Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles.

Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito.

Characterization of Entamoeba fatty acid elongases; validation as targets and provision of promising leads for new drugs against amebiasis.

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26-30 carbons) and/or medium (8-12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba.

Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.

Trophozoite fitness dictates the intestinal epithelial cell response to Giardia intestinalis infection.

Giardia intestinalis is a non-invasive, protozoan parasite infecting the upper small intestine of most mammals. Symptomatic infections cause the diarrhoeal disease giardiasis in humans and animals, but at least half of the infections are asymptomatic. However, the molecular underpinnings of these different outcomes of the infection are still poorly defined. Here, we studied the early transcriptional response to G. intestinalis trophozoites, the disease-causing life-cycle stage, in human enteroid-derived, 2-dimensional intestinal epithelial cell (IEC) monolayers. Trophozoites preconditioned in media that maximise parasite fitness triggered only neglectable inflammatory transcription in the IECs during the first hours of co-incubation. By sharp contrast, "non-fit" or lysed trophozoites induced a vigorous IEC transcriptional response, including high up-regulation of many inflammatory cytokines and chemokines. Furthermore, "fit" trophozoites could even suppress the stimulatory effect of lysed trophozoites in mixed infections, suggesting active G. intestinalis suppression of the IEC response. By dual-species RNA-sequencing, we defined the IEC and G. intestinalis gene expression programs associated with these differential outcomes of the infection. Taken together, our results inform on how G. intestinalis infection can lead to such highly variable effects on the host, and pinpoints trophozoite fitness as a key determinant of the IEC response to this common parasite.

Plasmodium falciparum utilizes pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.

A detailed kinetic model of glycolysis in Plasmodium falciparum-infected red blood cells for antimalarial drug target identification.

Upon infection by the malaria parasite Plasmodium falciparum, the glycolytic rate of a red blood cell increases up to 100-fold, possibly contributing to lactic acidosis and hypoglycemia in patients with severe malaria. This dramatic increase in glucose uptake and metabolism was correctly predicted by a newly constructed detailed enzyme kinetic model of glucose metabolism in the trophozoite-infected red blood cell. Subsequently, we expanded the model to simulate an infected red blood cell culture, including the different asexual blood-stage forms of the malaria parasite. The model simulations were in good agreement with experimental data, for which the measured parasitic volume was an important parameter. Upon further analysis of the model, we identified glucose transport as a drug target that would specifically affect infected red blood cells, which was confirmed experimentally with inhibitor titrations. This model can be a first step in constructing a whole-body model for glucose metabolism in malaria patients to evaluate the contribution of the parasite's metabolism to the disease state.

PTEN differentially regulates endocytosis, migration, and proliferation in the enteric protozoan parasite Entamoeba histolytica.

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes including cytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite.

PI Kinase-EhGEF2-EhRho5 axis contributes to LPA stimulated macropinocytosis in Entamoeba histolytica.

Entamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis.

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole.

Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite.

Novel cytoskeletal traits in the intestinal parasites (Squirmida, Platyproteum vivax) of Pacific peanut worms (Sipuncula, Phascolosoma agassizii).

The cytoskeletal organization of a squirmid, namely Platyproteum vivax, was investigated with confocal laser scanning microscopy (CLSM) to refine inferences about convergent evolution among intestinal parasites of marine invertebrates. Platyproteum inhabits Pacific peanut worms (Phascolosoma agassizii) and has traits that are similar to other lineages of myzozoan parasites, namely gregarine apicomplexans within Selenidium, such as conspicuous feeding stages, called "trophozoites," capable of dynamic undulations. SEM and CLSM of P. vivax revealed an inconspicuous flagellar apparatus and a uniform array of longitudinal microtubules organized in bundles (LMBs). Extreme flattening of the trophozoites and a consistently oblique morphology of the anterior end provided a reliable way to distinguish dorsal and ventral surfaces. CLSM revealed a novel system of microtubules oriented in the flattened dorsoventral plane. Most of these dorsoventral microtubule bundles (DVMBs) had a punctate distribution and were evenly spaced along a curved line spanning the longitudinal axis of the trophozoites. This configuration of microtubules is inferred to function in maintaining the flattened shape of the trophozoites and facilitate dynamic undulations. The novel traits in Platyproteum are consistent with phylogenomic data showing that this lineage is only distantly related to Selenidium and other marine gregarine apicomplexans with dynamic intestinal trophozoites.

Observations on the transmission of Dientamoeba fragilis and the cyst life cycle stage.

Little is known about the life cycle and mode of transmission of Dientamoeba fragilis. Recently it was suggested that fecal­oral transmission of cysts may play a role in the transmission of D. fragilis. In order to establish an infection, D. fragilis is required to remain viable when exposed to the pH of the stomach. In this study, we investigated the ability of cultured trophozoites to withstand the extremes of pH. We provide evidence that trophozoites of D. fragilis are vulnerable to highly acidic conditions. We also investigated further the ultrastructure of D. fragilis cysts obtained from mice and rats by transmission electron microscopy. These studies of cysts showed a clear cyst wall surrounding an encysted parasite. The cyst wall was double layered with an outer fibrillar layer and an inner layer enclosing the parasite. Hydrogenosomes, endoplasmic reticulum and nuclei were present in the cysts. Pelta-axostyle structures, costa and axonemes were identifiable and internal flagellar axonemes were present. This study therefore provides additional novel details and knowledge of the ultrastructure of the cyst stage of D. fragilis.

Andrographolide induced cytotoxicity and cell cycle arrest in Giardia trophozoites.

Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia, affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia, assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti-Giardia activity almost equivalent to the reference drug metronidazole, with an IC50 value of 4.99 µM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance.

Agar dehydration: a simple method for long-term storage of Acanthamoeba spp. collection at room temperature.

This study describes dehydration of agar containing cysts as a novel and inexpensive method for long-term storage of Acanthamoeba spp. collections at room temperature. Five hundred microliters of axenically cultured Acanthamoeba spp. trophozoites (106 cells/mL) in PYG media or 150 µl of amoeba suspension (106 cells or cysts/mL) from monoxenic plate culture was spread onto the surface of non-nutritive agar (NNA, 2-3-mm thick) without or with a layer of heat-inactivated Escherichia coli, respectively. The plates were sealed and incubated at 30 °C. After the encystment, the Parafilm® was removed, and the plates were kept at the same temperature until the NNA was completely dehydrated. The dehydrated cyst-containing NNA was cut in rectangles and stored in airtight tubes at room temperature for up to 3 years. Cyst viability was assessed by inoculating them in fresh NNA with a layer of E. coli and in PYG followed by incubation at 30 °C. One hundred percent of samples from all specimens (19) stored over the 3 years allowed new cultures to be re-established; however, two strains showed reduced viability, at 66.7% and 62.5%, after 2 years of room temperature storage. One hundred percent of the cyst samples produced axenically and maintained in dry NNA allowed the re-establishment of axenic cultures through direct incubation in PYG, with excystment occurring within 24 or 48 h. For the first time, we report the dehydration of cyst-containing agar as an economical and effective method for the long-term storage of Acanthamoeba spp. collections at room temperature. It enables the creation of large collections using reduced space and economical transport of Acanthamoeba strains, in addition to allowing better organization of the collection.

Entamoeba histolytica: EhADH, an Alix Protein, Participates in Several Virulence Events through Its Different Domains.

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1-400 aa), Linker (246-446 aa) and Adh (444-687 aa) to evaluate their role in virulence. The TrophozBro11-400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226-446 also increased the virulence properties, but with lower effect than the TrophozBro11-400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444-687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.

Imidazole Carbamates as a Promising Alternative for Treating Trichomoniasis: In Vitro Effects on the Growth and Gene Expression of Trichomonas vaginalis.

Metronidazole (MTZ) is the most common drug used against Trichomonas vaginalis (T. vaginalis) infections; however, treatment failures and high rates of recurrence of trichomoniasis have been reported, suggesting the presence of resistance in T. vaginalis to MTZ. Therefore, research into new therapeutic options against T. vaginalis infections has become increasingly urgent. This study investigated the trichomonacidal activity of a series of five imidazole carbamate compounds (AGR-1, AGR-2, AGR-3, AGR-4, and AGR-5) through in vitro susceptibility assays to determine the IC50 value of each compound. All five compounds demonstrated potent trichomonacidal activity, with IC50 values in the nanomolar range and AGR-2 being the most potent (IC50 400 nM). To gain insight into molecular events related to AGR-induced cell death in T. vaginalis, we analyzed the expression profiles of some metabolic genes in the trophozoites exposed to AGR compounds and MTZ. It was found that both AGR and MTZ compounds reduced the expression of the glycolytic genes (CK, PFK, TPI, and ENOL) and genes involved in metabolism (G6PD, TKT, TALDO, NADHOX, ACT, and TUB), suggesting that disturbing these key metabolic genes alters the survival of the T. vaginalis parasite and that they probably share a similar mechanism of action. Additionally, the compounds showed low cytotoxicity in the Caco-2 and HT29 cell lines, and the results of the ADMET analysis indicated that these compounds have pharmacokinetic properties similar to those of MTZ. The findings offer significant insights that can serve as a basis for future in vivo studies of the compounds as a potential new treatment against T. vaginalis.

In vitro efficacy testing of ivermectin against Tritrichomonas foetus trophozoites.

There are no recommended drugs to treat cattle infected with the protozoan Tritrichomonas foetus (TF). Ivermectin, widely used in the treatment of intestinal parasites, was found effective against some protozoa growing in vitro. Here, its effectiveness against a TF line was investigated. Trophozoites were incubated in media with increasing concentrations of ivermectin and mortality was determined after 24 h. Ivermectin killed cells with a mean maximum effective concentration (EC50) of 2.47 µg mL-1. The effective concentration of ivermectin was rather high for a formulation suitable for systemic treatment. However, topical treatment of animals against TF could still be considered and tested.

EhRho6-mediated actin degradation in Entamoeba histolytica is associated with compromised pathogenicity.

Entamoeba histolytica causes amoebiasis which is a major health concern in developing countries. E. histolytica pathogenicity has been implicated to a large repertoire of small GTPases which switch between the inactive GDP bound state and the active GTP bound state with the help of guanine nucleotide exchange factors (GEFs) and GTPase activating protein (GAPs). Rho family of small GTPases are well known to modulate the actin cytoskeletal dynamics which plays a major role in E. histolytica pathogenicity. Here, we report an atypical amoebic RhoGEF, and its preferred substrate EhRho6, which, upon overexpression abrogated the pathogenic behavior of the amoeba such as adhesion to host cell, monolayer destruction, erythrophagocytosis, and formation of actin dots. A causative immunoblot analysis revealed actin degradation in the EhRho6 overexpressing trophozoites that could be inhibited by blocking the amoebic proteasomal pathway. A careful analysis of the results from a previously published transcriptomics study, in conjunction with our observations, led to the identification of a clade of Rho GTPases in this pathogenic amoeba which we hypothesize to have implications during the amoebic encystation.

Identification of glycine betaine as a host-derived molecule required for the vegetative proliferation of the protozoan parasite Perkinsus olseni.

Perkinsus olseni is an industrially significant protozoan parasite of Manila clam, Ruditapes philippinarum. So far, various media, based on Dulbecco's Modified Eagle Medium and Ham's F-12 nutrient mixture with supplementation of fetal bovine serum (FBS), have been developed to proliferate the parasitizing trophozoite stage of P. olseni. The present study showed that P. olseni did not proliferate in FBS-deficient Perkinsus broth medium (PBMΔF), but proliferated well in PBMΔF supplemented with tissue extract of host Manila clams, indicating that FBS and Manila clam tissue contained molecule(s) required for P. olseni proliferation. Preliminary characterization suggested that the host-derived molecule(s) was a heat-stable molecule(s) with a molecular weight of less than 3 kDa, and finally a single molecule required for the proliferation was purified by high-performance liquid chromatography processes. High-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses identified this molecule as glycine betaine (=trimethylglycine), and the requirement of this molecule for P. olsseni proliferation was confirmed by an assay using chemically synthesized, standard glycine betaine. Although glycine betaine was required for the proliferation of all examined Perkinsus species, supplementation of glycine betaine precursors, such as choline and betaine aldehyde, enhanced the proliferation of 4 Perkinsus species (P. marinus, P. chesapeaki, P. mediterraneus and P. honshuensis), but not of 2 others (P. olseni and P. beihaiensis). Thus, it was concluded that the ability to biosynthesise glycine betaine from its precursors varied among Perkinsus species, and that P. olseni and P. beihaiensis lack the ability required to biosynthesize glycine betaine for proliferation.

Activity of compounds derived from benzofuroxan in Trichomonasvaginalis.

Trichomoniasis is a sexually transmitted infection caused by the protozoan Trichomonas vaginalis. Currently, trichomoniasis is treated with the class of nitroimidazoles, namely, metronidazole; however, resistant isolates and strains have been reported. The compounds derived from benzofuroxan are biologically active heterocycles. This study evaluated the in vitro antiparasitic activity of these compounds in trophozoites of T. vaginalis and determined the mean inhibitory concentration (IC50), minimum inhibitory concentration (MIC), mortality curve, and cytotoxicity. The compounds were named EH1, EH2, EH3, and EA2 and tested in various concentrations: 100 to 15 µM (EH1 and EH2); 100 to 5 µM (EH3); and 100 to 25 µM (EA2), respectively. The greatest efficacy was observed in the highest concentrations in 24 h, with inhibition of approximately 100% of trophozoites. Compounds EH2 and EH3 had the lowest MIC: EH2 (35 µM) and EH3 (45 µM), with IC50 of 11.33 µM and 6.83 µM, respectively. Compound EA2 was effective at the highest concentrations. The activity of the compounds in T. vaginalis started in the first hour of incubation with 90% inhibition; after 12 h, inhibition >95% was observed. Compound EH1 showed the lowest activity, with the highest activity between 12 and 24 h after incubation. These results demonstrate that benzofuroxan derivatives are promising compounds for the in vitro treatment of T. vaginalis.

In Vitro Evaluation of the Antiamoebic Activity of Kaempferol against Trophozoites of Entamoeba histolytica and in the Interactions of Amoebae with Hamster Neutrophils.

Entamoeba histolytica (E. histolytica) is a parasite in humans that provokes amoebiasis. The most employed drug is metronidazole (MTZ); however, some studies have reported that this drug induces genotoxic effects. Therefore, it is necessary to explore new compounds without toxicity that can eliminate E. histolytica. Flavonoids are polyphenolic compounds that have demonstrated inhibition of growth and dysregulation of amoebic proteins. Despite the knowledge acquired to date, action mechanisms are not completely understood. The present work evaluates the effect of kaempferol against E. histolytica trophozoites and in the interactions with neutrophils from hamster, which is a susceptibility model. Our study demonstrated a significant reduction in the amoebic viability of trophozoites incubated with kaempferol at 150 µM for 90 min. The gene expression analysis showed a significant downregulation of Pr (peroxiredoxin), Rr (rubrerythrin), and TrxR (thioredoxin reductase). In interactions with amoebae and neutrophils for short times, we observed a reduction in ROS (reactive oxygen species), NO (nitric oxide), and MPO (myeloperoxidase) neutrophil activities. In conclusion, we confirmed that kaempferol is an effective drug against E. histolytica through the decrease in E. histolytica antioxidant enzyme expression and a regulator of several neutrophil mechanisms, such as MPO activity and the regulation of ROS and NO.