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Malaria hypnozoites are dormant parasite stages that reside inside hepatocytes. Upon activation, these stages can resume growth, causing new episodes of blood stage malaria infection. This chapter describes a fast and sensitive protocol for the detection of bioluminescent (BL) hypnozoites in vitro. Using transgenic Plasmodium cynomolgi parasites that differentially express the BL reporter proteins firefly luciferase and the ultrabright NanoLuc, hypnozoites can be distinguished from liver stage schizonts. This robust method sets the stage for implementation in large-scale drug screening platforms with the aim to find new compounds that eliminate hypnozoites.

Plasmodium cynomolgi (Pc) is one of the few parasite species that forms quiescent liver stage parasites known as hypnozoites and is therefore a suitable model for Plasmodium vivax. Very little is known about liver stage dormancy, which hampers the search for compounds with anti-hypnozoite activity. Here, we present the development of a Pc in vitro infection model using stem cell-derived hepatocytes from Macaca fascicularis. IPS cells were established on feeder free condition and differentiated into hepatocytes via inducible overexpression of key transcription factors. The generated hepatocytes were infected with Pc sporozoites and hypnozoite formation as well as schizont development were confirmed by immunofluorescence. This system is a promising tool to study the mechanisms underlying liver stage dormancy and facilitate drug discovery against hypnozoites.

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria.

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic P. cynomolgi parasite line that contains Nanoluc driven by the constitutive hsp70 promoter, as well as firefly luciferase driven by the schizont-specific lisp2 promoter. The transgenic parasite line showed similar fitness and drug sensitivity profiles of selected compounds to wild type. We demonstrate robust bioluminescence-based detection of hypnozoites in 96-well and 384-well plate formats, setting the stage for implementation in large scale drug screens.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts., Present understanding of Plasmodium vivax biology is hampered by its inability to grow in vitro. Here, the authors developed an in vitro culture of its simian counterpart, P. cynomolgi, which shares morphological and phenotypic similarities with P. vivax, initiating a new phase in vivax research.

Non-human primates (NHP) have been used extensively in the identification of new antimalarial compounds. Especially the Plasmodium cynomolgi/rhesus monkey model was a very popular substitute to evaluate compounds for P. vivax liver- and blood stages. Nowadays we have replaced, as much as possible, the NHP in vivo testing with in vitro assays. In this review, an overview is given on malaria drug testing in NHP, the development of the in vitro models and especially the in vitro hypnozoite assay, in which compounds can be tested for activity against dormant liver stage parasites. Benefits and limitations of this model will be discussed.

Relapses of Plasmodium dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old P. cynomolgi liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al., 2017). We now extend these findings by transcriptome profiling of 9 to 10 day-old liver stage parasites, thus revealing for the first time the maturation of the dormant stage over time. Although progression of dormancy leads to a 10-fold decrease in transcription and expression of only 840 genes, including genes associated with housekeeping functions, we show that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites.

The emergence of drug-resistant malaria parasites continues to hamper efforts to control this lethal disease. Dihydroorotate dehydrogenase has recently been validated as a new target for the treatment of malaria, and a selective inhibitor (DSM265) of the Plasmodium enzyme is currently in clinical development. With the goal of identifying a backup compound to DSM265, we explored replacement of the SF5-aniline moiety of DSM265 with a series of CF3-pyridinyls while maintaining the core triazolopyrimidine scaffold. This effort led to the identification of DSM421, which has improved solubility, lower intrinsic clearance, and increased plasma exposure after oral dosing compared to DSM265, while maintaining a long predicted human half-life. Its improved physical and chemical properties will allow it to be formulated more readily than DSM265. DSM421 showed excellent efficacy in the SCID mouse model of P. falciparum malaria that supports the prediction of a low human dose (

Two Plasmodium PI4 kinase (PI4K) inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical-cure agents for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on their in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and compounds were dosed orally. Both the KDU691 and LMV599 compounds were fully protective when administered prophylactically, and the more potent compound LMV599 achieved protection as a single oral dose of 25 mg/kg of body weight. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 or 25 mg/kg of LMV599 did not prevent relapse, as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. Pharmacokinetic data show that LMV599 achieved plasma exposure that was sufficient to achieve efficacy based on our in vitro data. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess these compounds' activity on established hypnozoites and predict radical cure in vivo .

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.

SUMMARYThe primate malariaPlasmodium knowlesihas a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite–host interactions. The adaptation to long-termin vitrocontinuous blood stage culture in rhesus monkey,Macaca fascicularisand human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model. The growing evidence thatP. knowlesiis an important human zoonosis in South-East Asia has added relevance to former and future studies of this parasite species.

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase ( Plasmodium falciparum NDH2) and cytochrome bc 1 . Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites ( Plasmodium cynomolgi ) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Contains fulltext : 177492.pdf (Publisher’s version ) (Closed access) As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro -cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax ). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC 50 ] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi. Here, we present a protocol for long-term in vitro cultivation of P. cynomolgi-infected M. fascicularis primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment, we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation. The protocol presented may further enable investigations of hypnozoite biology and the search for compounds that kill hypnozoites or disrupt their quiescence.

Regulation of currently identified genes involved in pyruvate metabolism of Kluyveromyces lactis strain CBS 2359 was studied in glucose-limited, ethanol-limited and acetate-limited chemostat cultures and during a glucose pulse added to a glucose-limited steady-state culture. Enzyme activity levels of the pyruvate dehydrogenase complex, pyruvate decarboxylase, alcohol dehydrogenase, acetyl-CoA synthetase and glucose-6-phosphate dehydrogenase were determined in all steady-state cultures. In addition, the mRNA levels of KlADH1-4, KlACS1, KlACS2, KlPDA1, KlPDC1 and RAG1 were monitored under steady-state conditions and during glucose pulses. In K. lactis, as in Saccharomyces cerevisiae, enzymes involved in glucose utilization (glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, pyruvate decarboxylase) showed the highest expression levels on glucose, whereas enzymes required for ethanol or acetate consumption (alcohol dehydrogenase, acetyl-CoA synthetase) showed the highest enzyme activities on ethanol. In cases where mRNA levels were determined, these corresponded well with the corresponding enzyme activities, suggesting that regulation is mostly achieved at the transcriptional level. Surprisingly, the activity of the K. lactis pyruvate dehydrogenase complex appeared to be regulated at the level of KlPDA1 transcription. The conclusions from the steady-state cultures were corroborated by glucose pulse experiments. Overall, expression of the enzymes of pyruvate metabolism in the Crabtree-negative yeast K. lactis appeared to be regulated in the same way as in Crabtree-positive S. cerevisiae, with one notable exception: the PDA1 gene encoding the E1alpha subunit of the pyruvate dehydrogenase complex is expressed constitutively in S. cerevisiae.

We describe a PCR-confirmed case of Plasmodium knowlesi infection with a high parasitemia level and clinical signs of severe malaria in a migrant worker from Malaysian Borneo in the Netherlands. Investigations showed that commercially available rapid antigen tests for detection of human Plasmodium infections can detect P. knowlesi infections in humans.

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation ('Crabtree effect'). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostate cultures (D = 0.05-0.40 h-1) growth was entirely respiratory, without significant accumulation of ethanol or other metabolities. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D = 0.10 h-1) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D = 0.10 h-1) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at mu = 0.10 h to growth at mu max. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation.

The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite's life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite's mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria. © 2013 Macmillan Publishers Limited. All rights reserved.

Long-term in vitro cultures of blood-stage parasites are so far feasible only for Plasmodium falciparum and P. knowlesi. In this chapter, we describe short-term ex vivo culturing of P. cynomolgi and P. vivax. We also describe long-term in vitro culturing of P. knowlesi as well as some techniques for synchronizing parasites. Cultured parasites can be used for a variety of purposes, e.g., for in vitro drug assays and antibody-mediated growth inhibition assays.

Long-term in vitro cultures of blood-stage parasites are so far feasible only for Plasmodium falciparum and P. knowlesi. In this chapter, we describe short-term ex vivo culturing of P. cynomolgi and P. vivax. We also describe long-term in vitro culturing of P. knowlesi as well as some techniques for synchronizing parasites. Cultured parasites can be used for a variety of purposes, e.g., for in vitro drug assays and antibody-mediated growth inhibition assays.

The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs.

Contains fulltext : 96475.pdf (Publisher’s version ) (Open Access) BACKGROUND: Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites. METHODOLOGY: P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured. RESULTS: The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine. CONCLUSION: Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs.

Cytidine diphosphate diacylglycerol synthase (CDS) diverts phosphatidic acid towards the biosynthesis of CDP-DAG, an obligatory liponucleotide intermediate in anionic phospholipid biosynthesis. The 78 kDa predicted Plasmodium falciparum CDS (PfCDS) is recovered as a 50 kDa conserved C-terminal cytidylyltransferase domain (C-PfCDS) and a 28 kDa fragment that corresponds to the unusually long hydrophilic asparagine-rich N-terminal extension (N-PfCDS). Here, we show that the two fragments of PfCDS are the processed forms of the 78 kDa pro-form that is encoded from a single transcript with no alternate translation start site for C-PfCDS. PfCDS, which shares 54% sequence identity with Plasmodium knowlesi CDS (PkCDS), could substitute for PkCDS in P. knowlesi. Experiments to disrupt either the full-length or the N-terminal extension of PkCDS indicate that not only the C-terminal cytidylyltransferase domain but also the N-terminal extension is essential to Plasmodium spp. PkCDS and PfCDS introduced in P. knowlesi were processed in the parasite, suggesting a conserved parasite-dependent mechanism. The N-PfCDS appears to be a peripheral membrane protein and is trafficked outside the parasite to the parasitophorous vacuole. Although the function of this unusual N-PfCDS remains enigmatic, the study here highlights features of this essential gene and its biological importance during the intra-erythrocytic cycle of the parasite.

Plasmodium knowlesi is a primate malaria parasite that is phylogenetically close to the major human parasite Plasmodium vivax . P. knowlesi causes life-threatening disease in humans, infects a wide range of non-human primates and is one of few malaria parasites amenable to cyclical in vitro propagation. A robust in vivo and in vitro genetic manipulation system has been developed for this parasite, enabling in vitro–in vivo shuttling of transgenes, which (together with recent characterization of its genome and that of its macaque experimental host) offers unique opportunities to gain insight in molecular function and parasite–host interactions.

A new type of piroplasm, phylogenetically closest to Babesia microti-like parasites previously detected in Eurasian red squirrels (Sciurus vulgaris orientis), was identified in a rhesus monkey (Macaca mulatta) imported from China. After challenge with Plasmodium cynomolgi M strain blood-stage parasites, the rhesus monkey repeatedly showed markedly reduced levels of Plasmodium parasitemia when compared with animals not infected with this organism.

A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210–219) and RVDDVVNVSG (ScAcs1p; 574–583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1–0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells. The nucleotide sequence of a common 5269 bp fragment has been deposited in the EMBL Data Library under Accession No. AJ314837., Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI P/AGR/11135/98

A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.

Two Kluyveromyces lactis genes encoding acetyl co-enzyme A synthetase isoenzymes were isolated. One we named KlACS1, as it has high similarity to the ACS1 gene of Saccharomyces cerevisiae. The other gene, KlACS2, showed more similarity to S. cerevisiae ACS2 than to KlACS1 or ScACS1. This suggests that divergence of the two isogenes occurred before the evolutionary separation of the species and that the different functions have been conserved. In line with this idea is the regulation of transcription of the genes. The mode of regulation appeared to be maintained between ScACS1 and KlACS1 and between ScACS2 and KlACS2. The KlACS1 transcript was absent in glucose-grown cells, whereas transcription levels in ethanol- and acetate-grown cells were high. Disruption of the KlACS1 gene did not result in growth defects on glucose or ethanol. The growth rate on acetate, however, was reduced by a factor of two. KlACS2 was expressed at similar levels during growth on glucose and acetate, whereas expression on ethanol was slightly higher. A null mutant in this gene showed a reduced growth rate on all three carbon sources. Taken together, these data suggest that KlACS2 is used during growth on glucose and that KlACS1 is most dominant during growth on acetate. Strains in which both ACS genes are deleted could only be retrieved when a plasmid containing the ACS2 gene was present, suggesting that the double mutant is lethal. Tetrad analysis confirmed that non-viable spores with a deduced Klacs1Klacs2 genotype germinated but could not divide further. It therefore appears that, as in S. cerevisiae, the pyruvate dehydrogenase bypass formed by the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl co-enzyme A synthetase is essential for growth. These results are in apparent contradiction with the growth on glucose of a strain with a disruption in the only structural pyruvate decarboxylase gene of K. lactis. Residual enzyme activity might, however, account for this discrepancy, or Acs fulfils an additional as yet unknown function, separate from its enzymatic activity. The sequences of KlACS1 and KlACS2 have been deposited in the EMBL database under Accession Nos AF061265 and AF134491, respectively. Copyright © 2002 John Wiley & Sons, Ltd.

VASA is so far the only known gene in mammals whose expression is specific for the germ cell lineage. We investigated the presence of VASA mRNA and protein in a series of germ cell tumors of different histologic subtypes and anatomic location, as well as in nongerm cell tumors such as testicular lymphomas and Leydig cell tumors. We detected VASA mRNA (by quantitative RT-PCR) and protein (by immunohistochemical staining) in normal spermatogenesis, seminoma (both classic and spermatocytic), carcinoma in situ (the precursor of classic seminoma and nonseminoma), dysgerminoma, and gonadoblastoma. VASA immunostaining was relatively weak in seminomas and dysgerminomas compared with spermatocytic seminomas, despite similar mRNA levels, suggesting that VASA is regulated in part by post-transcriptional mechanisms. A higher staining intensity compared with the invasive counterparts was observed in the precursor lesions (ie, carcinoma in situ and gonadoblastoma). No VASA mRNA or protein was detectable in nonseminomatous germ cell tumors (such as embryonal carcinoma, teratoma, and yolk sac tumor) and derived cell lines, or nongerm cell tumors such as lymphoma or Leydig cell tumor. These results provide direct evidence that some germ cell tumors retain germ cell characteristics, whereas other tumors of germ cell origin result from differentiation and loss of germ cell identity. Furthermore, these findings suggest that VASA is likely to serve as a useful and highly specific biomarker for germ cell tumors, particularly classic and spermatocytic seminoma/dysgerminoma, including their precursor stages.