Food Vacuole Research Articles

The "pushmi-pullyu" of resistance to chloroquine in malaria.

Malarial infection continues to impart devastating health problems in the developing world. Treatment of malaria has involved chemotherapy since 168 BC, with the most prevalent and successful forms using plant alkaloids. Perhaps the greatest treatment success against malaria was by chloroquine, a synthetic derivative of the quinines found in the Cinchona tree bark. Chloroquine is able to kill parasites by interfering with haem metabolism in the parasite's digestive vacuole. The widespread use of chloroquine predictably resulted in the development of drug-resistant malaria and the most highly implicated resistance mediators are the transporter proteins P-glycoprotein (P-gp) homologue 1 (P-gh1) and Plasmodium falciparum chloroquine-resistance transporter (PfCRT), which reside on the parasite's digestive vacuole. The presence of PfCRT and P-gh1 on the vacuole membrane is analogous to the two-headed fictional creature known as the "Pushmi-Pullyu". P-gh1 (Pushmi) increases influx of chloroquine into the vacuole, while PfCRT (Pullmi) causes efflux of chloroquine from the vacuole. This review describes how drug-resistant malarial parasites co-ordinate chloroquine distribution through adaptive mutations to promote their survival in the presence of this cytotoxic drug.

Quantification of labile heme in live malaria parasites using a genetically encoded biosensor

Heme is ubiquitous, yet relatively little is known about the maintenance of labile pools of this cofactor, which likely ensures its timely bioavailability for proper cellular function. Quantitative analysis of labile heme is of fundamental importance to understanding how nature preserves access to the diverse chemistry heme enables, while minimizing cellular damage caused by its redox activity. Here, we have developed and characterized a protein-based sensor that undergoes fluorescence quenching upon heme binding. By genetically encoding this sensor in the human malarial parasite, Plasmodium falciparum, we have quantified cytosolic labile heme levels in intact, blood-stage parasites. Our findings indicate that a labile heme pool (∼1.6 µM) is stably maintained throughout parasite development within red blood cells, even during a period coincident with extensive hemoglobin degradation by the parasite. We also find that the heme-binding antimalarial drug chloroquine specifically increases labile cytosolic heme, indicative of dysregulation of this homeostatic pool that may be a relevant component of the antimalarial activity of this compound class. We propose that use of this technology under various environmental perturbations in P. falciparum can yield quantitative insights into fundamental heme biology.

Comparative histological description of the digestive and visual system development of larval chame Dormitator latifrons (Pisces: Eleotridae)

This study was focused on the morphological description of chame larvae from one to six days post-hatching (dph), in order to generate information that helps understanding their feeding ability in early stages. The larvae were obtained by hormonal induction of a broodstock, using implants of GnRH synthetic analogues, during the spawning season from September to November 2010. The samples were included in historesin, and stained with hematoxylin-eosin. It was described that, once the larva hatched has a large amount of yolk it is gradually absorbed (from 1 to 3 dph). At day four, there is an open oral cavity with dental structures, the eye development is complete and there was pigmentation on it, the gills are observable and the foregut was differentiated in anterior and posterior intestine, showing the presence of digestive vacuoles in the anterior intestine. By five dph the larvae has developed muscle fibers and a structure identified as the stomach. At sixth dph, it is possible to identify the pancreas, the intestinal folds, and the brush border membrane, and there were food particles and bacteria rests in the gut. Based on the above, it is concluded that the larvae of D. latifrons from the fourth dph fully developed organs and systems that help the search of food, so they are capable of starting with exogenous feeding.

A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs.

Mutational changes in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been associated with differential responses to a wide spectrum of biologically active compounds including current and former quinoline and quinoline-like antimalarial drugs. PfCRT confers altered drug responsiveness by acting as a transport system, expelling drugs from the parasite's digestive vacuole where these drugs exert, at least part of, their antiplasmodial activity. To preserve the efficacy of these invaluable drugs, novel functional tools are required for epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of 7-chloro-N-{2-[(propan-2-yl)amino]ethyl}quinolin-4-amine functionalized with the fluorochrome 7-nitrobenzofurazan (NBD) (henceforth termed Fluo-CQ). In the parasite, Fluo-CQ accumulates in the digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The differential handling of the fluorescent probe, combined with live cell imaging, provides a diagnostic tool for quick detection of those P.falciparum strains that carry a PfCRT variant associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-resistant to Fluo-CQ when the chemical probe was tested in various CQ-sensitive and -resistant parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a coupled assay based on methemoglobin and the NADPH-dependent glutathione reductase (GRs) from P.falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ on redox equilibrium and methemoglobin reduction via PfCRT-mediated drug efflux in the cytosol and then continuous redox-dependent shuttling between food vacuole and cytosol. Taking into account these physicochemical characteristics, a model was proposed to explain Fluo-CQ antimalarial effects involving the contribution of PfCRT-mediated transport, methemoglobin reduction, hematin binding, and NBD reduction activity catalyzed by PfGR in CQ-resistant versus CQ-sensitive parasites. Therefore, introduction of NBD fluorophore in drugs is not inert and should be taken into account in drug transport and imaging studies.

Effects of Bacillus cereus Endospores on Free-Living Protist Growth.

We studied the predator-prey interactions between heterotrophic protists and endospores of Bacillus cereus group bacteria, in order to gain insight on survival and dispersal of B. cereus endospores in the environment. It has been hypothesised that the spore stage protects against digestion by predating protists. Therefore, experiments were carried out to investigate the impact of B. cereus endospores and vegetative cells, as the only food source, on individual amoeboid, flagellated and ciliated protists. The presence of fluorescent-labelled intracellular bacteria confirmed that B. cereus endospores as well as vegetative cells were ingested by protists and appeared intact in the food vacuoles when observed by epifluorescence microscopy. Furthermore, protist growth and bacterial predation were followed by qPCR. Protists were able to grow on vegetative cells as well as endospores of B. cereus, despite the lower cell division rates observed for some protists when feeding on bacterial endospores. Survival and proliferation of ingested bacteria inside protists cells was also observed. Finally, B. cereus spore germination and growth was observed within all protists with higher abundance in the amoeboid protist after antibiotic treatment of the protist surface. These observations support that protists can act as a potential breeding ground for B. cereus endospores.

Dinotefuran-induced morphophysiological changes in semi-engorged females Rhipicephalus sanguineus Latreille, 1806 (Acari: Ixodidae) ticks: Ultra-structural evaluation

The present study demonstrated the effects of dinotefuran (active ingredient of the acaricide Protetor Pet®) on the ovary and midgut cells of semi engorged R. sanguineus females exposed to different concentrations of this chemical. For this, 120 semi-engorged females were divided into four treatment groups with 30 individuals each: group I or control (distilled water), group II (5000ppm), groups III (6250ppm) and group IV (8334ppm of dinotefuran). All the ticks were immersed in the different concentrations of dinotefuran or in distilled water for 5min and then dried and kept in BOD incubator for 7days. The results showed alterations mainly regarding the damaged cell structures, such as yolk granules, organelles and the plasma membrane of the germ cells. In addition, structures related with defense mechanisms were found, such as vacuoles, cytoskeletal filaments, and myelin figures in the germ cells. Damages in the generative cells of the midgut, alterations in the size of digestive cells, the number of endosomes, digestive vacuoles, digestive residues, lipid drops and organelles in the cytoplasm of the digestive cells and the presence of microvilli in the plasma membrane of these cells also demonstrate the progressive damages caused by the action of dinotefuran in the midgut and germ cells of R. sanguineus semi-engorged females. The concentrations applied partially impaired the digestive processes; and, without proper nutrition, all the ectoparasite’s physiologic events are prevented from occurring, leading the individual to death. The germ cells were also damaged, and probably would not be able to advance in their development (I–V) and complete the vitellogenesis, which would affect the fertility of the female and consequently impede the formation of a new individual.

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology.

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens.

The Synechococcus cell surface protein SwmA increases vulnerability to predation by flagellates and ciliates

AbstractThe genus Synechococcus, a major contributor to ocean productivity, exhibits wide genetic variability and a distinct biogeography of genetic subgroups. Synechococcus sp. strain WH8102 is a motile Sargasso Sea isolate belonging to Clade III in subcluster 5.1. Non‐flagellar motility in WH8102 depends upon two large cell surface proteins, SwmA and SwmB. In the marine plankton, Synechococcus can experience high rates of mortality from protist predators, suggesting strong selective pressure for the maintenance of anti‐predation defenses. We used knockout mutants deficient in large cell surface proteins (SwmA‐ and SwmB‐) to test the hypothesis that these proteins defend WH8102 against predation by ciliates and flagellates, important consumers of Synechococcus in aquatic ecosystems. Contrary to our hypothesis, we found that wild‐type WH8102 were preyed upon at higher rates than SwmA‐ by ciliates and by two of three tested flagellate species. Thus the SwmA protein, constitutively produced by WH8102, made this strain more rather than less vulnerable to predation. SwmA proteins link to form the S layer, a cell coating known to promote adhesion in other microbial systems, suggesting the predation vulnerability arises from increased attachment between consumers and prey. Given the SwmA‐related enhancement of predation rates, our findings suggest two possibilities for WH8102 and perhaps other Clade III Synechococcus: (1) factors other than protist predation select for production of the S layer; (2) ingestion actually increases fitness, by exposing digestion‐resistant cells to beneficial conditions in the food vacuole.

An evolutionary balance: conservation vs innovation in ciliate membrane trafficking

As most of eukaryotic diversity lies in single-celled protists, they represent unique opportunities to ask questions about the balance of conservation and innovation in cell biological features. Among free-living protists the ciliates offer ease of culturing, a rich array of experimental approaches, and versatile molecular tools, particularly in Tetrahymena thermophila and Paramecium tetraurelia. These attributes have been exploited by researchers to analyze a wealth of cellular structures in these large and complex cells. This mini-review focuses on 3 aspects of ciliate membrane dynamics, all linked with endolysosomal trafficking. First is nutrition based on phagocytosis and maturation of food vacuoles. Secondly, we discuss regulated exocytosis from vesicles that have features of both dense core secretory granules but also lysosome-related organelles. The third topic is the targeting, breakdown and resorption of parental nuclei in mating partners. For all 3 phenomena, it is clear that elements of the canonical membrane-trafficking system have been retained and in some cases repurposed. In addition, there is evidence that recently evolved, lineage-specific proteins provide determinants in these pathways.

In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.

Design, Synthesis and Evaluation of Bifunctional Acridinine-Naphthalenediimide Redox-Active Conjugates as Antimalarials.

A novel class of bifunctional molecules was synthesized integrating acridine (Ac) and redox-active naphthalenediimide (NDI) scaffolds directly and through a flexible linker (en). We evaluated in vitro antiplasmodial activity, physicochemical properties, and a possible mode of action. Theoretical studies suggested electronic segmentation between the electron-rich Ac and electron-deficient NDI scaffolds. Orthogonal Ac–NDI molecules showed activities in the micromolar to submicromolar range against a chloroquine (CQ)-sensitive strain of human malaria pathogen Plasmodium falciparum (maximum activity, IC50: 0.419 μM). The flexible Ac–en–NDI molecules were most potent and showed activity in the nanomolar range against both CQ-sensitive (with most effective compounds, IC50: 3.65 and 4.33 nM) as well as CQ-resistant (with most effective compounds, IC50: 52.20 and 28.53 nM) strains of P. falciparum. Significantly, with CQ-resistant strains, the activity of the most effective compounds was 1 order of magnitude better than that of standard drug CQ. Ac–en–NDI-conjugated molecules were significantly more potent than the individual NDI and Ac-based molecules. The structure–activity relationship (SAR) suggests that the flexible spacer (en) linking the Ac and NDI scaffolds plays a vital role in exhibiting improved potency. None of the molecules triggered hemolysis in culture, and the most potent compounds did not show cytotoxicity in vitro against mammalian fibroblast NIH3T3 cells at their respective IC50 values. The other significant outcome of this work is that some of the investigated molecules have the potential to affect multiple processes in the parasite including the hemozoin formation in digestive vacuoles (DVs), mitochondrial membrane potential, and the redox homeostasis of the parasite.

Light- and Electron-microscopical Study of Belonocystis marina sp. nov. (Eukaryota: incertae sedis)

BelonocystisRainer, 1968 is an enigmatic protist genus, which currently lacks any supergroup affiliation. The spherical cells of this organism move on the substratum using fine non-branching pseudopodia. The cell surface is surrounded with a spiky covering. Belonocystis marina sp. nov. was studied using light- and electron microscopy. It was clearly shown that the surface structures of Belonocystis were scales, not a capsule. The new species could be distinguished by the morphology of the scales, which had a bulbous base with three "skirts" (circular latticed structures) and a spike consisting of many twisted fibrils. Each scale was associated with a short cytoplasmic outgrowth. The organic nature of these scales was confirmed by energy-dispersive X-ray microanalysis. Large multinucleated stages were discovered in the life-cycle of this organism. A survey of the cell ultrastructure revealed all the common eukaryotic organelles, including mitochondria with tubular cristae. No microtubules were detected in ultrathin sections of pseudopodia. Examination of food vacuole contents confirmed that this organism was bacterivorous. The finding of Belonocystis marina is the first record of the genus in a marine habitat. Many similarities in the scale structure and fine structure of the cell between Belonocystis and Luffisphaera were discussed.

Compstatin Cp40 blocks hematin-mediated deposition of C3b fragments on erythrocytes: Implications for treatment of malarial anemia

During malarial anemia, 20 uninfected red blood cells (RBCs) are destroyed for every RBC infected by Plasmodium falciparum (Pf). Increasing evidence indicates an important role for complement in destruction of uninfected RBCs. Products of RBC lysis induced by Pf, including the digestive vacuole and hematin, activate complement and promote C3 fragment deposition on uninfected RBCs. C3-opsonized cells are then subject to extravascular destruction mediated by fixed tissue macrophages which express receptors for C3 fragments. The Compstatin family of cyclic peptides blocks complement activation at the C3 cleavage step, and is under investigation for treatment of complement-mediated diseases. We demonstrate, that under a variety of stringent conditions, second-generation Compstatin analogue Cp40 completely blocks hematin-mediated deposition of C3 fragments on naïve RBCs. Our findings indicate that prophylactic provision of Compstatin for malaria-infected individuals at increased risk for anemia may provide a safe and inexpensive treatment to prevent or substantially reduce malarial anemia.

Influence of LAR and VAR on Para-Aminopyridine Antimalarials Targetting Haematin in Chloroquine-Resistance.

Antimalarial chloroquine (CQ) prevents haematin detoxication when CQ-base concentrates in the acidic digestive vacuole through protonation of its p-aminopyridine (pAP) basic aromatic nitrogen and sidechain diethyl-N. CQ export through the variant vacuolar membrane export channel, PFCRT, causes CQ-resistance in Plasmodium falciparum but 3-methyl CQ (sontochin SC), des-ethyl amodiaquine (DAQ) and bis 4-aminoquinoline piperaquine (PQ) are still active. This is determined by changes in drug accumulation ratios in parasite lipid (LAR) and in vacuolar water (VAR). Higher LAR may facilitate drug binding to and blocking PFCRT and also aid haematin in lipid to bind drug. LAR for CQ is only 8.3; VAR is 143,482. More hydrophobic SC has LAR 143; VAR remains 68,523. Similarly DAQ with a phenol substituent has LAR of 40.8, with VAR 89,366. In PQ, basicity of each pAP is reduced by distal piperazine N, allowing very high LAR of 973,492, retaining VAR of 104,378. In another bis quinoline, dichlorquinazine (DCQ), also active but clinically unsatisfactory, each pAP retains basicity, being insulated by a 2-carbon chain from a proximal nitrogen of the single linking piperazine. While LAR of 15,488 is still high, the lowest estimate of VAR approaches 4.9 million. DCQ may be expected to be very highly lysosomotropic and therefore potentially hepatotoxic. In 11 pAP antimalarials a quadratic relationship between logLAR and logResistance Index (RI) was confirmed, while log (LAR/VAR) vs logRI for 12 was linear. Both might be used to predict the utility of structural modifications.

Four important cytological events needed to establish endosymbiosis of symbiotic Chlorella sp. to the alga-free Paramecium bursaria

SUMMARY Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole (PV) derived from the host digestive vacuole (DV) to protect from lysosomal fusion. Irrespective of the mutual relationships between P. bursaria and their symbiotic algae, the alga-free paramecia and symbiotic algae still have an ability to grow independently and can be experimentally reinfected by mixing them. This phenomenon provides an excellent opportunity to elucidate cell-to-cell interactions between protozoa and algae during establishment of the secondary endosymbiosis. However, the detailed algal reinfection process had been unclear. Therefore, using pulse-label of the alga-free paramecia with the isolated symbiotic algae and chase method, we found four important cytological events needed to establish endosymbiosis. (1) 3 min after mixing, some algae show resistance to the host lysosomal enzymes in the DVs even if the digested ones are coexisted. (2) 30 min after mixing, the alga starts to leave from the DV to appear in the cytoplasm by budding of the DV membrane. (3) Within 15 min after the algal appearance in the cytoplasm, the vacuole enclosing a single green alga differentiates into the PV from the DV, which gives protection from the host lysosomal fusion. (4) After that, the alga localizes beneath the host cell cortex. At about 24 h after mixing, the alga increases by cell division and establishes endosymbiosis. In this review article, we describe our recent studies on the four events during the algal reinfection process.

Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite’s Chloroquine Resistance Transporter

Mutations in the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite’s digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite’s hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite’s survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite’s hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite’s sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.

Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters.

Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.

Imaging flow cytometry for the screening of compounds that disrupt the Plasmodium falciparum digestive vacuole

Malaria, despite being one of the world’s oldest infectious diseases, remains difficult to eradicate because the parasite is rapidly developing resistance to frontline chemotherapies. Previous studies have shown that the parasite exhibits features resembling programmed cell death upon treatment with drugs that disrupt its digestive vacuole (DV), providing a phenotypic readout that can be detected using the imaging flow cytometer. Large compound collections can thus be screened to identify drugs that are able to disrupt the DV of the malaria parasite using this high-content high-throughput screening platform. As a proof-of-concept, 4440 compounds were screened using this platform in 4months and 254 hits (5.7% hit rate) were obtained. Additionally, 25 compounds (0.6% top hit rate) were observed to retain potent DV disruption activity that was comparable to the canonical DV disruptive drug chloroquine when tested at a ten-fold lower concentration from the original screen. This pilot study demonstrates the robustness and high-throughput capability of the imaging flow cytometer and we report herein the methodology of this screening assay.

Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa

Archigregarines, an early branching lineage within Apicomplexa, are a poorly-known group of invertebrate parasites. By their phylogenetic position, archigregarines are an important lineage to understand the functional transition that occurred between free-living flagellated predators to obligatory parasites in Apicomplexa. In this study, we provide new ultrastructural data and phylogenies based on SSU rDNA sequences using the type species of archigregarines, the Selenidiidae Selenidium pendulaGiard, 1884. We describe for the first time the syzygy and early gamogony at the ultrastructural level, revealing a characteristic nuclear multiplication with centrocones, cryptomitosis, filamentous network of chromatin, a cyst wall secretion and a 9+0 flagellar axoneme of the male gamete. S. pendula belongs to a monophyletic lineage that includes several other related species, all infecting Sedentaria Polychaeta (Spionidae, Sabellaridae, Sabellidae and Cirratulidae). All of these Selenidium species exhibit similar biological characters: a cell cortex with the plasma membrane - inner membrane complex - subpellicular microtubule sets, an apical complex with the conoid, numerous rhoptries and micronemes, a myzocytosis with large food vacuoles, a nuclear multiplication during syzygy and young gamonts. Two other distantly related Selenidium-like lineages infect Terebellidae and Sipunculida, underlying the ability of archigregarines to parasite a wide range of marine hosts.

CRISPR-Cas9-modified pfmdr1 protects Plasmodium falciparum asexual blood stages and gametocytes against a class of piperazine-containing compounds but potentiates artemisinin-based combination therapy partner drugs.

Emerging resistance to first-line antimalarial combination therapies threatens malaria treatment and the global elimination campaign. Improved therapeutic strategies are required to protect existing drugs and enhance treatment efficacy. We report that the piperazine-containing compound ACT-451840 exhibits single-digit nanomolar inhibition of the Plasmodium falciparum asexual blood stages and transmissible gametocyte forms. Genome sequence analyses of in vitro-derived ACT-451840-resistant parasites revealed single nucleotide polymorphisms in pfmdr1, which encodes a digestive vacuole membrane-bound ATP-binding cassette transporter known to alter P. falciparum susceptibility to multiple first-line antimalarials. CRISPR-Cas9 based gene editing confirmed that PfMDR1 point mutations mediated ACT-451840 resistance. Resistant parasites demonstrated increased susceptibility to the clinical drugs lumefantrine, mefloquine, quinine and amodiaquine. Stage V gametocytes harboring Cas9-introduced pfmdr1 mutations also acquired ACT-451840 resistance. These findings reveal that PfMDR1 mutations can impart resistance to compounds active against asexual blood stages and mature gametocytes. Exploiting PfMDR1 resistance mechanisms provides new opportunities for developing disease-relieving and transmission-blocking antimalarials.