Despite the enormous significance of malaria parasites for global health, some basic features of their ultrastructure remain obscure. Here, we apply high-resolution volumetric electron microscopy to examine and compare the ultrastructure of the transmissible male and female sexual blood stages of Plasmodium falciparum as well as the more intensively studied asexual blood stages revisiting previously described phenomena in 3D. In doing so, we challenge the widely accepted notion of a single mitochondrion by demonstrating the presence of multiple mitochondria in gametocytes. We also provide evidence for a gametocyte-specific cytostome, or cell mouth. Furthermore, we generate the first 3D reconstructions of the parasite's endoplasmic reticulum (ER) and Golgi apparatus as well as gametocyte-induced extraparasitic structures in the infected red blood cell. Assessing interconnectivity between organelles, we find frequent structural appositions between the nucleus, mitochondria, and apicoplast. We provide evidence that the ER is a promiscuous interactor with numerous organelles and the trilaminar pellicle of the gametocyte. Public availability of these volumetric electron microscopy resources will facilitate reinterrogation by others with different research questions and expertise. Taken together, we reconstruct the 3D ultrastructure of P. falciparum gametocytes at nanometre scale and shed light on the unique organellar biology of these deadly parasites.

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The "9+2" axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked if there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana, have an extensive RSP complement including two divergent RSP9 orthologs, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither ortholog is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 ortholog. deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked with the different mode of assembly of trypanosomatid versus Plasmodium flagella.

EDITORIAL article Front. Cell. Infect. Microbiol., 28 April 2021 | https://doi.org/10.3389/fcimb.2021.680555

Severe infections are a major stress on haematopoiesis, where the consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow (BM) niches; however, what role the BM microenvironment plays in mediating the effects of infection on HSCs remains an open question. Here, using a murine model of malaria and combining single-cell RNA sequencing, mathematical modelling, transplantation assays and intravital microscopy, we show that haematopoiesis is reprogrammed upon infection, whereby the HSC compartment turns over substantially faster than at steady-state and HSC function is drastically affected. Interferon is found to affect both haematopoietic and mesenchymal BM cells and we specifically identify a dramatic loss of osteoblasts and alterations in endothelial cell function. Osteo-active parathyroid hormone treatment abolishes infection-triggered HSC proliferation and—coupled with reactive oxygen species quenching—enables partial rescuing of HSC function. Haltalli et al. show that Plasmodium berghei infection induces interferon release, and affects haematopoietic stem cell proliferation and function, as well as osteoblasts and vascular integrity, in the bone marrow niche.

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal '9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.

BackgroundDespite recent successes at controlling malaria, progress has stalled with an estimated 219 million cases and 435,000 deaths in 2017 alone. Combined with emerging resistance to front line antimalarial therapies in Southeast Asia, there is an urgent need for new treatment options and novel approaches to halt the spread of malaria. Plasmodium, the parasite responsible for malaria propagates through mosquito transmission. This imposes an acute bottleneck on the parasite population and transmission-blocking interventions exploiting this vulnerability are recognized as vital for malaria elimination.Methods13,533 small molecules with known activity against Plasmodium falciparum asexual parasites were screened for additional transmission-blocking activity in an ex vivo Plasmodium berghei ookinete development assay. Active molecules were then counterscreened in dose response against HepG2 cells to determine their activity/cytotoxicity window and selected non-toxic representative molecules were fully profiled in a range of transmission and mosquito infection assays. Furthermore, the entire dataset was compared to other published screens of the same molecules against P. falciparum gametocytes and female gametogenesis.Results437 molecules inhibited P. berghei ookinete formation with an IC50 < 10 μM. of which 273 showed >10-fold parasite selectivity compared to activity against HepG2 cells. Active molecules grouped into 49 chemical clusters of three or more molecules, with 25 doublets and 94 singletons. Six molecules representing six major chemical scaffolds confirmed their transmission-blocking activity against P. falciparum male and female gametocytes and inhibited P. berghei oocyst formation in the standard membrane feeding assay at 1 μM. When screening data in the P. berghei development ookinete assay was compared to published screens of the same library in assays against P. falciparum gametocytes and female gametogenesis, it was established that each assay identified distinct, but partially overlapping subsets of transmission-blocking molecules. However, selected molecules unique to each assay show transmission-blocking activity in mosquito transmission assays.ConclusionThe P. berghei ookinete development assay is an excellent high throughput assay for efficiently identifying antimalarial molecules targeting early mosquito stage parasite development. Currently no high throughput transmission-blocking assay is capable of identifying all transmission-blocking molecules.

Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N-(3-chloro-4-fluorophenyl)-2-methyl-2-{[4-methyl-3-(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low-nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter-screen up to 25 μm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for in vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

We assessed a combination multi-stage malaria vaccine schedule in which RTS,S/AS01B was given concomitantly with viral vectors expressing multiple-epitope thrombospondin-related adhesion protein (ME-TRAP) in a 0-month, 1-month, and 2-month schedule. RTS,S/AS01B was given as either three full doses or with a fractional (1/5th) third dose. Efficacy was assessed by controlled human malaria infection (CHMI). Safety and immunogenicity of the vaccine regimen was also assessed. Forty-one malaria-naive adults received RTS,S/AS01B at 0, 4 and 8 weeks, either alone (Groups 1 and 2) or with ChAd63 ME-TRAP at week 0, and modified vaccinia Ankara (MVA) ME-TRAP at weeks 4 and 8 (Groups 3 and 4). Groups 2 and 4 received a fractional (1/5th) dose of RTS,S/AS01B at week 8. CHMI was delivered by mosquito bite 11 weeks after first vaccination. Vaccine efficacy was 6/8 (75%), 8/9 (88.9%), 6/10 (60%), and 5/9 (55.6%) of subjects in Groups 1, 2, 3, and 4, respectively. Immunological analysis indicated significant reductions in anti-circumsporozoite protein antibodies and TRAP-specific T cells at CHMI in the combination vaccine groups. This reduced immunogenicity was only observed after concomitant administration of the third dose of RTS,S/AS01B with the second dose of MVA ME-TRAP. The second dose of the MVA vector with a four-week interval caused significantly higher anti-vector immunity than the first and may have been the cause of immunological interference. Co-administration of ChAd63/MVA ME-TRAP with RTS,S/AS01B led to reduced immunogenicity and efficacy, indicating the need for evaluation of alternative schedules or immunization sites in attempts to generate optimal efficacy.

Current strategies for the mass administration of antimalarial drugs demand oral formulations to target the asexual Plasmodium stages in the peripheral bloodstream, whereas recommendations for future interventions stress the importance of also targeting the transmission stages of the parasite as it passes between humans and mosquitoes. Orally administered polyamidoamine (PAA) nanoparticles conjugated to chloroquine reached the blood circulation and cured Plasmodium yoelii-infected mice, slightly improving the activity of the free drug and inducing in the animals immunity against malaria. Liquid chromatography with tandem mass spectrometry analysis of affinity chromatography-purified PAA ligands suggested a high adhesiveness of PAAs to Plasmodium falciparum proteins, which might be the mechanism responsible for the preferential binding of PAAs to Plasmodium-infected erythrocytes vs. non-infected red blood cells. The weak antimalarial activity of some PAAs was found to operate through inhibition of parasite invasion, whereas the observed polymer intake by macrophages indicated a potential of PAAs for the treatment of certain coinfections such as Plasmodium and Leishmania. When fluorescein-labeled PAAs were fed to females of the malaria mosquito vectors Anopheles atroparvus and Anopheles gambiae, persistent fluorescence was observed in the midgut and in other insect’s tissues. These results present PAAs as a versatile platform for the encapsulation of orally administered antimalarial drugs and for direct administration of antimalarials to mosquitoes, targeting mosquito stages of Plasmodium.

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2–1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male–female and dual-asexual-sexual, with one promising N -((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population.

Severe infections are a major source of stress on haematopoiesis, where consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow niches, which have been shown to be altered during ageing and haematopoietic malignancies. Whether the bone marrow (BM) microenvironment plays a role in mediating the effects of infection on HSCs remains an open question. Here we used an experimental murine model of malaria coupled with intravital microscopy, single cell RNA-Seq, mathematical modelling and transplantation assays to obtain a quantitative understanding of the proliferation dynamics of haematopoietic stem and progenitor cells (HSPCs) during Plasmodium infection. We uncovered that during Plasmodium infection the HSC compartment turns over significantly faster than in steady state, and that a global interferon response and loss of functional HSCs are linked to alterations in BM endothelium function and osteoblasts number. Finally, interventions that targeted osteoblasts uncoupled HSC proliferation and function thus opening up new avenues for therapeutic interventions that may improve the health of survivors of severe infections.

A critical step towards malaria elimination will be the interruption of Plasmodium transmission from the human host to the mosquito. At the core of the transmission cycle lies Plasmodium sexual reproduction leading to zygote formation and mosquito midgut colonisation by ookinetes. Whilst in vitro ookinete culture from the murine and avian malaria parasites, Plasmodium berghei and P. gallinaceum , has greatly increased our knowledge of transmission biology; efforts to mimic the process in the human parasite P. falciparum have, to date, had only limited success. Using fluorescence microscopy and flow cytometry with antibodies specific to the male gametocyte and developing ookinetes, we sought to evaluate P. falciparum ookinete production using previously published in vitro protocols. We then compared in vitro versus in vivo ookinete production in both P. falciparum and P. berghei parasites, exploring potential barriers to complete development. Finally, we sought to test a wide range of literature-led culture conditions towards further optimisation of in vitro P. falciparum ookinete production. Despite extensive testing, our efforts to replicate published methods did not produce appreciable quantities of fully formed P. falciparum ookinetes in vitro . In parallel, however, gametocyte cultures that failed to differentiate fully in vitro successfully developed into ookinetes in vivo with an efficiency approximating that of P. berghei . Flow cytometry analysis showed that this disparity likely lies with the poor fertilization of P. falciparum gametes in vitro . Attempts to improve gametocyte fertility or define conditions more permissive to fertilisation/ookinete survival in vitro were also unsuccessful. Current in vitro conditions for P. falciparum ookinete production are not optimal for gamete fertilisation either due to the lack of parasite-species-specific mosquito factors missing from in vitro culture, or non-permissive cues contaminating culture preparations.

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 μM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection.

Over a century since Ronald Ross discovered that malaria is caused by the bite of an infectious mosquito it is still unclear how the number of parasites injected influences disease transmission. Currently it is assumed that all mosquitoes with salivary gland sporozoites are equally infectious irrespective of the number of parasites they harbour, though this has never been rigorously tested. Here we analyse >1000 experimental infections of humans and mice and demonstrate a dose-dependency for probability of infection and the length of the host pre-patent period. Mosquitoes with a higher numbers of sporozoites in their salivary glands following blood-feeding are more likely to have caused infection (and have done so quicker) than mosquitoes with fewer parasites. A similar dose response for the probability of infection was seen for humans given a pre-erythrocytic vaccine candidate targeting circumsporozoite protein (CSP), and in mice with and without transfusion of anti-CSP antibodies. These interventions prevented infection more efficiently from bites made by mosquitoes with fewer parasites. The importance of parasite number has widespread implications across malariology, ranging from our basic understanding of the parasite, how vaccines are evaluated and the way in which transmission should be measured in the field. It also provides direct evidence for why the only registered malaria vaccine RTS,S was partially effective in recent clinical trials.

Antimalarial compounds with dual therapeutic and transmission-blocking activity are desired as high-value partners for combination therapies. Here, we report the identification and characterization of hexahydroquinolines (HHQs) that show low nanomolar potency against both pathogenic and transmissible intra-erythrocytic forms of the malaria parasite Plasmodium falciparum. This activity translates into potent transmission-blocking potential, as shown by in vitro male gamete formation assays and reduced oocyst infection and prevalence in Anopheles mosquitoes. In vivo studies illustrated the ability of lead HHQs to suppress Plasmodium berghei blood-stage parasite proliferation. Resistance selection studies, confirmed by CRISPR-Cas9-based gene editing, identified the digestive vacuole membrane-spanning transporter PfMDR1 (P. falciparum multidrug resistance gene-1) as a determinant of parasite resistance to HHQs. Haemoglobin and haem fractionation assays suggest a mode of action that results in reduced haemozoin levels and might involve inhibition of host haemoglobin uptake into intra-erythrocytic parasites. Furthermore, parasites resistant to HHQs displayed increased susceptibility to several first-line antimalarial drugs, including lumefantrine, confirming that HHQs have a different mode of action to other antimalarials drugs for which PfMDR1 is known to confer resistance. This work evokes therapeutic strategies that combine opposing selective pressures on this parasite transporter as an approach to countering the emergence and transmission of multidrug-resistant P. falciparum malaria.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.

Haematopoiesis is the complex developmental process that maintains the turnover of all blood cell lineages. It critically depends on the correct functioning of rare, quiescent haematopoietic stem cells (HSCs) and more numerous, HSC-derived, highly proliferative and differentiating haematopoietic progenitor cells (HPCs). Infection is known to affect HSCs, with severe and chronic inflammatory stimuli leading to stem cell pool depletion, while acute, non-lethal infections exert transient and even potentiating effects. Both whether this paradigm applies to all infections and whether the HSC response is the dominant driver of the changes observed during stressed haematopoiesis remain open questions. We use a mouse model of malaria, based on natural, sporozoite-driven Plasmodium berghei infection, as an experimental platform to gain a global view of haematopoietic perturbations during infection progression. We observe coordinated responses by the most primitive HSCs and multiple HPCs, some starting before blood parasitaemia is detected. We show that, despite highly variable inter-host responses, primitive HSCs become highly proliferative, but mathematical modelling suggests that this alone is not sufficient to significantly impact the whole haematopoietic cascade. We observe that the dramatic expansion of Sca-1+ progenitors results from combined proliferation of direct HSC progeny and phenotypic changes in downstream populations. We observe that the simultaneous perturbation of HSC/HPC population dynamics is coupled with early signs of anaemia onset. Our data uncover a complex relationship between Plasmodium and its host's haematopoiesis and raise the question whether the variable responses observed may affect the outcome of the infection itself and its long-term consequences on the host.