Hydroxamate-based compounds are potent inhibitors of Toxoplasma gondii HDAC biological activity.

Toxoplasmosis is a critical health issue for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii that is found worldwide. Although efficient drugs are commonly used to treat toxoplasmosis, serious adverse events are common. Therefore, new compounds with potent anti-T. gondii activity are needed to provide better suited treatments. We have tested compounds designed to target specifically histone deacetylase enzymes. Among the 55 compounds tested, we identified three compounds showing a concentration of drug required for 50% inhibition (IC50) in the low 100 nM range with a selectivity index of more than 100. These compounds are not only active at inhibiting the growth of the parasite in vitro but also at preventing some of the consequences of the acute disease in vivo. Two of these hydroxamate based compound also induce a hyper-acetylation of the parasite histones while the parasitic acetylated tubulin level remains unchanged. These findings suggest that the enzymes regulating histone acetylation are potent therapeutic targets for the treatment of acute toxoplasmosis.

Mapping PP1c and Its Inhibitor 2 Interactomes Reveals Conserved and Specific Networks in Asexual and Sexual Stages of Plasmodium.

Malaria parasites require multiple phosphorylation and dephosphorylation steps to drive signaling pathways for proper differentiation and transformation. Several protein phosphatases, including protein phosphatase 1 (PP1), one of the main dephosphorylation enzymes, have been shown to be indispensable for the Plasmodium life cycle. The catalytic subunit of PP1 (PP1c) participates in cellular processes via dynamic interactions with a vast number of binding partners that contribute to its diversity of action. In this study, we used Plasmodium berghei transgenic parasite strains stably expressing PP1c or its inhibitor 2 (I2) tagged with mCherry, combined with the mCherry affinity pulldown of proteins from asexual and sexual stages, followed by mass spectrometry analyses. Mapped proteins were used to identify interactomes and to cluster functionally related proteins. Our findings confirm previously known physical interactions of PP1c and reveal enrichment of common biological processes linked to cellular component assembly in both schizonts and gametocytes to biosynthetic processes/translation in schizonts and to protein transport exclusively in gametocytes. Further, our analysis of PP1c and I2 interactomes revealed that nuclear export mediator factor and peptidyl-prolyl cis-trans isomerase, suggested to be essential in P. falciparum, could be potential targets of the complex PP1c/I2 in both asexual and sexual stages. Our study emphasizes the adaptability of Plasmodium PP1 and provides a fundamental study of the protein interaction landscapes involved in a myriad of events in Plasmodium, suggesting why it is crucial to the parasite and a source for alternative therapeutic strategies.

Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission.

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle.

The Drosophila melanogaster antimicrobial peptides Mtk-1 and Mtk-2 are active against the malarial parasite Plasmodium falciparum.

Antimicrobial peptides (AMPs) are important components of the vertebrate and invertebrate innate immune systems. Although AMPs are widely recognized for their broad-spectrum activity against bacteria, fungi, and viruses, their activity against protozoan parasites has not been investigated in detail. In this study, we tested 10 AMPs from three different insect species: the greater wax moth Galleria mellonella (cecropin A-D), the fruit fly Drosophila melanogaster (drosocin, Mtk-1 and Mtk-2), and the blow fly Lucilia sericata (LSerPRP-2, LSerPRP-3 and stomoxyn). We tested each AMP against the protozoan parasite Plasmodium falciparum which is responsible for the most severe form of malaria in humans. We also evaluated the impact of these insect AMPs on mouse and pig erythrocytes. Whereas all AMPs showed low hemolytic effects towards mouse and pig erythrocytes, only D. melanogaster Mtk-1 and Mtk-2 significantly inhibited the growth of P. falciparum at low concentrations. Mtk-1 and Mtk-2 could therefore be considered as leads for the development of antiparasitic drugs targeting the clinically important asexual blood stage of P. falciparum.

Analysis of the interactome of the Ser/Thr Protein Phosphatase type 1 in Plasmodium falciparum.

BACKGROUND: Protein Phosphatase 1 (PP1) is an enzyme essential to cell viability in the malaria parasite Plasmodium falciparum (Pf). The activity of PP1 is regulated by the binding of regulatory subunits, of which there are up to 200 in humans, but only 3 have been so far reported for the parasite. To better understand the P. falciparum PP1 (PfPP1) regulatory network, we here report the use of three strategies to characterize the PfPP1 interactome: co-affinity purified proteins identified by mass spectrometry, yeast two-hybrid (Y2H) screening and in silico analysis of the P. falciparum predicted proteome. RESULTS: Co-affinity purification followed by MS analysis identified 6 PfPP1 interacting proteins (Pips) of which 3 contained the RVxF consensus binding, 2 with a Fxx[RK]x[RK] motif, also shown to be a PP1 binding motif and one with both binding motifs. The Y2H screens identified 134 proteins of which 30 present the RVxF binding motif and 20 have the Fxx[RK]x[RK] binding motif. The in silico screen of the Pf predicted proteome using a consensus RVxF motif as template revealed the presence of 55 potential Pips. As further demonstration, 35 candidate proteins were validated as PfPP1 interacting proteins in an ELISA-based assay. CONCLUSIONS: To the best of our knowledge, this is the first study on PfPP1 interactome. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1. Their analysis indicates a high diversity of biological functions for PP1 in Plasmodium. Based on the present data and on an earlier study of the Pf interactome, a potential implication of Pips in protein folding/proteolysis, transcription and pathogenicity networks is proposed. The present work provides a starting point for further studies on the structural basis of these interactions and their functions in P. falciparum.

Relationship Between Pneumocystis carinii Burden and the Degree of Host Immunosuppression in an Airborne Transmission Experimental Model.

To quantitatively assess the risk of contamination by Pneumocystis depending on the degree of immunosuppression (ID) of the exposed rat hosts, we developed an animal model, where rats went through different doses of dexamethasone. Then, natural and aerial transmission of Pneumocystis carinii occurred during cohousing of the rats undergoing gradual ID levels (receivers) with nude rats developing pneumocystosis (seeders). Following contact between receiver and seeder rats, the P. carinii burden of receiver rats was determined by toluidine blue ortho staining and by qPCR targeting the dhfr monocopy gene of this fungus. In this rat model, the level of circulating CD4(+) and CD8(+) T lymphocytes remained significantly stable and different for each dose of dexamethasone tested, thus reaching the goal of a new stable and gradual ID rat model. In addition, an inverse relationship between the P. carinii burden and the level of circulating CD4(+) or CD8(+) T lymphocytes was evidenced. This rat model may be used to study other opportunistic pathogens or even co-infections in a context of gradual ID.

Genome wide in silico analysis of Plasmodium falciparum phosphatome.

BACKGROUND: Eukaryotic cellular machineries are intricately regulated by several molecular mechanisms involving transcriptional control, post-translational control and post-translational modifications of proteins (PTMs). Reversible protein phosphorylation/dephosphorylation process, which involves kinases as well as phosphatases, represents an important regulatory mechanism for diverse pathways and systems in all organisms including human malaria parasite, Plasmodium falciparum. Earlier analysis on P. falciparum protein-phosphatome revealed presence of 34 phosphatases in Plasmodium genome. Recently, we re-analysed P. falciparum phosphatome aimed at identifying parasite specific phosphatases. RESULTS: Plasmodium database (PlasmoDB 9.2) search, combined with PFAM and CDD searches, revealed 67 candidate phosphatases in P. falciparum. While this number is far less than the number of phosphatases present in Homo sapiens, it is almost the same as in other Plasmodium species. These Plasmodium phosphatase proteins were classified into 13 super families based on NCBI CDD search. Analysis of proteins expression profiles of the 67 phosphatases revealed that 44 phosphatases are expressed in both schizont as well as gametocytes stages. Fourteen phosphatases are common in schizont, ring and trophozoite stages, four phosphatases are restricted to gametocytes, whereas another three restricted to schizont stage. The phylogenetic trees for each of the known phosphatase super families reveal a considerable phylogenetic closeness amongst apicomplexan organisms and a considerable phylogenetic distance with other eukaryotic model organisms included in the study. The GO assignments and predicted interaction partners of the parasite phosphatases indicate its important role in diverse cellular processes. CONCLUSION: In the study presented here, we reviewed the P. falciparum phosphatome to show presence of 67 candidate phosphatases in P. falciparum genomes/proteomes. Intriguingly, amongst these phosphatases, we could identify six Plasmodium specific phosphatases and 33 putative phosphatases that do not have human orthologs, thereby suggesting that these phosphatases have the potential to be explored as novel antimalarial drug targets.

Plasmodium falciparum encodes a conserved active inhibitor-2 for Protein Phosphatase type 1: perspectives for novel anti-plasmodial therapy.

BACKGROUND: It is clear that the coordinated and reciprocal actions of kinases and phosphatases are fundamental in the regulation of development and growth of the malaria parasite. Protein Phosphatase type 1 is a key enzyme playing diverse and essential roles in cell survival. Its dephosphorylation activity/specificity is governed by the interaction of its catalytic subunit (PP1c) with regulatory proteins. Among these, inhibitor-2 (I2) is one of the most evolutionarily ancient PP1 regulators. In vivo studies in various organisms revealed a defect in chromosome segregation and cell cycle progression when the function of I2 is blocked. RESULTS: In this report, we present evidence that Plasmodium falciparum, the causative agent of the most deadly form of malaria, expresses a structural homolog of mammalian I2, named PfI2. Biochemical, in vitro and in vivo studies revealed that PfI2 binds PP1 and inhibits its activity. We further showed that the motifs 12KTISW16 and 102HYNE105 are critical for PfI2 inhibitory activity. Functional studies using the Xenopus oocyte model revealed that PfI2 is able to overcome the G2/M cell cycle checkpoint by inducing germinal vesicle breakdown. Genetic manipulations in P. falciparum suggest an essential role of PfI2 as no viable mutants with a disrupted PfI2 gene were detectable. Additionally, peptides derived from PfI2 and competing with RVxF binding sites in PP1 exhibit anti-plasmodial activity against blood stage parasites in vitro. CONCLUSIONS: Taken together, our data suggest that the PfI2 protein could play a role in the regulation of the P. falciparum cell cycle through its PfPP1 phosphatase regulatory activity. Structure-activity studies of this regulator led to the identification of peptides with anti-plasmodial activity against blood stage parasites in vitro suggesting that PP1c-regulator interactions could be a novel means to control malaria.

PhosphoTyrosyl phosphatase activator of Plasmodium falciparum: identification of its residues involved in binding to and activation of PP2A.

In Plasmodium falciparum (Pf), the causative agent of the deadliest form of malaria, a tight regulation of phosphatase activity is crucial for the development of the parasite. In this study, we have identified and characterized PfPTPA homologous to PhosphoTyrosyl Phosphatase Activator, an activator of protein phosphatase 2A which is a major phosphatase involved in many biological processes in eukaryotic cells. The PfPTPA sequence analysis revealed that five out of six amino acids involved in interaction with PP2A in human are conserved in P. falciparum. Localization studies showed that PfPTPA and PfPP2A are present in the same compartment of blood stage parasites, suggesting a possible interaction of both proteins. In vitro binding and functional studies revealed that PfPTPA binds to and activates PP2A. Mutation studies showed that three residues (V(283), G(292) and M(296)) of PfPTPA are indispensable for the interaction and that the G(292) residue is essential for its activity. In P. falciparum, genetic studies suggested the essentiality of PfPTPA for the completion of intraerythrocytic parasite lifecycle. Using Xenopus oocytes, we showed that PfPTPA blocked the G2/M transition. Taken together, our data suggest that PfPTPA could play a role in the regulation of the P. falciparum cell cycle through its PfPP2A regulatory activity.

Plasmodium falciparum inhibitor-3 homolog increases protein phosphatase type 1 activity and is essential for parasitic survival.

Growing evidence indicates that the protein regulators governing protein phosphatase 1 (PP1) activity have crucial functions because their deletion drastically affects cell growth and division. PP1 has been found to be essential in Plasmodium falciparum, but little is known about its regulators. In this study, we have identified a homolog of Inhibitor-3 of PP1, named PfI3. NMR analysis shows that PfI3 belongs to the disordered protein family. High affinity interaction of PfI3 and PfPP1 is demonstrated in vitro using several methods, with an apparent dissociation constant K(D) of 100 nm. We further show that the conserved (41)KVVRW(45) motif is crucial for this interaction as the replacement of the Trp(45) by an Ala(45) severely decreases the binding to PfPP1. Surprisingly, PfI3 was unable to rescue a yeast strain deficient in I3 (Ypi1). This lack of functional orthology was supported as functional assays in vitro have revealed that PfI3, unlike yeast I3 and human I3, increases PfPP1 activity. Reverse genetic approaches suggest an essential role of PfI3 in the growth and/or survival of blood stage parasites because attempts to obtain knock-out parasites were unsuccessful, although the locus of PfI3 is accessible. The main localization of a GFP-tagged PfI3 in the nucleus of all blood stage parasites is compatible with a regulatory role of PfI3 on the activity of nuclear PfPP1.

Gene profiling analysis reveals the contribution of CD24 and P2Y6R to the susceptibility of young rats to Plasmodium berghei infection.

Our previous studies have shown that Plasmodium berghei infection induces distinct clinical, parasitological and immunological states in young susceptible rats versus adult resistant rats. This susceptibility was mainly found to be related to inadequate cellular responses. In this study we first identified the altered genes in young susceptible rats. Unexpectedly, transcriptome analysis did not reveal any alteration of effector cytokines or their receptors. At day 13 p.i., six transcripts corresponding to faim3, mesothelin, gas3 (PMP22), gas7, CD24 and P2Y6R were significantly decreased in young infected rats when compared with adult infected rats. Because CD24 and P2Y6R participate in cellular immune responses, we next evaluated their role in the course of infection. Adoptive transfer experiments showed a transient but robust participation of CD24+ cells in the control of parasitaemia. The role of P2Y6R was investigated via its specific ability to be activated by Uridine di-Phosphate (UDP). Young rats treated with UDP partially restored the expression of P2Y6R, controlled parasitaemia and survived thereafter. In conclusion, this study contributes to the discovery of novel biomarkers in young susceptible rats and suggests that the decrease in their expression could be among the reasons for the development of severe pathology in malaria.

A potent HDAC inhibitor blocks Toxoplasma gondii tachyzoite growth and profoundly disrupts parasite gene expression.

INTRODUCTION: Toxoplasmosis is a major health issue worldwide, especially for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii. Although the drugs commonly used to treat toxoplasmosis are efficient, they present serious side effects and adverse events are common. Therefore, there is a need for the discovery of new compounds with potent anti-Toxoplasma gondii activity. METHODS: This study tested compounds designed to target enzymes that are involved in the epigenetic regulation of gene expression. RESULTS: Among the most active compounds, an HDAC inhibitor showing an IC50 of 30 nM with a selectivity index above 100 was identified. MC1742 was active at inhibiting the growth of the parasite in vitro but also at preventing the consequences of the acute disease in vivo. This compound induced hyper-acetylation of histones, while the acetylated tubulin level remained unchanged. After MC1742 treatment, the parasite expression profile was profoundly changed with the activation of genes preferentially expressed in the sexual stages that are normally repressed in the tachyzoite stage. CONCLUSIONS: These findings suggest that this compound disturbs the Toxoplasma gondii gene expression program, inducing parasite death.

Deciphering the Role of Protein Phosphatases in Apicomplexa: The Future of Innovative Therapeutics?

Parasites belonging to the Apicomplexa phylum still represent a major public health and world-wide socioeconomic burden that is greatly amplified by the spread of resistances against known therapeutic drugs. Therefore, it is essential to provide the scientific and medical communities with innovative strategies specifically targeting these organisms. In this review, we present an overview of the diversity of the phosphatome as well as the variety of functions that phosphatases display throughout the Apicomplexan parasites' life cycles. We also discuss how this diversity could be used for the design of innovative and specific new drugs/therapeutic strategies.

Plasmodium berghei leucine-rich repeat protein 1 downregulates protein phosphatase 1 activity and is required for efficient oocyst development.

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1-LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.

Plasmodium falciparum dynein light chain 1 interacts with actin/myosin during blood stage development.

Dynein light chain 1 (LC1), a member of the leucine-rich repeat protein family, has been shown to be engaged in controlling flagellar motility in Chlamydomonas reinhardtii and Trypanosoma brucei via its interaction with the dynein gamma heavy chain. In Plasmodium falciparum, we have identified the LC1 ortholog, designated Pfdlc1. Negative attempts to disrupt the dlc1 gene by reverse genetic approaches in both P. falciparum and P. berghei suggest either its essentiality for parasite survival or the inaccessibility of its locus. Expression studies revealed high levels of DLC1 protein in late trophozoites and schizonts, pointing to an unexpected role of this protein in blood-stage parasites as they do not have flagella. Interactions studies and co-immunoprecipitation experiments revealed that PfDLC1 was able to bind to P. falciparum myosin A and actin 1. The PfDLC1 interacting domains present in P. falciparum myosin A and actin 1 were mapped to sequences containing SDIE and/or EEMKT motifs present in the upper 50-kDa segment of the myosin A head domain and in the subdomain IV of actin 1, respectively. Detection of PfDLC1 by fluorescence tagging and immunofluorescence staining using specific antibodies showed a cytoplasmic location similar to actin and immunofluorescence studies showed a co-localization of PfDLC1 and myosin A. Taken together, these findings suggest that PfDLC1 might play an important role in P. falciparum erythrocytic stages by its interaction with myosin A and actin 1, known to be essential for parasite development.

The Multifaceted Role of Protein Phosphatase 1 in Plasmodium.

Protein phosphatase type 1 (PP1) forms a wide range of Ser/Thr-specific phosphatase holoenzymes which contain one catalytic subunit (PP1c), present in all eukaryotic cells, associated with variable subunits known as regulatory proteins. It has recently been shown that regulators take a leading role in the organization and the control of PP1 functions. Many studies have addressed the role of these regulators in diverse organisms, including humans, and investigated their link to diseases. In this review we summarize recent advances on the role of PP1c in Plasmodium, its interactome and regulators. As a proof of concept, peptides interfering with the regulator binding capacity of PP1c were shown to inhibit the growth of P. falciparum, suggesting their potential as drug precursors.

Differential Fractionation of Erythrocytes Infected by Plasmodium berghei.

The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected erythrocyte. The task remains delicate despite the use of enzymes or detergents theoretically capable of degrading specific membranes inside the infected cell. The remit of this protocol is to propose a method to isolate the erythrocyte cytosol and ghosts from the other compartments of the infected cell via a percoll gradient. Also, the lysis of the erythrocyte membrane is done using equinatoxin II, which has proven to be more effective at erythrocyte lysis regardless of the cell infection status, compared to the commonly used streptolysin. The parasitophorous vacuole (PV) content is collected after saponin lysis, before recovering membrane and parasite cytosol proteins by Triton X-100 lysis. The lysates thus obtained are analyzed by Western blot to assess the accuracy of the various extraction steps. This protocol allows the separation of the host compartment from the parasite compartments (PV and parasite), leading to potential studies of host proteins as well as parasite proteins exported to the host cell.

Plasmodium pseudo-Tyrosine Kinase-like binds PP1 and SERA5 and is exported to host erythrocytes.

Pseudokinases play key roles in many biological processes but they are poorly understood compared to active kinases. Eight putative pseudokinases have been predicted in Plasmodium species. We selected the unique pseudokinase belonging to tyrosine kinase like (TKL) family for detailed structural and functional analysis in P. falciparum and P. berghei. The primary structure of PfpTKL lacks residues critical for kinase activity, supporting its annotation as a pseudokinase. The recombinant pTKL pseudokinase domain was able to bind ATP, but lacked catalytic activity as predicted. The sterile alpha motif (SAM) and RVxF motifs of PfpTKL were found to interact with the P. falciparum proteins serine repeat antigen 5 (SERA5) and protein phosphatase type 1 (PP1) respectively, suggesting that pTKL has a scaffolding role. Furthermore, we found that PP1c activity in a heterologous model was modulated in an RVxF-dependent manner. During the trophozoite stages, PbpTKL was exported to infected erythrocytes where it formed complexes with proteins involved in cytoskeletal organization or host cell maturation and homeostasis. Finally, genetic analysis demonstrated that viable strains obtained by genomic deletion or knocking down PbpTKL did not affect the course of parasite intra-erythrocytic development or gametocyte emergence, indicating functional redundancy during these parasite stages.

Identification of novel quinazoline derivatives as potent antiplasmodial agents.

Despite the recent reductions in the global burden of malaria, this disease remains a devastating cause of death in tropical and subtropical regions. As there is no broadly effective vaccine for malaria, prevention and treatment still rely on chemotherapy. Unfortunately, emerging resistance to the gold standard artemisinin combination therapies means that new drugs with novel modes of action are urgently needed. In this context, Plasmodium histone modifying enzymes have emerged as potential drug targets, prompting us to develop and optimize compounds directed against such epigenetic targets. A panel of 51 compounds designed to target different epigenetic enzymes were screened for activity against Plasmodium falciparum parasites. Based on in vitro activity against drug susceptible and drug-resistant P. falciparum lines, selectivity index criterion and favorable pharmacokinetic properties, four compounds, one HDAC inhibitor (1) and three DNMT inhibitors (37, 43 and 45), were selected for preclinical studies in a mouse model of malaria. In vivo data showed that 37, 43 and 45 exhibited oral efficacy in the mouse model of Plasmodium berghei infection. These compounds represent promising starting points for the development of novel antimalarial drugs.

Characterization of a Protein Phosphatase Type-1 and a Kinase Anchoring Protein in Plasmodium falciparum.

With its multiple regulatory partners, the conserved Protein Phosphatase type-1 (PP1) plays a central role in many functions of the biology of eukaryotic cells, including Plasmodium falciparum. Here, we characterized a protein named PfRCC-PIP, as a major partner of PfPP1. We established its direct interaction in vitro and its presence in complex with PfPP1 in the parasite. The use of Xenopus oocyte model revealed that RCC-PIP can interact with the endogenous PP1 and act in synergy with suboptimal doses of progesterone to trigger oocyte maturation, suggesting a regulatory effect on PP1. Reverse genetic studies suggested an essential role for RCC-PIP since no viable knock-out parasites could be obtained. Further, we demonstrated the capacity of protein region containing RCC1 motifs to interact with the parasite kinase CDPK7. These data suggest that this protein is both a kinase and a phosphatase anchoring protein that could provide a platform to regulate phosphorylation/dephosphorylation processes.