacuole independent of proton translocation. We further show that intravacuolar accumulation of antimalarial chemotherapeutics is surprisingly resilient to severe deacidification of the vacuole and that modulation of V-ATPase activity does not affect parasite sensitivity toward these drugs.

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts.

Protein export and host membrane remodeling are crucial for multiple Plasmodium species to establish a niche in infected hosts. To better understand the contribution of these processes to successful parasite infection in vivo, we sought to find and characterize protein components of the intraerythrocytic Plasmodium berghei-induced membrane structures (IBIS) that form in the cytoplasm of infected erythrocytes. We identified proteins that immunoprecipitate with IBIS1, a signature member of the IBIS in P. berghei-infected erythrocytes. In parallel, we also report our data describing proteins that co-precipitate with the PTEX (Plasmodium translocon of exported proteins) component EXP2. To validate our findings, we examined the location of three candidate IBIS1-interactors that are conserved across multiple Plasmodium species, and we found they localized to IBIS in infected red blood cells and two further colocalized with IBIS1 in the liver-stage parasitophorous vacuole membrane. Successful gene deletion revealed that these two tryptophan-rich domain-containing proteins, termed here IPIS2 and IPIS3 (for intraerythrocytic Plasmodium-induced membrane structures), are required for efficient blood-stage growth. Erythrocytes infected with IPIS2-deficient schizonts in particular fail to bind CD36 as efficiently as wild-type P. berghei-infected cells and therefore fail to effectively sequester out of the circulating blood. Our findings support the idea that intra-erythrocytic membrane compartments are required across species for alterations of the host erythrocyte that facilitate interactions of infected cells with host tissues.

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.

A lipocalin mediates unidirectional heme biomineralization in malaria parasites.

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.

The parasitophorous vacuole of the blood-stage malaria parasite.

The pathology of malaria is caused by infection of red blood cells with unicellular Plasmodium parasites. During blood-stage development, the parasite replicates within a membrane-bound parasitophorous vacuole. A central nexus for host-parasite interactions, this unique parasite shelter functions in nutrient acquisition, subcompartmentalization and the export of virulence factors, making its functional molecules attractive targets for the development of novel intervention strategies to combat the devastating impact of malaria. In this Review, we explore the origin, development, molecular composition and functions of the parasitophorous vacuole of Plasmodium blood stages. We also discuss the relevance of the malaria parasite's intravacuolar lifestyle for successful erythrocyte infection and provide perspectives for future research directions in parasitophorous vacuole biology.

Identification of clustered organellar short (cos) RNAs and of a conserved family of organellar RNA-binding proteins, the heptatricopeptide repeat proteins, in the malaria parasite.

Gene expression in mitochondria of Plasmodium falciparum is essential for parasite survival. The molecular mechanisms of Plasmodium organellar gene expression remain poorly understood. This includes the enigmatic assembly of the mitochondrial ribosome from highly fragmented rRNAs. Here, we present the identification of clustered organellar short RNA fragments (cosRNAs) that are possible footprints of RNA-binding proteins (RBPs) in Plasmodium organelles. In plants, RBPs of the pentatricopeptide repeat (PPR) class produce footprints as a consequence of their function in processing organellar RNAs. Intriguingly, many of the Plasmodium cosRNAs overlap with 5'-ends of rRNA fragments. We hypothesize that these are footprints of RBPs involved in assembling the rRNA fragments into a functioning ribosome. A bioinformatics search of the Plasmodium nuclear genome identified a hitherto unrecognized organellar helical-hairpin-repeat protein family that we term heptatricopeptide repeat (HPR) proteins. We demonstrate that selected HPR proteins are targeted to mitochondria in P. berghei and that one of them, PbHPR1, associates with RNA, but not DNA in vitro. A phylogenetic search identified HPR proteins in a wide variety of eukaryotes. We hypothesize that HPR proteins are required for processing and stabilizing RNAs in Apicomplexa and other taxa.

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.

The Plasmodium berghei translocon of exported proteins reveals spatiotemporal dynamics of tubular extensions.

The erythrocyte is an extraordinary host cell for intracellular pathogens and requires extensive remodelling to become permissive for infection. Malaria parasites modify their host red blood cells through protein export to acquire nutrients and evade immune responses. Endogenous fluorescent tagging of three signature proteins of the Plasmodium berghei translocon of exported proteins (PTEX), heat shock protein 101, exported protein 2 (EXP2), and PTEX88, revealed motile, tubular extensions of the parasitophorous vacuole that protrude from the parasite far into the red blood cell. EXP2 displays a more prominent presence at the periphery of the parasite, consistent with its proposed role in pore formation. The tubular compartment is most prominent during trophozoite growth. Distinct spatiotemporal expression of individual PTEX components during sporogony and liver-stage development indicates additional functions and tight regulation of the PTEX translocon during parasite life cycle progression. Together, live cell imaging and correlative light and electron microscopy permitted previously unrecognized spatiotemporal and subcellular resolution of PTEX-containing tubules in murine malaria parasites. These findings further refine current models for Plasmodium-induced erythrocyte makeover.

In Vivo Function of PTEX88 in Malaria Parasite Sequestration and Virulence.

Malaria pathology is linked to remodeling of red blood cells by eukaryotic Plasmodium parasites. Central to host cell refurbishment is the trafficking of parasite-encoded virulence factors through the Plasmodium translocon of exported proteins (PTEX). Much of our understanding of its function is based on experimental work with cultured Plasmodium falciparum, yet direct consequences of PTEX impairment during an infection remain poorly defined. Using the murine malaria model parasite Plasmodium berghei, it is shown here that efficient sequestration to the pulmonary, adipose, and brain tissue vasculature is dependent on the PTEX components thioredoxin 2 (TRX2) and PTEX88. While TRX2-deficient parasites remain virulent, PTEX88-deficient parasites no longer sequester in the brain, correlating with abolishment of cerebral complications in infected mice. However, an apparent trade-off for virulence attenuation was spleen enlargement, which correlates with a strongly reduced schizont-to-ring-stage transition. Strikingly, general protein export is unaffected in PTEX88-deficient mutants that mature normally in vitro. Thus, PTEX88 is pivotal for tissue sequestration in vivo, parasite virulence, and preventing exacerbation of spleen pathology, but these functions do not correlate with general protein export to the host erythrocyte. The presented data suggest that the protein export machinery of Plasmodium parasites and their underlying mechanistic features are considerably more complex than previously anticipated and indicate challenges for targeted intervention strategies.

Towards genome-wide experimental genetics in the in vivo malaria model parasite Plasmodium berghei.

Plasmodium berghei was identified as a parasite of thicket rats (Grammomys dolichurus) and Anopheles dureni mosquitoes in African highland forests. Successful adaptation to a range of rodent and mosquito species established P. berghei as a malaria model parasite. The introduction of stable transfection technology, permitted classical reverse genetics strategies and thus systematic functional profiling of the gene repertoire. In the past 10 years following the publication of the P. berghei genome sequence, many new tools for experimental genetics approaches have been developed and existing ones have been improved. The infection of mice is the principal limitation towards a genome-wide repository of mutant parasite lines. In the past few years, there have been some promising and most welcome developments that allow rapid selection and isolation of recombinant parasites while simultaneously minimising animal usage. Here, we provide an overview of all the currently available tools and methods.

Two putative protein export regulators promote Plasmodium blood stage development in vivo.

Protein export is considered an essential feature of malaria parasite blood stage development. Here, we examined five components of the candidate Plasmodium translocon of exported proteins (PTEX), a complex thought to mediate protein export across the parasitophorous vacuole membrane into the host cell. Using the murine malaria model parasite Plasmodium berghei, we succeeded in generating parasite lines lacking PTEX88 and thioredoxin 2 (TRX2). Repeated attempts to delete the remaining three translocon components failed, suggesting essential functions for EXP2, PTEX150, and heat shock protein 101 (HSP101) during blood stage development. To analyze blood infections of the null-mutants, we established a flow cytometry-assisted intravital competition assay using three novel high fluorescent lines (Bergreen, Beryellow, and Berred). Although blood stage development of parasites lacking TRX2 was affected, the deficit was much more striking in PTEX88 null-mutants. The multiplication rate of PTEX88-deficient parasites was strongly reduced resulting in out-competition by wild-type parasites. Endogenous tagging revealed that TRX2::tag resides in distinct punctate organelles of unknown identity. PTEX88::tag shows a diffuse intraparasitic pattern in blood stage parasites. In trophozoites, PTEX88::tag also localized to previously unrecognized extensions reaching from the parasite surface into the erythrocyte cytoplasm. Together, our results indicate auxiliary roles for TRX2 and PTEX88 and central roles for EXP2, PTEX150, and HSP101 during P. berghei blood infection.