Isabelle Florent

Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase

A Plasmodium falciparum single copy gene predicting a 122 kDa protein belonging to the Ml family of zincmetallopeptidases was previously reported and related to erythrocytic schizont proteins of 96 (p96) and 68 (p68) kDa. By using protease inhibitors during parasite harvest and enzyme preparations, and polyclonal antibodies specific for 2 peptidic domains deduced from the gene, we identified the 120 kDa precursor and demonstrated its processing into p96 and p68. The N-terminal ends of p96 and p68 were mapped between glycine-123 and lysine-163, both proteins thus containing the catalytic domain. The purified enzyme, here named PfA-M1 (p96/p68), displayed strict aminopeptidase activity, optimal at pH 74, with broad substrate spectrum. Its inhibition and reactivation profiles were typical of zinc-metalloaminopeptidases. By Western blotting, PfA-M1 was detected in trophozoites, in addition to schizonts, but not in early rings. PfA-M1 was localized by indirect immunofluorescence confocal microscopy. In trophozoites, the labelling was diffuse in the parasite cytoplasm, with accumulations around the food vacuole. In schizonts, it turned progressively to a vesicle-like pattern, ending as a clear spot in released merozoites. The involvement of PfA-M1 in haemoglobin breakdown and erythrocyte reinvasion is discussed in light of the dual functions recently reported for several P. falciparum proteases.

A PLASMODIUM FALCIPARUM AMINOPEPTIDASE GENE BELONGING TO THE M1 FAMILY OF ZINC-METALLOPEPTIDASES IS EXPRESSED IN ERYTHROCYTIC STAGES

A new single copy gene has been isolated from Plasmodium falciparum, by immunoscreening a genomic DNA expression library. The gene appears devoid of introns, displays the classical A + T richness and codon usage of P. falciparum genes, and is transcribed into a 4 kb mRNA in erythrocytic stages. The deduced amino acid sequence corresponds to a 1056 residue protein (122 kDa) containing the canonical HExxHx18E signature of zinc-metallopeptidase active sites of the M1 family at position 467-490, a downstream conserved tyrosine residue involved in catalysis in position 551, and the GAMEN conserved motif characteristic of aminopeptidases in the M1 family, at position 431-435. The greatest similarities were found with aminopeptidases N of Escherichia coli and Haemophilius influenza (more than 80% identical residues in the canonical signature of the active site) but significant similarities centred on the active site region exist with all other members of the M1 family such as other prokaryotic aminopeptidases, eukaryotic aminopeptidases A and N and leukotriene A4 hydrolases (40-50% identical residues in the canonical signature of the active site). A polyclonal serum raised to a synthetic peptide deduced from the gene labelled schizont proteins of 96 and 68 kDa purified to homogeneity and both displaying aminopeptidase activity, as well as cytoplasmic structures in schizont stages.

Probiotics for the Control of Parasites: An Overview

Probiotics are defined as live organisms, which confer benefits to the host. Their efficiency was demonstrated for the treatment of gastrointestinal disorders, respiratory infections, and allergic symptoms, but their use is mostly limited to bacterial and viral diseases. During the last decade, probiotics as means for the control of parasite infections were reported covering mainly intestinal diseases but also some nongut infections, that are all of human and veterinary importance. In most cases, evidence for a beneficial effect was obtained by studies using animal models. In a few cases, cellular interactions between probiotics and pathogens or relevant host cells were also investigated using in vitro culture systems. However, molecular mechanisms mediating the beneficial effects are as yet poorly understood. These studies indicate that probiotics might indeed provide a strain-specific protection against parasites, probably through multiple mechanisms. But more unravelling studies are needed to justify probiotic utilisation in therapeutics.

Design, synthesis and antimalarial activity of novel, quinoline-Based, zinc metallo-aminopeptidase inhibitors

PfA-M1, a neutral zinc aminopeptidase of Plasmodium falciparum, is a new potential target for the discovery of antimalarials. The design and synthesis of a library of 45 quinoline-based inhibitors of PfA-M1 is reported. The best inhibitor displays an IC(50) of 854 nM. The antimalarial activity on a CQ-resistant strain and the specificity towards mammalian aminopeptidase N are also discussed.

Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole

BackgroundThe Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.MethodsBy using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.ResultsThe high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.ConclusionsThe PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

Assessing functional annotation transfers with inter-species conserved coexpression: application to Plasmodium falciparum

BackgroundPlasmodium falciparum is the main causative agent of malaria. Of the 5 484 predicted genes of P. falciparum, about 57% do not have sufficient sequence similarity to characterized genes in other species to warrant functional assignments. Non-homology methods are thus needed to obtain functional clues for these uncharacterized genes. Gene expression data have been widely used in the recent years to help functional annotation in an intra-species way via the so-called Guilt By Association (GBA) principle.ResultsWe propose a new method that uses gene expression data to assess inter-species annotation transfers. Our approach starts from a set of likely orthologs between a reference species (here S. cerevisiae and D. melanogaster) and a query species (P. falciparum). It aims at identifying clusters of coexpressed genes in the query species whose coexpression has been conserved in the reference species. These conserved clusters of coexpressed genes are then used to assess annotation transfers between genes with low sequence similarity, enabling reliable transfers of annotations from the reference to the query species. The approach was used with transcriptomic data sets of P. falciparum, S. cerevisiae and D. melanogaster, and enabled us to propose with high confidence new/refined annotations for several dozens hypothetical/putative P. falciparum genes. Notably, we revised the annotation of genes involved in ribosomal proteins and ribosome biogenesis and assembly, thus highlighting several potential drug targets.ConclusionsOur approach uses both sequence similarity and gene expression data to help inter-species gene annotation transfers. Experiments show that this strategy improves the accuracy achieved when using solely sequence similarity and outperforms the accuracy of the GBA approach. In addition, our experiments with P. falciparum show that it can infer a function for numerous hypothetical genes.

Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

Cycloartane triterpenes from the leaves of Neoboutonia macrocalyx L.

Neoboutonia macrocalyx is used by people in south western Uganda around Kibale National Park in the treatment of malaria. Phytochemical investigation on the leaves of this plant led to the isolation of nine cycloartane triterpenes (1-9) and one phenanthrene; 7-methoxy-2,8 dimethyl-9,10-dihydrophenantherene-3,6 diol (10) along with three known compounds which included 22-de-O-acetyl-26-deoxyneoboutomellerone (11), mellerin B (12) and 6-hydroxystigmast-4-en-3-one (13). The chemical structures of the compounds were established mainly through a combination of spectroscopic techniques. The isolated compounds were evaluated for antiplasmodial activity against the chloroquine-resistant FcB1/Colombia strain of Plasmodium falciparum and for cytotoxicity against the KB (nasopharyngeal epidermoid carcinoma) and MRC-5 (human diploid embryonic lung) cells. Seven out of 13 compounds exhibited good antiplasmodial activity with IC50 of ⩽5μg/ml with two compounds exhibiting low cytotoxicity and five compounds having significant cytotoxicity.

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.

Access to new endoperoxide derivatives by electrochemical oxidation of substituted 3-azabicyclo[4.1.0]hept-4-enes.

A series of substituted 3-azabicyclo[4.1.0]hept-4-ene derivatives were prepared and analysed by cyclic voltammetry. Preparative aerobic electrochemical oxidation reactions were then carried out. Three original endoperoxides were isolated, characterised and subjected to antimalarial and cytotoxicity activity assays.