Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase phoD as a novel determinant of resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites, coupled with the lack of potent inhibitors necessitates the discovery of novel conserved druggable targets for the generation of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of novel and conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of evolutionarily-related Babesia spp. ( B. bovis and B. divergens ). We identified a potent antibabesial inhibitor from the Malaria Box, MMV019266. We were able to select for resistance to this compound in two species of Babesia, achieving 10-fold or greater resistance after ten weeks of intermittent selection. After sequencing of multiple independently derived lines in the two species, we identified mutations in a single conserved gene in both species: a membrane-bound metallodependent phosphatase (putatively named PhoD). In both species, the mutations were found in the phoD-like phosphatase domain, proximal to the predicted ligand binding site. Using reverse genetics, we validated that mutations in PhoD confer resistance to MMV019266. We have also demonstrated that PhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of PhoD alter the sensitivity to MMV019266 in the parasite: overexpression of PhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting PhoD is a resistance mechanism. Together, we have generated a robust pipeline for identification of resistance loci, and identified PhoD as a novel determinant of resistance in Babesia species. Highlights: Use of two species for in vitro evolution identifies a high confidence locus associated with resistance Resistance mutation in phoD was validated using reverse genetics in B. divergens Perturbation of phoD using function genetics results in changes in the level of resistance to MMV019266Epitope tagging reveals localization to the ER/apicoplast, a conserved localization with a similar protein in diatoms Together, phoD is a novel resistance determinant in multiple Babesia spp .

Co-option of Plasmodium falciparum PP1 for egress from host erythrocytes.

Asexual proliferation of the Plasmodium parasites that cause malaria follows a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCα, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCα and find that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.

Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes.

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing Plasmodium spp. and Toxoplasma gondii, a major cause of opportunistic infections. Plasmodium spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications stages, are not followed by karyokinesis. T. gondii divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite Babesia bigemina and the pig parasite Cystoisospora suis. B. bigemina produces two daughters per division round by a "binary fission" mechanism whereas C. suis produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), Sarcocystis neurona, which also undergoes endopolygeny but differs from C. suis by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal vs. external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota.

Targeting Plasmodium Proteases to Block Malaria Parasite Escape and Entry.

Proliferation of malaria parasites in a host requires mechanisms to spread between red blood cells (RBCs). We discuss here the implications for biology and antimalarial drug development of companion studies that establish the requirement of two Plasmodium spp. proteases of the plasmepsin family in parasite egress from, and invasion into, RBCs.

PfCDPK1 mediated signaling in erythrocytic stages of Plasmodium falciparum.

Calcium Dependent Protein Kinases are key effectors of calcium signaling in malaria parasite. PfCDPK1 is critical for asexual development of Plasmodium falciparum, but its precise function and substrates remain largely unknown. Using a conditional knockdown strategy, we here establish that this kinase is critical for the invasion of host erythrocytes. Furthermore, using a multidisciplinary approach involving comparative phosphoproteomics we gain insights into the underlying molecular mechanisms. We identify substrates of PfCDPK1, which includes proteins of Inner Membrane Complex and glideosome-actomyosin motor assembly. Interestingly, PfCDPK1 phosphorylates PfPKA regulatory subunit (PfPKA-R) and regulates PfPKA activity in the parasite, which may be relevant for the process of invasion. This study delineates the signaling network of PfCDPK1 and sheds light on mechanisms via which it regulates invasion.Calcium dependent protein kinase 1 (CDPK1) plays an important role in asexual development of Plasmodium falciparum. Using phosphoproteomics and conditional knockdown of CDPK1, the authors here identify CDPK1 substrates and a cross-talk between CDPK1 and PKA, and show the role of CDPK1 in parasite invasion.

Host Cell Tropism and Adaptation of Blood-Stage Malaria Parasites: Challenges for Malaria Elimination.

Plasmodium falciparum and Plasmodium vivax account for most of the mortality and morbidity associated with malaria in humans. Research and control efforts have focused on infections caused by P. falciparum and P. vivax, but have neglected other malaria parasite species that infect humans. Additionally, many related malaria parasite species infect nonhuman primates (NHPs), and have the potential for transmission to humans. For malaria elimination, the varied and specific challenges of all of these Plasmodium species will need to be considered. Recent advances in molecular genetics and genomics have increased our knowledge of the prevalence and existing diversity of the human and NHP Plasmodium species. We are beginning to identify the extent of the reservoirs of each parasite species in humans and NHPs, revealing their origins as well as potential for adaptation in humans. Here, we focus on the red blood cell stage of human infection and the host cell tropism of each human Plasmodium species. Determinants of tropism are unique among malaria parasite species, presenting a complex challenge for malaria elimination.

Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony.

Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection1. DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly2,3. However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.

Extensive Shared Chemosensitivity between Malaria and Babesiosis Blood-Stage Parasites.

The apicomplexan parasites that cause malaria and babesiosis invade and proliferate within erythrocytes. To assess the potential for common antiparasitic treatments, we measured the sensitivities of multiple species of Plasmodium and Babesia parasites to the chemically diverse collection of antimalarial compounds in the Malaria Box library. We observed that these parasites share sensitivities to a large fraction of the same inhibitors and we identified compounds with strong babesiacidal activity.

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling.

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca(2+) Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca(2+) indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca(2+) signaling in the model apicomplexan Toxoplasma gondii In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca(2+) We define the pool of Ca(2+) regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca(2+) signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca(2+) The enhancers identified are capable of releasing intracellular Ca(2+) stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum Inhibition of Ca(2+)-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca(2+) stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca(2+), underscoring the importance of these pathways and the therapeutic potential of their inhibition.

Parasite Calcineurin Regulates Host Cell Recognition and Attachment by Apicomplexans.

Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse-genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition, and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans.

Malaria. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion.

Efforts to identify host determinants for malaria have been hindered by the absence of a nucleus in erythrocytes, which precludes genetic manipulation in the cell in which the parasite replicates. We used cultured red blood cells derived from hematopoietic stem cells to carry out a forward genetic screen for Plasmodium falciparum host determinants. We found that CD55 is an essential host factor for P. falciparum invasion. CD55-null erythrocytes were refractory to invasion by all isolates of P. falciparum because parasites failed to attach properly to the erythrocyte surface. Thus, CD55 is an attractive target for the development of malaria therapeutics. Hematopoietic stem cell-based forward genetic screens may be valuable for the identification of additional host determinants of malaria pathogenesis.

Host-parasite interactions that guide red blood cell invasion by malaria parasites.

PURPOSE OF REVIEW: Malaria is caused by the infection and proliferation of parasites from the genus Plasmodium in red blood cells (RBCs). A free Plasmodium parasite, or merozoite, released from an infected RBC must invade another RBC host cell to sustain a blood-stage infection. Here, we review recent advances on RBC invasion by Plasmodium merozoites, focusing on specific molecular interactions between host and parasite. RECENT FINDINGS: Recent work highlights the central role of host-parasite interactions at virtually every stage of RBC invasion by merozoites. Biophysical experiments have for the first time measured the strength of merozoite-RBC attachment during invasion. For P. falciparum, there have been many key insights regarding the invasion ligand PfRh5 in particular, including its influence on host species tropism, a co-crystal structure with its RBC receptor basigin, and its suitability as a vaccine target. For P. vivax, researchers identified the origin and emergence of the parasite from Africa, demonstrating a natural link to the Duffy-negative RBC variant in African populations. For the simian parasite P. knowlesi, zoonotic invasion into human cells is linked to RBC age, which has implications for parasitemia during an infection and thus malaria. SUMMARY: New studies of the molecular and cellular mechanisms governing RBC invasion by Plasmodium parasites have shed light on various aspects of parasite biology and host cell tropism, and indicate opportunities for malaria control.

Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum.

Red blood cell invasion by the malaria parasite Plasmodium falciparum relies on a complex protein network that uses low and high affinity receptor-ligand interactions. Signal transduction through the action of specific kinases is a control mechanism for the orchestration of this process. In the present study we report on the phosphorylation of the CPD (cytoplasmic domain) of P. falciparum Rh2b (reticulocyte homologue protein 2b). First, we identified Ser3233 as the sole phospho-acceptor site in the CPD for in vitro phosphorylation by parasite extract. We provide several lines of evidence that this phosphorylation is mediated by PfCK2 (P. falciparum casein kinase 2): phosphorylation is cAMP independent, utilizes ATP as well as GTP as phosphate donors, is inhibited by heparin and tetrabromocinnamic acid, and is mediated by purified PfCK2. We raised a phospho-specific antibody and showed that Ser3233 phosphorylation occurs in the parasite prior to host cell egress. We analysed the spatiotemporal aspects of this phosphorylation using immunoprecipitated endogenous Rh2b and minigenes expressing the CPD either at the plasma or rhoptry membrane. Phosphorylation of Rh2b is not spatially restricted to either the plasma or rhoptry membrane and most probably occurs before Rh2b is translocated from the rhoptry neck to the plasma membrane.

Review of the mechanism of processive actin filament elongation by formins.

We review recent structural and biophysical studies of the mechanism of action of formins, proteins that direct the assembly of unbranched actin filaments for cytokinetic contractile rings and other cellular structures. Formins use free actin monomers to nucleate filaments and then remain bound to the barbed ends of these filaments as they elongate. In addition to variable regulatory domains, formins typically have formin homology 1 (FH1) and formin homology 2 (FH2) domains. FH1 domains have multiple binding sites for profilin, an abundant actin monomer binding protein. FH2 homodimers encircle the barbed end of a filament. Most FH2 domains inhibit actin filament elongation, but FH1 domains concentrate multiple profilin-actin complexes near the end of the filament. FH1 domains transfer actin very rapidly onto the barbed end of the filament, allowing elongation at rates that exceed the rate of elongation by the addition of free actin monomers diffusing in solution. Binding of actin to the end of the filament provides the energy for the highly processive movement of the FH2 as a filament adds thousands of actin subunits. These biophysical insights provide the context to understand how formins contribute to actin assembly in cells. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Energetic requirements for processive elongation of actin filaments by FH1FH2-formins.

Formin-homology (FH) 2 domains from formin proteins associate processively with the barbed ends of actin filaments through many rounds of actin subunit addition before dissociating completely. Interaction of the actin monomer-binding protein profilin with the FH1 domain speeds processive barbed end elongation by FH2 domains. In this study, we examined the energetic requirements for fast processive elongation. In contrast to previous proposals, direct microscopic observations of single molecules of the formin Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed that profilin is not required for formin-mediated processive elongation of growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin release the gamma-phosphate of ATP on average >2.5 min after becoming incorporated into filaments. Therefore, the release of gamma-phosphate from actin does not drive processive elongation. We compared experimentally observed rates of processive elongation by a number of different FH2 domains to kinetic computer simulations and found that actin subunit addition alone likely provides the energy for fast processive elongation of filaments mediated by FH1FH2-formin and profilin. We also studied the role of FH2 structure in processive elongation. We found that the flexible linker joining the two halves of the FH2 dimer has a strong influence on dissociation of formins from barbed ends but only a weak effect on elongation rates. Because formins are most vulnerable to dissociation during translocation along the growing barbed end, we propose that the flexible linker influences the lifetime of this translocative state.

A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion.

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.

The role of the FH1 domain and profilin in formin-mediated actin-filament elongation and nucleation.

BACKGROUND: Formin proteins nucleate actin filaments de novo and stay associated with the growing barbed end. Whereas the formin-homology (FH) 2 domains mediate processive association, the FH1 domains-in concert with the actin-monomer-binding protein profilin-increase the rate of barbed-end elongation. The mechanism by which this effect is achieved is not well understood. RESULTS: We used total internal reflection fluorescence microscopy to measure the effect of profilin on the elongation of single actin filaments associated with FH1FH2 constructs (derived from the formin Bni1p from S. cerevisiae) with FH1 domains containing one to eight profilin-binding polyproline tracks. Over a large range of profilin concentrations (0.5-25 microM), the rate of barbed-end elongation increases with the number of polyproline tracks in the FH1 domain. The binding of profilin-actin to the FH1 domain is the rate-limiting step (up to rates of at least 88 s(-1)) in FH1-mediated transfer of actin subunits to the barbed end. Dissociation of formins from barbed ends growing in the presence of profilin is proportional to the elongation rate. Profilin profoundly inhibits nucleation by FH2 and FH1FH2 constructs, but profilin-actin bound to FH1 might contribute weakly to nucleation. CONCLUSIONS: To achieve fast elongation, formin FH1 domains bind profilin-actin complexes and deliver them rapidly to the barbed end associated with the FH2 domain. Because subunit addition promotes dissociation of FH2 domains from growing barbed ends, FH2 domains must pass through a state that is prone to dissociation during each cycle of actin subunit addition coupled to formin translocation.