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Sitamaquine is a 8-aminoquinoline in development for the treatment of visceral leishmaniasis by oral route, no activity being observed on the experimental cutaneous leishmaniasis experimental models. Recent data explain how sitamaquine accumulate in Leishmania parasites, however its molecular targets remain to be identified. An advantage of sitamaquine is its short elimination half-life, preventing a rapid resistance emergence. The antileishmanial action of its metabolites is not known. The selection of a sitamaquine-resistant clone of L. donovani in laboratory and the phase II clinical trials pointing out some adverse effects such as methemoglobinemia and nephrotoxicity are considered for a further development decision. more...

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. more...

Our current view of the evolutionary history, coding and adaptive capacities of Apicomplexa, protozoan parasites of a wide range of metazoan, is currently strongly biased toward species infecting humans, as data on early diverging apicomplexan lineages infecting invertebrates is extremely limited. Here, we characterized the genome of the marine eugregarine Porospora gigantea, intestinal parasite of Lobsters, remarkable for the macroscopic size of its vegetative feeding forms (trophozoites) and its gliding speed, the fastest so far recorded for Apicomplexa. Two highly syntenic genomes named A and B were assembled. Similar in size (~ 9 Mb) and coding capacity (~ 5300 genes), A and B genomes are 10.8% divergent at the nucleotide level, corresponding to 16-38 My in divergent time. Orthogroup analysis across 25 (proto)Apicomplexa species, including Gregarina niphandrodes, showed that A and B are highly divergent from all other known apicomplexan species, revealing an unexpected breadth of diversity. Phylogenetically these two species branch sisters to Cephaloidophoroidea, and thus expand the known crustacean gregarine superfamily. The genomes were mined for genes encoding proteins necessary for gliding, a key feature of apicomplexans parasites, currently studied through the molecular model called glideosome. Sequence analysis shows that actin-related proteins and regulatory factors are strongly conserved within apicomplexans. In contrast, the predicted protein sequences of core glideosome proteins and adhesion proteins are highly variable among apicomplexan lineages, especially in gregarines. These results confirm the importance of studying gregarines to widen our biological and evolutionary view of apicomplexan species diversity, and to deepen our understanding of the molecular bases of key functions such as gliding, well known to allow access to the intracellular parasitic lifestyle in Apicomplexa., (© 2022. The Author(s).) more...

Blastogregarines are poorly studied parasites of polychaetes superficially resembling gregarines, but lacking syzygy and gametocyst stages in the life cycle. Furthermore, their permanent multinuclearity and gametogenesis by means of budding considerably distinguish them from other parasitic Apicomplexa such as coccidians and hematozoans. The affiliation of blastogregarines has been uncertain: different authors considered them highly modified gregarines, an intermediate apicomplexan lineage between gregarines and coccidians, or an isolated group of eukaryotes altogether. Here, we report the ultrastructure of two blastogregarine species, Siedleckia nematoides and Chattonaria mesnili, and provide the first molecular data on their phylogeny based on SSU, 5.8S, and LSU rDNA sequences. Morphological analysis reveals that blastogregarines possess both gregarine and coccidian features. Several traits shared with archigregarines likely represent the ancestral states of the corresponding cell structures for parasitic apicomplexans: a distinctive tegument structure and myzocytotic feeding with a well-developed apical complex. Unlike gregarines but similar to coccidians however, the nuclei of male blastogregarine gametes are associated with two kinetosomes. Molecular phylogenetic analyses reveal that blastogregarines are an independent, early diverging lineage of apicomplexans. Overall, the morphological and molecular evidence congruently suggests that blastogregarines represent a separate class of Apicomplexa., (Copyright © 2018 Elsevier GmbH. All rights reserved.) more...

Microscopic and phylogenetic analyses were performed on endocommensal astome ciliates retrieved from the middle intestine of a marine cirratulid polychaete, Cirriformia tentaculata, collected in the bay of Roscoff (English Channel, Northwest French coast) and on the Southwest English coast. Three morphotypes of the astome genus Durchoniella were identified, two corresponding to described species (the type species Durchoniella brasili (Léger and Duboscq, 1904) De Puytorac, 1954 and Durchoniella legeriduboscqui De Puytorac, 1954) while a third morphotype remains undescribed. Their small subunit (SSU) rRNA gene sequences showed at least 97.2% identity and phylogenetic analyses grouped them at the base of the subclass Scuticociliatia (Oligohymenophorea), as a sister lineage to all astomes from terrestrial oligochaete annelids. Ultrastructural examination by transmission electron microscopy and fluorescence in situ hybridization analyses revealed the presence of endocytoplasmic cocci and rod-shaped bacteria surrounded by a very thin membrane. These endocytoplasmic bacteria may play a role in the association between endocommensal astome ciliates and cirratulid polychaetes inhabiting in anoxic coastal sediments., (Copyright © 2017 Elsevier GmbH. All rights reserved.) more...

Background: Gregarines are a group of early branching Apicomplexa parasitizing invertebrate animals. Despite their wide distribution and relevance to the understanding the phylogenesis of apicomplexans, gregarines remain understudied: light microscopy data are insufficient for classification, and electron microscopy and molecular data are fragmentary and overlap only partially., Methods: Scanning and transmission electron microscopy, PCR, DNA cloning and sequencing (Sanger and NGS), molecular phylogenetic analyses using ribosomal RNA genes (18S (SSU), 5.8S, and 28S (LSU) ribosomal DNAs (rDNAs))., Results and Discussion: We present the results of an ultrastructural and molecular phylogenetic study on the marine gregarine Ancora sagittata from the polychaete Capitella capitata followed by evolutionary and taxonomic synthesis of the morphological and molecular phylogenetic evidence on eugregarines. The ultrastructure of Ancora sagittata generally corresponds to that of other eugregarines, but reveals some differences in epicytic folds (crests) and attachment apparatus to gregarines in the family Lecudinidae, where Ancora sagittata has been classified. Molecular phylogenetic trees based on SSU (18S) rDNA reveal several robust clades (superfamilies) of eugregarines, including Ancoroidea superfam. nov., which comprises two families (Ancoridae fam. nov. and Polyplicariidae) and branches separately from the Lecudinidae; thus, all representatives of Ancoroidea are here officially removed from the Lecudinidae. Analysis of sequence data also points to possible cryptic species within Ancora sagittata and the inclusion of numerous environmental sequences from anoxic habitats within the Ancoroidea. LSU (28S) rDNA phylogenies, unlike the analysis of SSU rDNA alone, recover a well-supported monophyly of the gregarines involved (eugregarines), although this conclusion is currently limited by sparse taxon sampling and the presence of fast-evolving sequences in some species. Comparative morphological analyses of gregarine teguments and attachment organelles lead us to revise their terminology. The terms "longitudinal folds" and "mucron" are restricted to archigregarines, whereas the terms "epicystic crests" and "epimerite" are proposed to describe the candidate synapomorphies of eugregarines, which, consequently, are considered as a monophyletic group. Abolishing the suborders Aseptata and Septata, incorporating neogregarines into the Eugregarinida, and treating the major molecular phylogenetic lineages of eugregarines as superfamilies appear as the best way of reconciling recent morphological and molecular evidence. Accordingly, the diagnosis of the order Eugregarinida Léger, 1900 is updated., Competing Interests: The authors declare that they have no competing interests. more...

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., (Copyright © 2016 Elsevier GmbH. All rights reserved.) more...

The efficacy of chloroquine, once the drug of choice in the fight against Plasmodium falciparum, is now severely limited due to widespread resistance. Amodiaquine is one of the most potent antimalarial 4-aminoquinolines known and remains effective against chloroquine-resistant parasites, but toxicity issues linked to a quinone-imine metabolite limit its clinical use. In search of new compounds able to retain the antimalarial activity of amodiaquine while circumventing quinone-imine metabolite toxicity, we have synthesized five 4-aminoquinolines that feature rings lacking hydroxyl groups in the side chain of the molecules and are thus incapable of generating toxic quinone-imines. The new compounds displayed high in vitro potency (low nanomolar IC50), markedly superior to chloroquine and comparable to amodiaquine, against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, accompanied by low toxicity to L6 rat fibroblasts and MRC5 human lung cells, and metabolic stability comparable or higher than that of amodiaquine. Computational studies indicate a unique mode of binding of compound 4 to heme through the HOMO located on a biphenyl moeity, which may partly explain the high antiplasmodial activity observed for this compound. more...

We have previously shown that secreted phospholipases A2 (sPLA2s) from animal venoms inhibit the in vitro development of Plasmodium falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA2s in host defense against P. falciparum has not been investigated. We show here that 4 out of 10 human sPLA2s, namely, hGX, hGIIF, hGIII, and hGV, exhibit potent in vitro anti-Plasmodium properties with half-maximal inhibitory concentrations (IC50s) of 2.9 ± 2.4, 10.7 ± 2.1, 16.5 ± 9.7, and 94.2 ± 41.9 nM, respectively. Other human sPLA2s, including hGIIA, are inactive. The inhibition is dependent on sPLA2 catalytic activity and primarily due to hydrolysis of plasma lipoproteins from the parasite culture. Accordingly, purified lipoproteins that have been prehydrolyzed by hGX, hGIIF, hGIII, and hGV are more toxic to P. falciparum than native lipoproteins. However, the total enzymatic activities of human sPLA2s on purified lipoproteins or plasma did not reflect their inhibitory activities on P. falciparum. For instance, hGIIF is 9-fold more toxic than hGV but releases a lower quantity of nonesterified fatty acids (NEFAs). Lipidomic analyses of released NEFAs from lipoproteins demonstrate that sPLA2s with anti-Plasmodium properties are those that release polyunsaturated fatty acids (PUFAs), with hGIIF being the most selective enzyme. NEFAs purified from lipoproteins hydrolyzed by hGIIF were more potent at inhibiting P. falciparum than those from hGV, and PUFA-enriched liposomes hydrolyzed by sPLA2s were highly toxic, demonstrating the critical role of PUFAs. The selectivity of sPLA2s toward low- and high-density (LDL and HDL, respectively) lipoproteins and their ability to directly attack parasitized erythrocytes further explain their anti-Plasmodium activity. Together, our findings indicate that 4 human sPLA2s are active against P. falciparum in vitro and pave the way to future investigations on their in vivo contribution in malaria pathophysiology., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.) more...

Background: In the past decades, many studies focused on the cell motility of apicomplexan invasive stages as they represent a potential target for chemotherapeutic intervention. Gregarines (Conoidasida, Gregarinasina) are a heterogeneous group that parasitize invertebrates and urochordates, and are thought to be an early branching lineage of Apicomplexa. As characteristic of apicomplexan zoites, gregarines are covered by a complicated pellicle, consisting of the plasma membrane and the closely apposed inner membrane complex, which is associated with a number of cytoskeletal elements. The cell cortex of eugregarines, the epicyte, is more complicated than that of other apicomplexans, as it forms various superficial structures., Results: The epicyte of the eugregarines, Gregarina cuneata, G. polymorpha and G. steini, analysed in the present study is organised in longitudinal folds covering the entire cell. In mature trophozoites and gamonts, each epicytic fold exhibits similar ectoplasmic structures and is built up from the plasma membrane, inner membrane complex, 12-nm filaments, rippled dense structures and basal lamina. In addition, rib-like myonemes and an ectoplasmic network are frequently observed. Under experimental conditions, eugregarines showed varied speeds and paths of simple linear gliding. In all three species, actin and myosin were associated with the pellicle, and this actomyosin complex appeared to be restricted to the lateral parts of the epicytic folds. Treatment of living gamonts with jasplakinolide and cytochalasin D confirmed that actin actively participates in gregarine gliding. Contributions to gliding of specific subcellular components are discussed., Conclusions: Cell motility in gregarines and other apicomplexans share features in common, i.e. a three-layered pellicle, an actomyosin complex, and the polymerisation of actin during gliding. Although the general architecture and supramolecular organisation of the pellicle is not correlated with gliding rates of eugregarines, an increase in cytoplasmic mucus concentration is correlated. Furthermore, our data suggest that gregarines utilize several mechanisms of cell motility and that this is influenced by environmental conditions. more...

Our work on targeting redox equilibria of malarial parasites propagating in red blood cells has led to the selection of six 1,4-naphthoquinones, which are active at nanomolar concentrations against the human pathogen Plasmodium falciparum in culture and against Plasmodium berghei in infected mice. With respect to safety, the compounds do not trigger hemolysis or other signs of toxicity in mice. Concerning the antimalarial mode of action, we propose that the lead benzyl naphthoquinones are initially oxidized at the benzylic chain to benzoyl naphthoquinones in a heme-catalyzed reaction within the digestive acidic vesicles of the parasite. The major putative benzoyl metabolites were then found to function as redox cyclers: (i) in their oxidized form, the benzoyl metabolites are reduced by NADPH in glutathione reductase-catalyzed reactions within the cytosols of infected red blood cells; (ii) in their reduced forms, these benzoyl metabolites can convert methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Studies on a fluorinated suicide-substrate indicate as well that the glutathione reductase-catalyzed bioactivation of naphthoquinones is essential for the observed antimalarial activity. In conclusion, the antimalarial naphthoquinones are suggested to perturb the major redox equilibria of the targeted infected red blood cells, which might be removed by macrophages. This results in development arrest and death of the malaria parasite at the trophozoite stage. more...

The new Ru(II) chloroquine complexes [Ru(eta(6)-arene)(CQ)Cl2] (CQ = chloroquine; arene = p-cymene 1, benzene 2), [Ru(eta(6)-p-cymene)(CQ)(H2O)2][BF4]2 (3), [Ru(eta(6)-p-cymene)(CQ)(en)][PF6]2 (en = ethylenediamine) (4), and [Ru(eta(6)-p-cymene)(eta(6)-CQDP)][BF4]2 (5, CQDP = chloroquine diphosphate) have been synthesized and characterized by use of a combination of NMR and FTIR spectroscopy with DFT calculations. Each complex is formed as a single coordination isomer: In 1-4, chloroquine binds to ruthenium in the eta(1)-N mode through the quinoline nitrogen atom, whereas in 5 an unprecedented eta(6) bonding through the carbocyclic ring is observed. 1, 2, 3, and 5 are active against CQ-resistant (Dd2, K1, and W2) and CQ-sensitive (FcB1, PFB, F32, and 3D7) malaria parasites (Plasmodium falciparum); importantly, the potency of these complexes against resistant parasites is consistently higher than that of the standard drug chloroquine diphosphate. 1 and 5 also inhibit the growth of colon cancer cells, independently of the p53 status and of liposarcoma tumor cell lines with the latter showing increased sensitivity, especially to 1 (IC50 8 microM); this is significant because this type of tumor does not respond to currently employed chemotherapies. more...

We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A(2) (sPLA(2)s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA(2)s in malaria, we investigated the interactions between bee venom phospholipase A(2) (bvPLA(2)), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100x IC(50)), bvPLA(2) binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA(2)/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA(2)s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria. more...

Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. more...

Lecudina tuzetae is a parasitic protozoan (Gregarine, Apicomplexa) living in the intestine of a marine polychaete annelid, Nereis diversicolor. Using electron and fluorescence microscopy, we have characterized the dynamic changes in microtubule organization during the sexual phase of the life cycle. The gametocyst excreted from the host worm into seawater consists of two (one male and one female) gamonts in which cortical microtubule arrays are discernible. Each gamont undergoes multiple nuclear divisions without cytokinesis, resulting in the formation of large multinucleate haploid cells. After cellularization, approximately 1000 individual gametes are produced from each gamont within 24 h. Female gametes are spherical and contain interphase cytoplasmic microtubule arrays emanating from a gamma-tubulin-containing site. In male gametes, both interphase microtubules and a flagellum with "6 + 0" axonemal microtubules extend from the same microtubule-organizing site. At the beginning of spore formation, each zygote secretes a wall to form a sporocyst. Following meiotic and mitotic divisions, each sporocyst gives rise to eight haploid cells that ultimately differentiate into sporozoites. The ovoid shaped sporocyst is asymmetric and forms at least two distinctive microtubule arrays: spindle microtubules and microtubule bundles originating from the protruding apical end corresponding to the dehiscence pole of the sporocyst. Because antibodies raised against mammalian centrosome components, such as gamma-tubulin, pericentrin, Cep135, and mitosis-specific phosphoproteins, react strongly with the microtubule-nucleating sites of Lecudina, this protozoan is likely to share common centrosomal antigens with higher eukaryotes., (Copyright 2005 Wiley-Liss, Inc) more...

We have demonstrated that the 80 kDa POP Tc80 (prolyl oligopeptidase of Trypanosoma cruzi) is involved in the process of cell invasion, since specific inhibitors block parasite entry into non-phagocytic mammalian host cells. In contrast with other POPs, POP Tc80 is capable of hydrolysing large substrates, such as fibronectin and native collagen. In this study, we present the cloning of the POPTc80 gene, whose deduced amino acid sequence shares considerable identity with other members of the POP family, mainly within its C-terminal portion that forms the catalytic domain. Southern-blot analysis indicated that POPTc80 is present as a single copy in the genome of the parasite. These results are consistent with mapping of POPTc80 to a single chromosome. The active recombinant protein (rPOP Tc80) displayed kinetic properties comparable with those of the native enzyme. Novel inhibitors were assayed with rPOP Tc80, and the most efficient ones presented values of inhibition coefficient Ki < or = 1.52 nM. Infective parasites treated with these specific POP Tc80 inhibitors attached to the surface of mammalian host cells, but were incapable of infecting them. Structural modelling of POP Tc80, based on the crystallized porcine POP, suggested that POP Tc80 is composed of an alpha/beta-hydrolase domain containing the catalytic triad Ser548-Asp631-His667 and a seven-bladed beta-propeller non-catalytic domain. Docking analysis suggests that triple-helical collagen access to the catalytic site of POP Tc80 occurs in the vicinity of the interface between the two domains. more...

Trophozoite cysteine protease (TCP) activity, isolated from Plasmodium falciparum soluble 100,000 g extracts, displayed native falcipain-1 kinetic parameters towards peptidyl substrates. The labelling of either isolated TCP or soluble 100,000 g extracts by a cystatin-derived probe (biotinyl-Leu-Val-Gly-CHN2) revealed a single band of ca. 30 kDa by SDS-PAGE, which was resolved into four spots displaying isoelectric points (pI) from 4.7 to 5.3 after two-dimensional separation. The molecular mass and pI correspond to those of falcipain-3, falcipain-2, falcipain-2' and falcipain-1, respectively. The two central spots were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry as falcipain-2 and falcipain-2'. This activity-based probe represents a potential tool for profiling active falcipains in parasites. more...

Trypanosoma cruzi expresses oligopeptidase B and cathepsin B that have important functions in the interaction with mammalian host cells. In this study, we demonstrated that sera from both chagasic rabbits and humans have specific antibodies to highly purified native oligopeptidase B and cathepsin B. Levels of antibodies to cathepsin B were higher than those observed to oligopeptidase B by absorbance values recorded upon ELISA. We next showed that 90% and 30% of sera from individuals with mucocutaneous leishmaniasis have antibodies that recognize oligopeptidase B and cathepsin B as antigens, respectively. In addition, 55% and 40% of sera from kala-azar patients have antibodies to oligopeptidase B and cathepsin B, respectively. Sera from malaria patients did not recognize the proteases as antigens. Despite high levels of specific antibodies, sera from T. cruzi-infected patients did not inhibit the activities of either oligopeptidase B or cathepsin B. Furthermore, sera or IgG purified from either infected or non-infected individuals enhanced the enzymatic activity of the secreted oligopeptidase B. Oligopeptidase B secreted by trypomastigotes and cathepsin B released upon parasite lysis retain their enzymatic activities and may be associated with Chagas' disease pathogenesis by hydrolyzing host proteins and inducing host immune responses. more...

A number of new Au(I) and Au(III) complexes of chloroquine (CQ) have been prepared, characterized, and evaluated in vitro against several strains of Plasmodium falciparum. [(CQ)Au(PPh(3))][NO(3)] (2) was synthesized by reaction of AuCl(PPh(3)) with AgNO(3) followed by treatment with CQ. Similar reactions of AuCl(PR(3)) (R = Me, Et) with KPF(6) and CQ yielded [(CQ)Au(PMe(3))][PF(6)] (3), and [(CQ)Au(PEt(3))][PF(6)] (4), respectively. KAuCl(4) reacted with CQ to produce the Au(III) complex [(CQ)(2)Au(Cl)(2)]Cl (5), which in turn formed [(CQ)Au(Cl)(SR)(Et(2)O)]Cl (6) by reaction with 1-thio-beta-d-glucose-2,3,4,6-tetraacetate (SRH). The new compounds were characterized by a combination of elemental analysis, fast atom bombardment mass spectrometry (FAB-MS), and NMR spectroscopy. All the complexes display in vitro activity against CQ-sensitive and CQ-resistant strains of Plasmodium falciparum. The highest activity for this series was obtained for complex 4, which is 5 times more active than chloroquine diphosphate (CQDP) against the CQ-resistant strain FcB1. On preincubation of noninfected red blood cells with complexes 1, 5, and 6, protection against subsequent infection was observed in some cases. No clear structure-activity correlations could be established for this series of compounds. more...

Antibacterial, antiparasitidal and antiviral properties have recently been attributed to members of the secreted phospholipases A(2) (sPLA(2)s) superfamily. Seven sPLA(2)s from groups IA, IB, IIA and III, were tested here in different culture conditions for inhibition of the in vitro intraerythrocytic development of Plasmodium falciparum, the causative agent of the most severe form of human malaria. In the presence of human serum, all sPLA(2)s were inhibitory, with three out of seven exhibiting IC(50)<0.1 nM. In all cases, inhibition could be induced by enzymatic pre-treatment of the serum. By contrast, no effect was observed when parasites were grown in a semi-defined medium (AlbuMAX II) devoid of lipoproteins and containing 10 times less phospholipids than the medium with human serum, strongly suggesting that hydrolysis of serum generating toxic lipid by-products, rather than a direct interaction of the sPLA(2) with the infected erythrocyte, is a general feature of the anti-Plasmodium properties of sPLA(2)s. Furthermore, in serum, six out of the seven sPLA(2)s were toxic against both trophozoite and schizont stages of the parasite development, contrasting with the trophozoite-selective bee venom enzyme's toxicity. Deciphering the molecular mechanisms at play in the phenotypic singularity of the bee venom enzyme toxicity might offer new prospects in antimalarial fight. more...

Trypanosoma cruzi is an intracellular protozoan parasite able to invade a wide variety of mammalian cells. To have access to the target organs/cells, the parasite must cross the basal laminae and the extracellular matrix (ECM). We previously characterized an 80-kDa proteinase (Tc80) secreted by the infective trypomastigotes that hydrolyzes native collagens and might be involved in infection by degrading ECM components. Here, we present evidence indicating a role for Tc80 in the invasion of nonphagocytic cells. Tc80 was classified as a member of the prolyl oligopeptidase (POP) family of serine proteases and was also found to hydrolyze fibronectin. Selective inhibitors for POP Tc80 were synthesized that blocked parasite entry into cells. Blockage occurred when trypomastigotes were preincubated with irreversible inhibitors but not after host cell preincubation, and the blockage correlated with inhibition of POP Tc80 activity in treated parasites. These data and the enzyme location inside a vesicular compartment close to the flagellar pocket, a specialized domain in endocytosis/exocytosis, strongly suggest a role for POP Tc80 in the maturation of parasite protein(s) and/or, after secretion, in a local action on parasite or host cell/ECM components required for invasion. more...

Secreted phospholipases A(2) (sPLA(2)s) from snake and insect venoms and from mammalian pancreas are structurally related enzymes that have been associated with several toxic, pathological, or physiological processes. We addressed the issue of whether toxic sPLA(2)s might exert specific effects on the Plasmodium falciparum intraerythrocytic development. We showed that both toxic and non-toxic sPLA(2)s are lethal to P. falciparum grown in vitro, with large discrepancies between respective IC(50) values; IC(50) values from toxic PLA(2)s ranged from 1.1 to 200 pm, and IC(50) values from non-toxic PLA(2)s ranged from 0.14 to 1 microm. Analysis of the molecular mechanisms responsible for cytotoxicity of bee venom PLA(2) (toxic) and hog pancreas PLA(2) (non-toxic) demonstrated that, in both cases, enzymatic hydrolysis of serum phospholipids present in the culture medium was responsible for parasite growth arrest. However, bee PLA(2)-lipolyzed serum induced stage-specific inhibition of P. falciparum development, whereas hog PLA(2)-lipolyzed serum killed parasites at either stage. Sensitivity to bee PLA(2)-treated serum appeared restricted to the 19-26-h period of the 48 h parasite cycle. Analysis of the respective role of the different lipoprotein classes as substrates of bee PLA(2) showed that enzyme treatment of high density lipoproteins, low density lipoproteins, and very low density lipoproteins/chylomicrons fractions induces cytotoxicity of either fraction. In conclusion, our results demonstrate that toxic and non-toxic PLA(2)s 1) are cytotoxic to P. falciparum via hydrolysis of lipoprotein phospholipids and 2) display different killing processes presumably involving lipoprotein by-products recognizing different targets on the infected red blood cell. more...

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. more...