Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth.

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

Effect of magnetic field on the growth of the cultured Entamoeba histolytica isolated from patients in Palestine.

Static magnetic field (SMF) is generated in vicinity of moving charge or current passing through conductor. In this study, we aimed to investigate the effect of SMF on the growth of the cultured Entamoeba histolytica (E. histolytica) trophozoites. Different SMF strengths with maximum value equals 30 mT (mT) was applied on the E.histolytica for different periods of times: 0 h, 24 h, 48 h, and 72 h. A modified diphasic liver infusion agar medium was used for culturing E. histolytica in vitro. The results showed the successful stabilization of culture of E. histolytica trophozoites. If we kept the sample for longer time, e. g. 14 days, the growth rate decreases to zero. When applying 10 mT and 15 mT SMF on the sample, it is found that the cultivated E. histolytica trophozoites dies after 4 and 2 days respectively. The experiments suggested that the SMF inhibited the growth and the propagation of E. histolytica cells. In addition, it completely killed all the cells in a short time interval which depend on the SMF strength. It is concluded that the SMFs inhibits the growth of E. histolytica and change the morphology of these cells. Thus, we recommend to use SMF as treatment to mitigate the growth of E. histolytica.

Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds.

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300-400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC50 values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.

Unveiling the role of EVs in anaerobic parasitic protozoa.

The anaerobic or microaerophilic protozoan parasites such as the enteric human pathogens Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, Blastocystis hominis and urogenital tract parasites Trichomonas vaginalis are able to survival in an environment with oxygen deprivation. Despite living in hostile environments these pathogens adopted different strategies to survive within the hosts. Among them, the release of extracellular vesicles (EVs) has become an active endeavor in the study of pathogenesis for these parasites. EVs are heterogenous, membrane-limited structures that have played important roles in cellular communication, transferring information through cargo and modulating the immune system of the host. In this review, we described several aspects of the recently characterized EVs of the anaerobic protozoa, including their role in adhesion, modulation of the immune response and omics analysis to understand the potential of these EVs in the pathogenesis of these diseases caused by anaerobic parasites.

Eukaryotic translation initiation factor 5A and its posttranslational modifications play an important role in proliferation and potentially in differentiation of the human enteric protozoan parasite Entamoeba histolytica.

The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein and is essential in all eukaryotes. However, the specific roles of eIF5A in translation and in other biological processes remain elusive. In the present study, we described the role of eIF5A, its posttranslational modifications (PTM), and the biosynthetic pathway needed for the PTM in Entamoeba histolytica, the protozoan parasite responsible for amoebic dysentery and liver abscess in humans. E. histolytica encodes two isotypes of eIF5A and two isotypes of enzymes, deoxyhypusine synthase (DHS), responsible for their PTM. Both of the two eIF5A isotypes are functional, whereas only one DHS (EhDHS1, but not EhDHS2), is catalytically active. The DHS activity increased ~2000-fold when EhDHS1 was co-expressed with EhDHS2 in Escherichia coli, suggesting that the formation of a heteromeric complex is needed for full enzymatic activity. Both EhDHS1 and 2 genes were required for in vitro growth of E. histolytica trophozoites, indicated by small antisense RNA-mediated gene silencing. In trophozoites, only eIF5A2, but not eIF5A1, gene was actively transcribed. Gene silencing of eIF5A2 caused compensatory induction of expression of eIF5A1 gene, suggesting interchangeable role of the two eIF5A isotypes and also reinforcing the importance of eIF5As for parasite proliferation and survival. Furthermore, using a sibling species, Entamoeba invadens, we found that eIF5A1 gene was upregulated during excystation, while eIF5A2 was downregulated, suggesting that eIF5A1 gene plays an important role during differentiation. Taken together, these results have underscored the essentiality of eIF5A and DHS, for proliferation and potentially in the differentiation of this parasite, and suggest that the hypusination associated pathway represents a novel rational target for drug development against amebiasis.

Signals and signal transduction pathways in Entamoeba histolytica during the life cycle and when interacting with bacteria or human cells.

Entamoeba histolytica is the etiological agent of amebiasis in humans. This ameba parasite resides as a commensal in the intestine where it shares intestinal resources with the bacterial microbiome. In the intestinal ecosystem, the ameba encysts and eventually develops disease by invading the tissues. E. histolytica possesses cell surface receptors for the proper sensing of signals involved in encystation or sustaining parasite interaction with bacteria and human cells. Among those receptors are the Gal/GalNAc lectin, G protein-coupled receptors, and transmembrane kinases. In addition there are recently discovered, promising proteins, including orthologs of Toll-type receptors and ß trefoil lectins. These proteins trigger a wide variety of signal transduction pathways; however, most of the players involved in the signaling pathways evoked in this parasite are unknown. This review provides an overview of amoebic receptors and their role in encystation, adherence to bacteria or human cells, as well as the reported intracellular signal transduction processes that they can trigger. This knowledge is essential for understanding the lifestyle of E. histolytica and its cytopathic effect on bacteria and human cells that are responsible for infection.

Entamoeba stage conversion: progress and new insights.

Entamoeba histolytica, an anaerobic protozoan, is an important global health problem. This parasite has a biphasic life cycle consisting of a dormant cyst stage which is environmentally resistant and transmits the infection, and the proliferative trophozoite stage which is motile and causes invasive disease. The stage conversion process remains poorly understood despite being central to amoebic biology. In this review, we will highlight recent progress in our understanding of Entamoeba stage conversion including dissecting transcriptome analysis in development, characterization of transcriptional networks, demonstration of epigenetic regulation, and role of small molecules that regulate Entamoeba development.

Rabeprazole inhibits several functions of Entamoeba histolytica related with its virulence.

Amoebiasis is a human parasitic disease caused by Entamoeba histolytica. The parasite can invade the large intestine and other organs such as liver; resistance to the host tissue oxygen is a condition for parasite invasion and survival. Thioredoxin reductase of E. histolytica (EhTrxR) is a critical enzyme mainly involved in maintaining reduced the redox system and detoxifying the intracellular oxygen; therefore, it is necessary for E. histolytica survival under both aerobic in vitro and in vivo conditions. In the present work, it is reported that rabeprazole (Rb), a drug widely used to treat heartburn, was able to inhibit the EhTrxR recombinant enzyme. Moreover, Rb affected amoebic proliferation and several functions required for parasite virulence such as cytotoxicity, oxygen reduction to hydrogen peroxide, erythrophagocytosis, proteolysis, and oxygen and complement resistances. In addition, amoebic pre-incubation with sublethal Rb concentration (600 µM) promoted amoebic death during early liver infection in hamsters. Despite the high Rb concentration used to inhibit amoebic virulence, the wide E. histolytica pathogenic-related functions affected by Rb strongly suggest that its molecular structure can be used as scaffold to design new antiamoebic compounds with lower IC50 values.

Characterization of Extracellular Vesicles from Entamoeba histolytica Identifies Roles in Intercellular Communication That Regulates Parasite Growth and Development.

Extracellular vesicles (EVs) secreted by eukaryotic and prokaryotic cells to transport lipids, proteins, and nucleic acids to the external environment have important roles in cell-cell communication through cargo transfer. We identified and characterized EVs from Entamoeba histolytica, a protozoan parasite and a human pathogen. Conditioned medium from amebic parasites contained particles consistent with the expected size and morphology of EVs. Mass spectrometry was used to characterize the EV proteome and showed that it was enriched in common exosome marker proteins, including proteins associated with vesicle formation, cell signaling, and metabolism, as well as cytoskeletal proteins. Additionally, the EVs were found to selectively package small RNAs (sRNA), which were protected within the vesicles against RNase treatment. Sequencing analysis of the sRNA contained in EVs revealed that the majority were 27 nucleotides (nt) in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba RNA interference (RNAi) pathway proteins, including Argonaute, were also present in amebic EVs. Interestingly, we found that the amebic EVs impacted intercellular communication between parasites and altered encystation efficiency. EVs isolated from encysting parasites promoted encystation in other parasites, whereas EVs from metabolically active trophozoites impeded encystation. Overall, the data reveal that Entamoeba secrete EVs that are similar in size and shape to previously characterized exosomes from other organisms and that these EVs contain a defined protein and small RNA cargo and have roles in intercellular communication among parasites and influence growth kinetics.

Crystal structure of O-Acetylserine sulfhydralase (OASS) isoform 3 from Entamoeba histolytica: Pharmacophore-based virtual screening and validation of novel inhibitors.

The l-cysteine is crucial for growth, survival, defense against oxidative stress, and pathogenesis of Entamoeba histolytica. The de novo biosynthesis of l-cysteine in E. histolytica, has a two-step pathway, where O-acetylserine sulfhydrylase (OASS) catalyses the last step by converting OAS to l-cysteine. This pathway is absent in humans and hence represents a promising target for novel therapeutics. E. histolytica expresses three isoforms of OASS and knockdown studies showed the importance of these enzymes for the survival of the pathogen. Here, we report the crystal structure of OASS isoform 3 from E. histolytica to 1.54 Å resolution. The active site geometries and kinetics of EhOASS3 and EhOASS1 structures were found to be very similar. Small-molecule libraries were screened against EhOASS3 and compounds were shortlisted based on the docking scores. F3226-1387 showed best inhibition with IC50 of 38 µM against EhOASS3 and was able to inhibit the growth of the organism to 72%.

Heteroleptic NiII complexes: Synthesis, structural characterization, computational studies and amoebicidal activity evaluation.

In this work, we present the synthesis, characterization, electrochemical studies, DFT calculations, and in vitro amoebicidal effect of seven new heteroleptic NiII coordination compounds. The crystal structures of [H2(pdto)](NO3)2 and [Ni(pdto)(NO3)]PF6 are presented, pdto = 2,2'-[1,2-ethanediylbis-(sulfanediyl-2,1-ethanediyl)]dipyridine. The rest of the compounds have general formulae: [Ni(pdto)(NN)](PF6) where N-N = 2,2'-bipyridine (bpy), 4,4'-dimethyl-2,2'-bipyridine (44dmbpy), 5,5'-dimethyl-2,2'-bipyridine (55dmbpy), 1,10-phenanthroline (phen), 4,7-dimethyl-1,10-phenanthroline (47dmphen) and 5,6-dimethyl-1,10-phenanthroline (56dmphen). The size of NN ligand and its substituents modulate the compound electronic features and influence their antiproliferative efficiency against Entamoeba histolytica. 56dmphen derivative, shows the biggest molar volume and presents a powerful amoebicidal activity (IC50 = 1.2 µM), being seven times more effective than the first-line drug for human amoebiasis metronidazole. Also, increases the reactive oxygen species concentration within the trophozoites. This could be the trigger of the E. histolytica growth inhibition. The antiparasitic effect is described using NiII electron density, molar volume, estimated by DFT, as well as the experimental redox potential and diffusion coefficients. In general, amoebicidal efficiency is directly proportional to the increment of the molar volume and decreases when the redox potential becomes more positive.

Vitamin supplementation increases the virulence of Entamoeba histolytica grown axenically.

As a consequence of axenic growth and the elimination of accompanying bacterial flora, Entamoeba histolytica virulence decreases rapidly, and pathogenicity is lost. This paper evaluated the impact of vitamin supplementation on the pathogenicity of E. histolytica. Growth of E. histolytica trophozoites, cultured axenically in PEHPS (a Spanish acronym for the main ingredients - casein peptone, liver, pancreas extract and bovine serum) medium, with or without vitamins, exhibited a similar growth rate. However, the vitamin-enriched PEHPS preparations expressed 2.65 times more haemolytic activity (at 60 min: 98 vs 48%, P < 0.05), 2.5 times more phospholipase A2 activity at 150 min of incubation and generated more hepatic abscesses (88 vs 60%, P = 0.05) than the preparations without vitamins. The haemolytic and phospholipase A2 activity for the PEHPS - V preparations were restored following vitamin supplementation with A and D. These data highlight, for the first time, that vitamins and specifically vitamin A and D were essential for the recovery of amoebic virulence, lost through axenic growth.

Anti-amoebic activity of a cecropin-melittin hybrid peptide (CM11) against trophozoites of Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite that is located in the lumen of the human intestine and can attack the epithelium. Antimicrobial peptides (AMPs) are effective against the wide range of microorganisms, such as bacteria, fungi, viruses, yeasts, and protozoa. The CM11 is a chimeric peptide that is derived from bee venom and butterfly compounds. In this study, the cytotoxic effect of CM11 on Human colonic carcinoma (Caco­2) cells and E. histolytica were assayed in various concentrations of peptide and metronidazole. The MTT results showed that the highest percentage of cytotoxicity on Caco­2 cells was in 24 µg/ml of CM11 peptide at 24 h and 48 h, which was 49.8%, and 44.3%, respectively. In the metronidazole group, the highest cytotoxicity with 40 µg/ml concentration was observed after 24 h and 48 h, with 43.5%, and 42.1%, respectively. The highest rate of apoptosis induced by CM11 on Caco­2 was 53.9% and 51.4% after 24 h and 48 h, respectively; however, these rates were 19.1% and 33.4% in the metronidazole group. The effect of peptide and metronidazole on E. histolytica at 24 h and 48 h showed that at the highest concentration of CM11 peptide (24 µg/ml) the cytotoxic effect was 93.7% and 94.9% and for metronidazole (40 µg/ml) was 65.5% and 74.3%, respectively. In coculture, 63.5% and 57.7% of parasites were killed in the highest concentration of CM11 and metronidazole, respectively. The results of this study revealed that CM11 peptide has a high toxicity on E. histolytica, and the use of antimicrobial peptides in the future can be considered as anti-amoebic compounds.

Entamoeba histolytica Calreticulin Induces the Expression of Cytokines in Peripheral Blood Mononuclear Cells Isolated From Patients With Amebic Liver Abscess.

Calreticulin (CRT) is a highly conserved protein in the endoplasmic reticulum that plays important roles in the regulation of key cellular functions. Little is known about the participation of E. histolytica CRT (EhCRT) in the processes of pathogenicity or in the modulation of the host immune response. The aim of this study was to evaluate the role of CRT in the proliferation and the cytokine profile in peripheral blood mononuclear cells (PBMCs) from patients with amebic liver abscess (ALA) during the acute phase (AP-ALA) of the disease compared to patients during the resolution phase (R-ALA). The PBMCs from each participant were cocultured with EhCRT and tested by the colorimetric method to evaluate their proliferation index (PI). The supernatants were subjected to an enzyme-linked immunosorbent assay (ELISA) to evaluate the concentration of cytokines. The mean values of all groups were compared using the independent t-test. When the PIs of individuals without diagnosis of liver abscess (NEG) were compared, there were no statistically significant differences in the proliferation of PBMCs between patients with AP-ALA and R-ALA when stimulated with EhCRT or concanavalin A (ConA). However, the levels of interleukins [IL-6, IL-10, granulocyte colony stimulating factor (GCSF), and transforming growth factor ß1 (TGFß1)] were higher in patients with AP-ALA, whereas in patients with R-ALA, higher levels of interferon gamma (IFNγ) were detected. These results suggest that EhCRT acts as a mitogen very similar to the activity of ConA. In addition, EhCRT is an excellent immunogen for the specific activation of PBMCs, inducing the differential expression of ILs depending on the outcome of disease, determining the type of immune response: a Th2 cytokine profile during the acute phase and a Th1 profile during the resolution phase.

Entamoeba histolytica: Quantitative Proteomics Analysis Reveals Putative Virulence-Associated Differentially Abundant Membrane Proteins.

Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.

Stress-induced nuclear depletion of Entamoeba histolytica 3'-5' exoribonuclease EhRrp6 and its role in growth and erythrophagocytosis.

The 3'-5' exoribonuclease Rrp6 is a key enzyme in RNA homeostasis involved in processing and degradation of many stable RNA precursors, aberrant transcripts, and noncoding RNAs. We previously have shown that in the protozoan parasite Entamoeba histolytica, the 5'-external transcribed spacer fragment of pre-rRNA accumulates under serum starvation-induced growth stress. This fragment is a known target of degradation by Rrp6. Here, we computationally and biochemically characterized EhRrp6 and found that it contains the catalytically important EXO and HRDC domains and exhibits exoribonuclease activity with both unstructured and structured RNA substrates, which required the conserved DEDD-Y catalytic-site residues. It lacked the N-terminal PMC2NT domain for binding of the cofactor Rrp47, but could functionally complement the growth defect of a yeast rrp6 mutant. Of note, no Rrp47 homologue was detected in E. histolytica Immunolocalization studies revealed that EhRrp6 is present both in the nucleus and cytosol of normal E. histolytica cells. However, growth stress induced its complete loss from the nuclei, reversed by proteasome inhibitors. EhRrp6-depleted E. histolytica cells were severely growth restricted, and EhRrp6 overexpression protected the cells against stress, suggesting that EhRrp6 functions as a stress sensor. Importantly EhRrp6 depletion reduced erythrophagocytosis, an important virulence determinant of E. histolytica This reduction was due to a specific decrease in transcript levels of some phagocytosis-related genes (Ehcabp3 and Ehrho1), whereas expression of other genes (Ehcabp1, Ehcabp6, Ehc2pk, and Eharp2/3) was unaffected. This is the first report of the role of Rrp6 in cell growth and stress responses in a protozoan parasite.

Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica.

Messenger RNA 3'-end polyadenylation is an important regulator of gene expression in eukaryotic cells. In our search for new ways of treating parasitic infectious diseases, we looked at whether or not alterations in polyadenylation might control the survival of Entamoeba histolytica (the agent of amoebiasis in humans). We used molecular biology and computational tools to characterize the mRNA cleavage factor EhCFIm25, which is essential for polyadenylation in E. histolytica. By using a strategy based on the systematic evolution of ligands by exponential enrichment, we identified single-stranded RNA aptamers that target EhCFIm25. The results of RNA-protein binding assays showed that EhCFIm25 binds to the GUUG motif in vitro, which differs from the UGUA motif bound by the homologous human protein. Accordingly, docking experiments and molecular dynamic simulations confirmed that interaction with GUUG stabilizes EhCFIm25. Incubating E. histolytica trophozoites with selected aptamers inhibited parasite proliferation and rapidly led to cell death. Overall, our data indicate that targeting EhCFIm25 is an effective way of limiting the growth of E. histolytica in vitro. The present study is the first to have highlighted the potential value of RNA aptamers for controlling this human pathogen.

Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target.

The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.

Amoebicidal activity of curcumin on Entamoeba histolytica trophozoites.

OBJECTIVES: This study was undertaken to investigate the amoebicidal potential of curcumin on Entamoeba histolytica, as well as its synergistic effect with metronidazole. METHODS: Entamoeba histolytica trophozoites were exposed to 100, 200 and 300 µm of curcumin, for 6, 12 and 24 h. Consequently, the viability of cells was determined by trypan blue exclusion test. All specimens were further analysed by scanning electron microscopy. For drug combination experiment, the Chou-Talalay method was used. KEY FINDINGS: Curcumin affected the growth and cell viability in a time- and dose-dependent manner. The higher inhibitory effects were observed with 300 µm at 24 h; 65.5% of growth inhibition and only 28.8% of trophozoites were viable. Additionally, curcumin also altered adhesion and the morphology of the trophozoites. Scanning electron microscopy revealed treated trophozoites with damages on the membrane, size alterations and parasites with loss of cellular integrity. In addition, the combination of curcumin + metronidazole exhibited a synergistic effect; the activity of both drugs was improved. CONCLUSIONS: This is the first report evaluating the effectiveness of curcumin against E. histolytica. Our results suggest that CUR could be considered for evaluation in future pharmacological studies as a promising amoebicidal agent or as complementary therapy.

Entamoeba histolytica Cyclooxygenase-Like Protein Regulates Cysteine Protease Expression and Virulence.

The intestinal protozoan parasite Entamoeba histolytica (Eh) causes amebiasis associated with severe diarrhea and/or liver abscess. Eh pathogenesis is multifactorial requiring both parasite virulent molecules and host-induced innate immune responses. Eh-induced host pro-inflammatory responses plays a critical role in disease pathogenesis by causing damage to tissues allowing parasites access to systemic sites. Eh cyclooxygenase (EhCox) derived prostaglandin E2 stimulates the chemokine IL-8 from mucosal epithelial cells that recruits neutrophils to the site of infection to exacerbate disease. At present, it is not known how EhCox is regulated or whether it affects the expression of other proteins in Eh. In this study, we found that gene silencing of EhCox (EhCoxgs) markedly increased endogenous cysteine protease (CP) protein expression and virulence without altering CP gene transcripts. Live virulent Eh pretreated with arachidonic acid substrate to enhance PGE2 production or aspirin to inhibit EhCox enzyme activity or addition of exogenous PGE2 to Eh had no effect on EhCP activity. Increased CP enzyme activity in EhCoxgs was stable and significantly enhanced erythrophagocytosis, cytopathic effects on colonic epithelial cells and elicited pro-inflammatory cytokines in mice colonic loops. Acute infection with EhCoxgs in colonic loops increased inflammation associated with high levels of myeloperoxidase activity. This study has identified EhCox protein as one of the important endogenous regulators of cysteine protease activity. Alterations of CP activity in response to Cox gene silencing may be a negative feedback mechanism in Eh to limit proteolytic activity during colonization that can inadvertently trigger inflammation in the gut.