Role of human group IIA secreted phospholipase A2 in malaria pathophysiology: Insights from a transgenic mouse model.

We previously showed that injection of recombinant human group IIA secreted phospholipase A2 (hGIIA sPLA2) to Plasmodium chabaudi-infected mice lowers parasitaemia by 20%. Here, we show that transgenic (TG) mice overexpressing hGIIA sPLA2 have a peak of parasitaemia about 30% lower than WT littermates. During infection, levels of circulating sPLA2, enzymatic activity and plasma lipid peroxidation were maximal at day-14, the peak of parasitaemia. Levels of hGIIA mRNA increased in liver but not in spleen and blood cells, suggesting that liver may contribute as a source of circulating hGIIA sPLA2. Before infection, baseline levels of leukocytes and pro-inflammatory cytokines were higher in TG mice than WT littermates. Upon infection, the number of neutrophils, lymphocytes and monocytes increased and were maximal at the peak of parasitaemia in both WT and TG mice, but were higher in TG mice. Similarly, levels of the Th1 cytokines IFN-γ and IL-2 increased in WT and TG mice, but were 7.7- and 1.7-fold higher in TG mice. The characteristic shift towards Th2 cytokines was observed during infection in both WT and TG mice, with increased levels of IL-10 and IL-4 at day-14. The current data are in accordance with our previous in vitro findings showing that hGIIA kills parasites by releasing toxic lipids from oxidized lipoproteins. They further show that hGIIA sPLA2 is induced during mouse experimental malaria and has a protective in vivo role, lowering parasitaemia by likely releasing toxic lipids from oxidized lipoproteins but also indirectly by promoting a more sustained innate immune response.

Antimalarial Activity of Human Group IIA Secreted Phospholipase A2 in Relation to Enzymatic Hydrolysis of Oxidized Lipoproteins.

The level of human group IIA secreted phospholipase A2 (hGIIA sPLA2) is increased in the plasma of malaria patients, but its role is unknown. In parasite culture with normal plasma, hGIIA is inactive against Plasmodium falciparum, contrasting with hGIIF, hGV, and hGX sPLA2s, which readily hydrolyze plasma lipoproteins, release nonesterified fatty acids (NEFAs), and inhibit parasite growth. Here, we revisited the anti-Plasmodium activity of hGIIA under conditions closer to those of malaria physiopathology where lipoproteins are oxidized. In parasite culture containing oxidized lipoproteins, hGIIA sPLA2 was inhibitory, with a 50% inhibitory concentration value of 150.0 ± 40.8 nM, in accordance with its capacity to release NEFAs from oxidized particles. With oxidized lipoproteins, hGIIF, hGV, and hGX sPLA2s were also more potent, by 4.6-, 2.1-, and 1.9-fold, respectively. Using specific immunoassays, we found that hGIIA sPLA2 is increased in plasma from 41 patients with malaria over levels for healthy donors (median [interquartile range], 1.6 [0.7 to 3.4] nM versus 0.0 [0.0 to 0.1] nM, respectively; P < 0.0001). Other sPLA2s were not detected. Malaria plasma, but not normal plasma, contains oxidized lipoproteins and was inhibitory to P. falciparum when spiked with hGIIA sPLA2 Injection of recombinant hGIIA into mice infected with P. chabaudi reduced the peak of parasitemia, and this was effective only when the level of plasma peroxidation was increased during infection. In conclusion, we propose that malaria-induced oxidation of lipoproteins converts these into a preferential substrate for hGIIA sPLA2, promoting its parasite-killing effect. This mechanism may contribute to host defense against P. falciparum in malaria where high levels of hGIIA are observed.

Azetidine-Containing Alkaloids Produced by a Quorum-Sensing Regulated Nonribosomal Peptide Synthetase Pathway in Pseudomonas aeruginosa.

Pseudomonas aeruginosa displays an impressive metabolic versatility, which ensures its survival in diverse environments. Reported herein is the identification of rare azetidine-containing alkaloids from P. aeruginosa PAO1, termed azetidomonamides, which are derived from a conserved, quorum-sensing regulated nonribosomal peptide synthetase (NRPS) pathway. Biosynthesis of the azetidine motif has been elucidated by gene inactivation, feeding experiments, and biochemical characterization in vitro, which involves a new S-adenosylmethionine-dependent enzyme to produce azetidine 2-carboxylic acid as an unusual building block of NRPS. The mutants of P. aeruginosa unable to produce azetidomonamides had an advantage in growth at high cell density in vitro and displayed rapid virulence in Galleria mellonella model, inferring functional roles of azetidomonamides in the host adaptation. This work opens the avenue to study the biological functions of azetidomonamides and related compounds in pathogenic and environmental bacteria.

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.

The Redox Cycler Plasmodione Is a Fast-Acting Antimalarial Lead Compound with Pronounced Activity against Sexual and Early Asexual Blood-Stage Parasites.

Previously, we presented the chemical design of a promising series of antimalarial agents, 3-[substituted-benzyl]-menadiones, with potent in vitro and in vivo activities. Ongoing studies on the mode of action of antimalarial 3-[substituted-benzyl]-menadiones revealed that these agents disturb the redox balance of the parasitized erythrocyte by acting as redox cyclers-a strategy that is broadly recognized for the development of new antimalarial agents. Here we report a detailed parasitological characterization of the in vitro activity profile of the lead compound 3-[4-(trifluoromethyl)benzyl]-menadione 1c (henceforth called plasmodione) against intraerythrocytic stages of the human malaria parasite Plasmodium falciparum We show that plasmodione acts rapidly against asexual blood stages, thereby disrupting the clinically relevant intraerythrocytic life cycle of the parasite, and furthermore has potent activity against early gametocytes. The lead's antiplasmodial activity was unaffected by the most common mechanisms of resistance to clinically used antimalarials. Moreover, plasmodione has a low potential to induce drug resistance and a high killing speed, as observed by culturing parasites under continuous drug pressure. Drug interactions with licensed antimalarial drugs were also established using the fixed-ratio isobologram method. Initial toxicological profiling suggests that plasmodione is a safe agent for possible human use. Our studies identify plasmodione as a promising antimalarial lead compound and strongly support the future development of redox-active benzylmenadiones as antimalarial agents.

Synthesis of New 4-Aminoquinolines and Evaluation of Their In Vitro Activity against Chloroquine-Sensitive and Chloroquine-Resistant Plasmodium falciparum.

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.

In vitro anti-Plasmodium falciparum properties of the full set of human secreted phospholipases A2.

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.

Indole alkaloids from Muntafara sessilifolia with antiplasmodial and cytotoxic activities.

Four vobasinyl-iboga bisindole and one 2-acyl monomeric indole alkaloids were isolated from the stem bark of Muntafara sessilifolia along with eleven known compounds. Their structures and relative stereochemistry were elucidated on the basis of spectroscopic data including 1D and 2D NMR and mass spectrometry (MS). All isolated compounds were evaluated in vitro for antiplasmodial activity against the chloroquine-resistant strain FcB1 of Plasmodium falciparum, and for cytotoxicity against the human lung cell line MRC-5 and the rat skeletal muscle cell line L-6. 3'-Oxo-tabernaelegantine A exhibited antiplasmodial activity (4.4 µM IC(50)) associated with non-significant cytotoxicity (selectivity index of 48). Tabernaelegantine B and D displayed the highest cytotoxicity with IC(50) values of 0.47 and 1.89 µM on MRC-5 cells, and 0.42 and 2.7 µM on L-6 cells, respectively.

Glutathione reductase-catalyzed cascade of redox reactions to bioactivate potent antimalarial 1,4-naphthoquinones--a new strategy to combat malarial parasites.

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.

Synthesis, characterization, and in vitro antimalarial and antitumor activity of new ruthenium(II) complexes of chloroquine.

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.

Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.

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.

Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-Plasmodium toxicity.

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.

Isolation and characterization of Psalmopeotoxin I and II: two novel antimalarial peptides from the venom of the tarantula Psalmopoeus cambridgei.

Two novel peptides that inhibit the intra-erythrocyte stage of Plasmodium falciparum in vitro were identified in the venom of the Trinidad chevron tarantula, Psalmopoeus cambridgei. Psalmopeotoxin I (PcFK1) is a 33-residue peptide and Psalmopeotoxin II (PcFK2) has 28-amino acid residues; both have three disulfide bridges and belong to the Inhibitor Cystine Knot superfamily. The cDNAs encoding both peptides were cloned, and nucleotide sequence analysis showed that the peptides are synthesized with typical signal peptides and pro-sequences that are cleaved at a basic doublet before secretion of the mature peptides. The IC(5O) of PcFK1 for inhibiting P. falciparum growth was 1.59+/-1.15 microM and that of PcFK2 was 1.15+/-0.95 microM. PcFK1 was adsorbed strongly to uninfected erythrocytes, but PcFK2 was not. Neither peptide has significant hemolytic activity at 10 microM. Electrophysiological recordings in isolated frog and mouse neuromuscular preparations revealed that the peptides (at up to 9.3 microM) do not affect neuromuscular transmission or quantal transmitter release. PcFK1 and PcFK2 do not affect the growth or viability of human epithelial cells, nor do they have any antifungal or antibacterial activity at 20 microM. Thus, PcFK1 and PcFK2 seem to interact specifically with infected erythrocytes. They could therefore be promising tools for antimalaria research and be the basis for the rational development of antimalarial drugs.

Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serum-dependent.

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.