Identification of Multiple Domains of Entamoeba histolytica Intermediate Subunit Lectin-1 with Hemolytic and Cytotoxic Activities.

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica have roles in the pathogenicity of intestinal amoebiasis. Igl1, the intermediate subunit lectin-1 of E. histolytica, has been shown to have both hemolytic and cytotoxic activities that reside in the C-terminus of the protein. To identify the amino acid regions responsible for these activities, recombinant proteins were prepared and used in hemolytic and cytotoxic assays. The results revealed that Igl1 has multiple domains with hemolytic and cytotoxic activities and that amino acids 787-846, 968-1028 and 1029-1088 are involved in these activities. The hemolytic activities of the fragments were partly inhibited by mannose, galactose and N-acetylgalactosamine, and glucose showed lower or negligible inhibitory effects for the activities. This is the first report of a protozoan protein with hemolytic and cytotoxic activities in multiple domains.

An Unusual U2AF2 Inhibits Splicing and Attenuates the Virulence of the Human Protozoan Parasite Entamoeba histolytica.

E. histolytica is the etiological agent of intestinal amebiasis and liver abscesses, which still poses public health threat globally. Metronidazole is the drug of choice against amebiasis. However, metronidazole-resistant amoebic clinical isolates and strains have been reported recently, challenging the efforts for amebiasis eradication. In search of alternative treatments, E. histolytica transcriptomes have shown the association of genes involved in RNA metabolism with the virulence of the parasite. Among the upregulated genes in amoebic liver abscesses are the splicing factors EhU2AF2 and a paralog of EhSF3B1. For this reason and because EhU2AF2 contains unusual KH-QUA2 (84KQ) motifs in its lengthened C-terminus domain, here we investigated how the role of EhU2AF2 in pre-mRNA processing impacts the virulence of the parasite. We found that 84KQ is involved in splicing inhibition/intron retention of several virulence and non-virulence-related genes. The 84KQ domain interacts with the same domain of the constitutive splicing factor SF1 (SF1KQ), both in solution and when SF1KQ is bound to branchpoint signal RNA probes. The 84KQ-SF1KQ interaction prevents splicing complex E to A transition, thus inhibiting splicing. Surprisingly, the deletion of the 84KQ domain in EhU2AF2 amoeba transformants increased splicing and enhanced the in vitro and in vivo virulence phenotypes. We conclude that the interaction of the 84KQ and SF1KQ domains, probably involving additional factors, tunes down Entamoeba virulence by favoring intron retention.

Modified amoebic liver abscess: Case report / Absceso hepático amebiano modificado: reporte de un caso

Abstract We present the case of a 56-year-old black female patient from a rural area in the Morón municipality, Ciego de Ávila province, Cuba. She was admitted with symptoms of dysentery with several days of evolution and a later episode of high fever, compromised general status, and abdominal pain located in the right hypochondrium. Analytical studies reported leukocytosis with a predominance of polymorphonuclear cells, Entamoeba histolytica was found in the stool study. Abdominal ultrasound reported a mixed image of 110 x 84 mm in the upper right lobe of the liver, as confirmed by computed tomography. This image was interpreted as a possible liver abscess. The patient received antimicrobial treatment for four weeks without a good response, thus requiring surgical intervention. She evolved favorably and was discharged after 21 days.

Resumen Se presenta el caso de una paciente de raza negra de 56 años procedente de área rural de Morón, provincia Ciego de Ávila (Cuba), quien ingresa por cuadro clínico de disentería de varios días de evolución acompañado de fiebre, compromiso de su estado general y dolor abdominal en el hipocondrio derecho. Los estudios analíticos de laboratorio mostraron leucocitosis con predominio de neutrófilos y presencia de trofozoitos de Entamoeba histolytica en la materia fecal. La ecografía de abdomen reporto una imagen mixta de 110 x 84 mm en el lóbulo derecho del hígado y la tomografía confirmó la lesión que se interpretó como un posible absceso hepático. Se inició tratamiento antimicrobiano por un periodo de 4 semanas sin adecuada respuesta por lo que requirió tratamiento quirúrgico. Su evolución fue favorable con egreso a los 21 días.

Ascorbic Acid Ameriolates Liver Damage by Myeloperoxidase Oxidative Products in a Hamster Model of Amoebic Liver Abscess.

Entamoeba histolytica is a protozoan-pathogen-causing amoebic liver abscess (ALA). After amoeba establishment in the liver, it causes abundant infiltrate of neutrophils. Liver tissue damage by neutrophils results in part from anti-amoebic oxidative intermediates, including reactive oxygen species (ROS), reactive nitrogen species (RNS), and hypochlorous acid (HOCl), derived from the myeloperoxidase (MPO) enzyme. Ascorbic acid (ASC) is an antioxidant that acts as a scavenger for ROS and NOS-derived free radicals. No previous information regarding the effect of ASC concerning the participation of MPO in an experimental model of ALA in hamsters has been reported. Thus, the aim of the present work was to analyze the effect of ASC on acute ALA development and to measure the activity and gene expression of the MPO enzyme. Hamsters were treated with ASC (800 mg/kg) and then intrahepatically inoculated with E. histolytica trophozoites. Animals were sacrificed at 3, 6, and 12 h post-inoculation (p.i.), and liver samples were collected. The percentage of lesions, amoeba in situ count, MPO activity, and mpo gene expression were ascertained. Compared to ALA hamsters without ASC treatment as the control group (CT), the ALA group treated with ASC had a significant decrease in liver lesions (all p.i. hours) and viable amoeba count (12 h p.i.) and an increase in MPO activity (12 h p.i.) and mpo gene expression (6 h/12 h p.i.). These data suggest that ASC ameliorated liver damage caused by oxidizing products via modulation of mpo expression and activity.

Furanocoumarins from Ruta chalepensis with Amebicide Activity.

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.

Queuine Is a Nutritional Regulator of Entamoeba histolytica Response to Oxidative Stress and a Virulence Attenuator.

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in [Formula: see text] Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in [Formula: see text], parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.IMPORTANCEEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.

Prevalence of intestinal parasitic infections and associated factors among patients attending at Sanja Primary Hospital, Northwest Ethiopia: An institutional-based cross-sectional study.

BACKGROUND: Intestinal Parasitic Infections are the most prevalent diseases in the world, predominantly in developing countries. It is estimated that more than two billion people are affected globally, mostly in tropical and sub-tropical parts of the world. Ethiopia is one of the countries in Africa with a high prevalence of intestinal parasites. However, there is a limited study conducted in the study area. Hence, this study was to assess the prevalence and associated factors of intestinal parasitosis among patients attending at Sanja Primary Hospital, Northwest Ethiopia. METHODS: An institutional-based cross-sectional study was conducted at Sanja Primary Hospital from January 1 to August 20, 2019. Stool samples were collected from 1240 study participants and analyzed by direct wet mount and formal ether concentration techniques. Furthermore, sociodemographic and explanatory variables were collected using a face-to-face interview. Data were entered into Epi data version 4.4.2.1 and transferred to SPSS version 23 for analysis. Bivariate and multivariate binary logistic regression models were fitted to identify associated factors of intestinal parasitic infections. Adjusted Odds Ratio (AOR) with 95% confidence interval (CI) was considered to ascertain the significance of the association. RESULTS: The overall prevalence of intestinal parasitic infection was 52.9% (95% CI: 50.2%-55.5%). Entamoeba histolytica/dispar (21.5%) was the leading cause of intestinal parasitosis followed by Hookworm species (13.3%). Furthermore, the rate of double and triple parasitic infections was observed in 6.1% and 0.5% of study participants respectively. Being Illiterate (AOR: 2.87, 95% CI: 1.06-7.47, p = 0.038), swimming habits of more than 4 times a month (AOR = 2.91, 95% CI 1.62-5.24, p< 0.001) and not washing hands before a meal (AOR: 3.92, 95% CI: 1.74-8.83, p = 0.001) were the key factors significantly associated with intestinal parasitic infection. CONCLUSIONS: The present study showed that the prevalence of intestinal parasitosis is high in the study area. Therefore, there is a need for an integrated control program, including improving personal, environmental sanitation and health education should be given to have a lasting impact on transmission.

Factors Associated with High Rates of Recurrence of Amebic Liver Abscess (ALA) in North India.

Recurrence of amebic liver abscess (ALA), once considered unusual, is increasingly being reported, despite proper management. Realizing the endemicity of ALA in the study setup, this 2-year follow-up study was conducted to investigate the recurrent cases and study the associated factors. A total of 101 confirmed cases of ALA were followed up for a period of 2 years. Recurrent cases were studied for associated bacterial flora, presence of resistance genes (nim), level of matrix metalloproteinase 3 and MMP-9, and genotypes of Entamoeba histolytica and statistically compared with the nonrecurrent cases as controls. Recurrence rates of 8.9% (nine patients) were detected. The presence of Prevotella along with an increased level of MMP-9 in abscess fluid and large size of abscesses (11 × 10.8 cm) was found to be significantly associated with recurrence in ALA. Among the nine cases, the presence of nimE gene was detected in two (22.2%) patients. The genotyping of E. histolytica strains showed that in seven (77.7%) cases, the genotype of E. histolytica was the same in the primary and recurrent samples. This study reports a high rate of recurrence in the cases of ALA, hinting toward the gradual development of clinical resistance toward the commonly used drug. The presence of nim gene and Prevotella in abscess fluid along with increased MMP-9 levels and large abscess size could be important predictors of recurrent ALA.

Low molecular weight protein tyrosine phosphatase (LMW-PTP2) protein can potentially modulate virulence of the parasite Entamoeba histolytica.

The Entamoeba histolytica parasite is the causative agent of amebiasis, infecting approximately 1% of the world population and causing 100,000 deaths per year. It binds to Fibronectin (FN), activating signaling pathways regulated by kinases and phosphatases. EhLMW-PTPs genes from E. histolytica encode for Low Molecular Weight Tyrosine Phosphatases expressed in trophozoites and amoebic cysts. The role of these phosphatases in the virulence of the parasite has not yet been well characterized. Our results showed a differential expression of the EhLMW-PTPs, at the mRNA and protein levels, in an asynchronous trophozoites culture. Furthermore, we observed that trophozoites transfected that overexpressed EhLMW-PTP2 phagocytized fewer erythrocytes, possibly due to decreased phagocytic cups, and showed deficiencies in adherence to FN and less cytopathic effect. These analyzes suggest that the parasite's EhLMW-PTPs have an essential role in the mechanisms of proliferation, adhesion, and phagocytosis, regulating its pathogenicity.

The atypical protein arginine methyltrasferase of Entamoeba histolytica (EhPRMTA) is involved in cell proliferation, heat shock response and in vitro virulence.

Protein arginine methylation regulates several cellular events, including epigenetics, splicing, translation, and stress response, among others. This posttranslational modification is catalyzed by protein arginine methyltransferases (PRMTs), which according to their products are classified from type I to type IV. The type I produces monomethyl arginine and asymmetric dimethyl arginine; in mammalian there are six families of this PRMT type (PRMT1, 2, 3, 4, 6, and 8). The protozoa parasite Entamoeba histolytica has four PRMTs related to type I; three of them are similar to PRMT1, but the other one does not show significant homology to be grouped in any known PRMT family, thus we called it as atypical PRMT (EhPRMTA). Here, we showed that EhPRMTA does not contain several of the canonical amino acid residues of type I PRMTs, confirming that it is an atypical PRMT. A specific antibody against EhPRMTA localized this protein in cytoplasm. The recombinant EhPRMTA displayed catalytic activity on commercial histones and the native enzyme modified its expression level during heat shock and erythrophagocytosis. Besides, the knockdown of EhPRMTA produced an increment in cell growth, and phagocytosis, but decreases cell migration and the survival of trophozoites submitted to heat shock, suggesting that this protein is involved in regulate negatively or positively these events, respectively. Thus, results suggest that this methyltransferase regulates some cellular functions related to virulence and cell surviving.

Rab7D small GTPase is involved in phago-, trogocytosis and cytoskeletal reorganization in the enteric protozoan Entamoeba histolytica.

Rab small GTPases regulate membrane traffic between distinct cellular compartments of all eukaryotes in a tempo-spatially specific fashion. Rab small GTPases are also involved in the regulation of cytoskeleton and signalling. Membrane traffic and cytoskeletal regulation play pivotal role in the pathogenesis of Entamoeba histolytica, which is a protozoan parasite responsible for human amebiasis. E. histolytica is unique in that its genome encodes over 100 Rab proteins, containing multiple isotypes of conserved members (e.g., Rab7) and Entamoeba-specific subgroups (e.g., RabA, B, and X). Among them, E. histolytica Rab7 is the most diversified group consisting of nine isotypes. While it was previously demonstrated that EhRab7A and EhRab7B are involved in lysosome and phagosome biogenesis, the individual roles of other Rab7 members and their coordination remain elusive. In this study, we characterised the third member of Rab7, Rab7D, to better understand the significance of the multiplicity of Rab7 isotypes in E. histolytica. Overexpression of EhRab7D caused reduction in phagocytosis of erythrocytes, trogocytosis (meaning nibbling or chewing of a portion) of live mammalian cells, and phagosome acidification and maturation. Conversely, transcriptional gene silencing of EhRab7D gene caused opposite phenotypes in phago/trogocytosis and phagosome maturation. Furthermore, EhRab7D gene silencing caused reduction in the attachment to and the motility on the collagen-coated surface. Image analysis showed that EhRab7D was occasionally associated with lysosomes and prephagosomal vacuoles, but not with mature phagosomes and trogosomes. Finally, in silico prediction of structural organisation of EhRab7 isotypes identified unique amino acid changes on the effector binding surface of EhRab7D. Taken together, our data suggest that EhRab7D plays coordinated counteracting roles: a inhibitory role in phago/trogocytosis and lyso/phago/trogosome biogenesis, and an stimulatory role in adherence and motility, presumably via interaction with unique effectors. Finally, we propose the model in which three EhRab7 isotypes are sequentially involved in phago/trogocytosis.

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.

Extracellular vesicles released by anaerobic protozoan parasites: Current situation.

Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism for transferring information between cells and organisms across all three kingdoms of life. Parasitic unicellular eukaryotes use EVs as vehicles for intercellular communication and host manipulation. Pathogenic protozoans are able to modulate the immune system of the host and establish infection by transferring a wide range of molecules contained in different types of EVs. In addition to effects on the host, EVs are able to transfer virulence factors, drug-resistance genes and differentiation factors between parasites. In this review we cover the current knowledge on EVs from anaerobic or microaerophilic extracellular protozoan parasites, including Trichomonas vaginalis, Tritrichomonas foetus, Giardia intestinalis and Entamoeba histolytica, with a focus on their potential role in the process of infection. The role of EVs in host: parasite communication adds a new level of complexity to our understanding of parasite biology, and may be a key to understand the complexity behind their mechanism of pathogenesis.

Direct and high-throughput assays for human cell killing through trogocytosis by Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebiasis. Pathogenesis is associated with profound damage to human tissues. We previously showed that amoebae kill human cells through trogocytosis. Trogocytosis is likely to underlie tissue damage during infection, although the mechanism is still unknown. Trogocytosis is difficult to assay quantitatively, which makes it difficult to study. Here, we developed two new, complementary assays to measure trogocytosis by quantifying human cell death. One assay uses CellTiterGlo, a luminescent readout for ATP, as a proxy for cell death. We found that the CellTiterGlo could be used to detect death of human cells after co-incubation with amoebae, and that it was sensitive to inhibition of actin or the amoeba surface Gal/GalNAc lectin, two conditions that are known to inhibit amoebic trogocytosis. The other assay uses two fluorescent nuclear stains to directly differentiate live and dead human cells by microscopy, and is also sensitive to inhibition of amoebic trogocytosis through interference with actin. Both assays are simple and inexpensive, can be used with suspension and adherent human cell types, and are amenable to high-throughput approaches. These new assays are tools to improve understanding of trogocytosis and amoebiasis pathogenesis.

The actin cytoskeleton orchestra in Entamoeba histolytica.

Years of evolution have kept actin conserved throughout various clades of life. It is an essential protein starring in many cellular processes. In a primitive eukaryote named Entamoeba histolytica, actin directs the process of phagocytosis. A finely tuned coordination between various actin-binding proteins (ABPs) choreographs this process and forms one of the virulence factors for this protist pathogen. The ever-expanding world of ABPs always has space to accommodate new and varied types of proteins to the earlier existing repertoire. In this article, we report the identification of 390 ABPs from Entamoeba histolytica. These proteins are part of diverse families that have been known to regulate actin dynamics. Most of the proteins are primarily uncharacterized in this organism; however, this study aims to annotate the ABPs based on their domain arrangements. A unique characteristic about some of the ABPs found is the combination of domains present in them unlike any other reported till date. Calponin domain-containing proteins formed the largest group among all types with 38 proteins, followed by 29 proteins with the infamous BAR domain in them, and 23 proteins belonging to actin-related proteins. The other protein families had a lesser number of members. Presence of exclusive domain arrangements in these proteins could guide us to yet unknown actin regulatory mechanisms prevalent in nature. This article is the first step to unraveling them.

Role of inflammasomes in innate host defense against Entamoeba histolytica.

Intestinal amebiasis is the disease caused by the extracellular protozoan parasite Entamoeba histolytica (Eh) that induces a dynamic and heterogeneous interaction profile with the host immune system during disease pathogenesis. In 90% of asymptomatic infection, Eh resides with indigenous microbiota in the outer mucus layer of the colon without prompting an immune response. However, for reasons that remain unclear, in a minority of the Eh-infected individuals, this fine tolerated relationship is switched to a pathogenic phenotype and advanced to an increasingly complex host-parasite interaction. Eh disease susceptibility depends on parasite virulence factors and their interactions with indigenous bacteria, disruption of the mucus bilayers, and adherence to the epithelium provoking host immune cells to evoke a robust pro-inflammatory response mediated by inflammatory caspases and inflammasome activation. To understand Eh pathogenicity and innate host immune responses, this review highlights recent advances in our understanding of how Eh induces outside-in signaling via Mϕs to activate inflammatory caspases and inflammasome to regulate pro-inflammatory responses.

Entamoeba histolytica and pathogenesis: A calcium connection.

Calcium signaling plays a key role in many essential processes in almost all eukaryotic systems. It is believed that it may also be an important signaling system of the protist parasite Entamoeba histolytica. Motility, adhesion, cytolysis, and phagocytosis/trogocytosis are important steps in invasion and pathogenesis of E. histolytica, and Ca2+ signaling is thought to be associated with these processes leading to tissue invasion. There are a large number of Ca2+-binding proteins (CaBPs) in E. histolytica, and a number of these proteins appear to be associated with different steps in pathogenesis. The genome encodes 27 EF-hand-containing CaBPs in addition to a number of other Ca2+-binding domain/motif-containing proteins, which suggest intricate calcium signaling network in this parasite. Unlike other eukaryotes, a typical calmodulin-like protein has not been seen in E. histolytica. Though none of the CaBPs display sequence similarity with a typical calmodulin, extensive structural similarity has been seen in spite of lack of significant functional overlap with that of typical calmodulins. One of the unique features observed in E. histolytica is the identification of CaBPs (EhCaBP1, EhCaBP3) that have the ability to directly bind actin and modulate actin dynamics. Direct interaction of CaBPs with actin has not been seen in any other system. Pseudopod formation and phagocytosis are some of the processes that require actin dynamics, and some of the amoebic CaBPs (EhC2Pk, EhCaBP1, EhCaBP3, EhCaBP5) participate in this process. None of these E. histolytica CaBPs have any homolog in organisms other than different species of Entamoeba, suggesting a novel Ca2+ signaling pathway that has evolved in this genus.