IgG antibody response against Plasmodium falciparum aminopeptidase 1 antigen in Gabonese children living in Makokou and Franceville.

The search for novel chemical classes of anti-malarial compounds to cope with the current state of chemoresistance of malaria parasites has led to the identification of Plasmodium falciparum aminopeptidase 1 (PfA-M1) as a new therapeutic target. PfA-M1, known to be involved in the hemoglobin digestion cascade which helps to provide most of the amino acids necessary to the parasite's metabolism, is currently considered as a promising target for anti-malarial chemotherapy. However, its immunogenic properties have not yet been tested in the Gabonese population. In Gabon, the prevalence of malaria remains three times higher in semi-urban areas (60·12%) than in urban areas (17·06%). We show that malaria-specific PfA-M1 antibodies are present in children and increase with the level of infection. Children living in semi-urban areas have higher anti-PfA-M1 antibody titers (0·14 ± 0·02 AU) than those living in urban areas (0·08 ± 0·02 AU, P = 0·03), and their antibody titers increase with age (P < 0·0001). Moreover, anti-PfA-M1 antibody titers decrease in children with hyperparasitemia (0·027 ± 0·055 AU) but they remain high in children with low parasite density (0·21 ± 0·034 AU, P = 0·034). In conclusion, our results suggest that malaria-specific PfA-M1 antibodies may play an important role in the immune response of the host against P. falciparum in Gabonese children. Further studies on the role of PfA-M1 during anemia are needed.

Diversity and biological activities of the bacterial community associated with the marine sponge Phorbas tenacior (Porifera, Demospongiae).

UNLABELLED: The diversity of the cultivable microbiota of the marine sponge Phorbas tenacior frequently found in the Mediterranean Sea was investigated, and its potential as a source of antimicrobial, antioxidant and antiplasmodial compounds was evaluated. The cultivable bacterial community was studied by isolation, cultivation and 16S rRNA gene sequencing. Twenty-three bacterial strains were isolated and identified in the Proteobacteria (α or γ classes) and Actinobacteria phyla. Furthermore, three different bacterial morphotypes localized extracellularly within the sponge tissues were revealed by microscopic observations. Bacterial strains were assigned to seven different genera, namely Vibrio, Photobacterium, Shewanella, Pseudomonas, Ruegeria, Pseudovibrio and Citricoccus. The strains affiliated to the same genus were differentiated according to their genetic dissimilarities using random amplified polymorphic DNA (RAPD) analyses. Eleven bacterial strains were selected for evaluation of their bioactivities. Three isolates Pseudovibrio P1Ma4, Vibrio P1MaNal1 and Citricoccus P1S7 revealed antimicrobial activity; Citricoccus P1S7 and Vibrio P1MaNal1 isolates also exhibited antiplasmodial activity, while two Vibrio isolates P1Ma8 and P1Ma5 displayed antioxidant activity. These data confirmed the importance of Proteobacteria and Actinobacteria associated with marine sponges as a reservoir of bioactive compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first report on the diversity of the cultivable bacteria associated with the marine sponge Phorbas tenacior, frequently found in the Mediterranean Sea. Evaluation of the antiplasmodial, antimicrobial and antioxidant activities of the isolates has been investigated and allowed to select bacterial strains, confirming the importance of Proteobacteria and Actinobacteria as sources of bioactive compounds.

Focal monopolar pulsed field ablation from within the great cardiac vein for idiopathic premature ventricular contractions after failed radiofrequency ablation.

BACKGROUND: Idiopathic epicardial premature ventricular contractions (PVCs) originating from the left ventricular summit are difficult to eliminate. OBJECTIVE: To describe feasibility and procedural safety of focal monopolar biphasic pulsed field ablation (F-PFA) from within the great cardiac vein (GCV) for the treatment of idiopathic epicardial PVCs. METHODS: In 4 pigs, F-PFA (CENTAURI, Cardiofocus) was applied from within the GCV followed by macroscopic gross analysis. In 4 patients with previously failed radiofrequency ablation, electroanatomic mapping was used to guide F-PFA from within the GCV and the ventricular outflow tracts. Coronary angiography and optical coherence tomography (OCT) were performed in 2 patients. RESULTS: In pigs, F-PFA from within the GCV (5mm away from the coronary arteries) resulted in myocardial lesions with a maximal depth of 4mm which was associated with non-obstructive transient coronary spasms. In patients, sequential delivery of F-PFA in the ventricular outflow tracts and from within the GCV eliminated the PVCs. During F-PFA delivery from within the GCV with prophylactic nitroglycerin application, coronary angiography showed no coronary spasm when F-PFA was delivered >5mm away from the coronary artery and a transient coronary spasm without changes in a subsequent OCT, when F-PFA was delivered directly on the coronary artery. Intracardiac echo and computer tomography integration was used to monitor F-PFA delivery from within the GCV. There were no immediate or short-term complications. CONCLUSION: Sequential mapping-guided F-PFA from endocardial ventricular outflow tracts and from within the GCV is feasible with a favourable procedural safety profile for the treatment of epicardial PVC.

Bioinformatic strategies to provide functional clues to the unknown genes in Plasmodium falciparum genome.

The fight against Plasmodium falciparum, the species responsible for 90% of the lethal forms of human malaria, took a new direction with the publication of its genome in 2002. However, the hopes that the genome should help bringing to the foreground the expected new "vaccines candidates" or "targets of new medicines" were disappointed by the low number of genes that could be functionally annotated--less than 40% upon the genome publication, just over 50% eight years later. This 10% gain of knowledge was made possible by the efforts of the entire scientific community in many directions which include: the production of transcriptomic and proteomic profiles at various stages of the parasite development and in response to drug or stress treatments; the proteomic study of subcellular compartments; the sequencing of numerous Plasmodium related species (allowing whole genome comparisons) and the sequencing of numerous P. falciparum strains (allowing investigations of gene polymorphism). In parallel with this production of experimental biological data, the development of original mining tools adapted to the P falciparum specificities quickly appeared as a priority, as the performances of "classical" bioinformatic tools, used successfully for other genomes, had limited efficacy. This was the aim of the PlasmoExplore project launched in 2007. This brief review does not cover all efforts made by the international community to decipher the P falciparum genome but focuses on improvements and novel mining methods investigated by the PlasmoExplore consortium, and some of the lessons we could learn from these efforts.

Discovery of new targets for antimalarial chemotherapy.

The understanding of the biology and the biochemistry of malaria parasites has considerably increased over the past two decades with the discovery of many potential targets for new antimalarial drugs. The decrypted genomes of several Plasmodium species and the new post-genomic tools further enriched our "reservoir" of targets and increased our ability to validate potential drug targets or to study the entire parasite metabolism. This review discusses targets involved in calcium metabolism, protein prenylation and apicoplast functions that have emerged by different approaches.

A family of genes related to a new expression site-associated gene in Trypanosoma equiperdum.

Two genes, belonging to a new expression site-associated gene family of six to eight members in Trypanosoma equiperdum and Trypanosoma brucei, have been cloned from a T. equiperdum variant. One of them, called ESAG-9c, is contained in the 1.78-C expression site and is found just upstream of the 5' barren region. The other one, called ESAG-9u, is unique in the family, is not telomere linked, and apparently is not expression site related. A 2-kb poly(A)+ mRNA is detected with probes for this ESAG-9 family in all T. equiperdum variants examined. By using polymerase chain reaction and restriction fragment length polymorphism techniques, it has been possible to distinguish between ESAG-9c and ESAG-9u and to show that ESAG-9c is transcribed in an expression site-specific manner. However, ESAG-9u (or another gene in the family having identical characteristics) is transcribed in all variants, regardless of the expression site used by these variants. Thus, this ESAG-9 family contains at least one gene that is under expression site control but might have other genes that are not. The function of these ESAG-9 genes is unknown. Transcripts homologous to ESAG-9 were detected in T. brucei bloodstream forms but not in procyclics.

On the role of repeated sequences 5' to variant surface glycoprotein genes in African trypanosomes.

In African trypanosomes, the DNA region situated upstream from all active and some silent variant surface glycoprotein genes (VSG genes) has a repetitive structure. This region is composed of a variable number of tandem repeats of an A + T-rich sequence which lacks the recognition sites for most commonly used restriction endonucleases, and is thus called 'barren region'. The length of the barren regions varies in different trypanosome variants from 0.2 to many kb. We have characterized the barren region upstream from the active VSG gene in two independent Trypanosoma equiperdum variants expressing the same VSG gene in the same expression site. To analyse the junction point between the expression site and the inserted gene, these two barren regions were cloned and sequenced. The longer barren region contains 14 repeats and the other contains two repeats. In both cases the junction point has been shown to lie within a repeat but different repeats were used in each case. These results argue that the repeats are important for the insertion of the duplicated-transposed gene into the expression site and that any repeat can be used.

Cloning of Plasmodium falciparum protein disulfide isomerase homologue by affinity purification using the antiplasmodial inhibitor 1,4-bis[3-[N-(cyclohexyl methyl)amino]propyl]piperazine..

A series of 10 1,4-bis(3-aminopropyl)piperazine compounds was found to display antiplasmodial activity with 50% growth inhibition between 30 and 250 nM, on three Plasmodium falciparum strains differently sensitive to chloroquine. By affinity chromatography using one of these compounds, a 52-kDa protein was isolated from P. falciparum, microsequenced and cloned. It corresponded to a single copy gene encoding a 453 amino acid protein displaying the typical features of protein disulfide isomerases, a thiol metabolizing enzyme belonging to the thiol: disulfide oxidoreductase superfamily, which was not previously described in malarial species.

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.

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.

The trypanosome VSG expression site encodes adenylate cyclase and a leucine-rich putative regulatory gene.

African trypanosomes are protozoan parasites that evade the host immune system by varying their dense antigenic coat. The Variant Surface Glycoprotein (VSG) is expressed exclusively from telomere-linked expression sites that contain in addition to the VSG gene, a number of open reading frames termed Expression Site Associated Genes (ESAGs). Here we demonstrate by complementation of a yeast mutant deleted for adenylate cyclase (cyr-1), that an ESAG from Trypanosoma equiperdum encodes an adenylate cyclase. Furthermore, we report that adjacent to adenylate cyclase in the expression site, is a separate open reading frame that encodes a protein sequence motif similar to the leucine-rich repeat regulatory domain of Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylate cyclases. The finding of two adjacent open reading frames homologous to a single enzyme in yeast suggests that the two expression site encoded proteins may interact to regulate adenylate cyclase activity during the course of an infection.

Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase.

Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase

Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.