Protein Phosphatase PP2C Identification in Entamoeba spp.

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

Entamoeba histolytica exploits the autophagy pathway in macrophages to trigger inflammation in disease pathogenesis.

The mechanism whereby Entamoeba histolytica (Eh) binding with macrophages at the intercellular junction triggers aggressive pro-inflammatory responses in disease pathogenesis is not well understood. The host intracellular protein degradation process autophagy and its regulatory proteins are involved in maintenance of cellular homeostasis and excessive inflammatory responses. In this study we unraveled how Eh hijacks the autophagy process in macrophages to dysregulate pro-inflammatory responses. Direct contact of live Eh with macrophages activated caspase-6 that induced rapid proteolytic degradation of the autophagy ATG16L1 protein complex independent of NLRP3 inflammasome and caspase-3/8 activation. Crohn's disease susceptible ATG16L1 T300A variant was highly susceptible to Eh-mediated degradation that augmented pro-inflammatory cytokines in mice. Quantitative proteomics revealed downregulation of autophagy and vesicle-mediated transport and upregulation of cysteine-type endopeptidase pathways in response to Eh. We conclude during Eh-macrophage outside-in signaling, ATG16L1 protein complex plays an overlooked regulatory role in shaping the pro-inflammatory landscape in amebiasis.

Case report: multiple and atypical amoebic cerebral abscesses resistant to treatment.

BACKGROUND: The parasite Entamoeba histolytica is the causal agent of amoebiasis, a worldwide emerging disease. Amebic brain abscess is a form of invasive amebiasis that is both rare and frequently lethal. This condition always begins with the infection of the colon by E. histolytica trophozoites, which subsequently travel through the bloodstream to extraintestinal tissues. CASE PRESENTATION: We report a case of a 71-year-old female who reported an altered state of consciousness, disorientation, sleepiness and memory loss. She had no history of hepatic or intestinal amoebiasis. A preliminary diagnosis of colloidal vesicular phase neurocysticercosis was made based on nuclear magnetic resonance imaging (NMRI). A postsurgery immunofluorescence study was positive for the 140 kDa fibronectin receptor of E. histolytica, although a serum analysis by ELISA was negative for IgG antibodies against this parasite. A specific E. histolytica 128 bp rRNA gene was identified by PCR in biopsy tissue. The final diagnosis was cerebral amoebiasis. The patient underwent neurosurgery to eliminate amoebic abscesses and was then given a regimen of metronidazole, ceftriaxone and dexamethasone for 4 weeks after the neurosurgery. However, a rapid decline in her condition led to death. CONCLUSIONS: The present case of an individual with a rare form of cerebral amoebiasis highlights the importance of performing immunofluorescence, NMRI and PCR if a patient has brain abscess and a poorly defined diagnosis. Moreover, the administration of corticosteroids to such patients can often lead to a rapid decline in their condition.

Immune Response to the Enteric Parasite Entamoeba histolytica.

Entamoeba histolytica is a protozoan parasite responsible for amoebiasis, a disease with a high prevalence in developing countries. Establishing an amoebic infection involves interplay between pathogenic factors for invasion and tissue damage, and immune responses for protecting the host. Here, we review the pathogenicity of E. histolytica and summarize the latest knowledge on immune response and immune evasion mechanisms during amoebiasis.

Gastric and Caecal Amoebiasis in a Red Kangaroo (Macropus rufus) with Disseminated Toxoplasmosis.

A 1-year-old male red kangaroo (Macropus rufus) with an acute clinical history of lethargy, depression and increased respiratory rate was presented for necropsy examination. Gross lesions in the digestive tract were a distended stomach with watery content and multifocal raised ulcers covered by fibrinous pseudomembranes in the forestomach and caecum. On histopathology, there was necrotizing and ulcerative gastritis and typhlitis with intralesional amoebic trophozoites and Toxoplasma gondii zoites. Lesions due to T. gondii infection were observed in multiple organs and diagnosis was confirmed by fluorescent antibody test and immunohistochemistry. Both toxoplasmosis and gastric amoebiasis are diseases described in macropods. In this case report, we describe concurrent disease caused by both protozoa in a red kangaroo. Lesions of amoebiasis were also observed in the caecum. Both toxoplasmosis and amoebiasis should be considered as differential diagnoses of ulcerative lesions in the gastrointestinal tract in macropods during necropsy examination. Amoebiasis should be suspected especially when ulcerative lesions are observed in the forestomach.

Entamoeba histolytica Interaction with Enteropathogenic Escherichia coli Increases Parasite Virulence and Inflammation in Amebiasis.

Epidemiological studies suggest frequent association of enteropathogenic bacteria with Entamoeba histolytica during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic Escherichia coli (strain DH5α) could modify the virulence of E. histolytica to cause disease in animal models of amebiasis. In vitro studies showed a 2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted with EPEC but not with E. coli DH5α for 2.5 h. This was associated with increased E. histolytica proteolytic activity as revealed by zymogram analysis and degradation of the E. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 substrate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP-A1, -A2, -A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked upregulation of E. histolytica virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inoculated with either E. histolytica-EPEC or E. histolytica-E. coli DH5α. Histopathology of livers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reactions, ischemia, and necrosis with high expression of il-1ß, ifn-γ, and tnf-α proinflammatory cytokine genes compared with that in livers of E. histolytica-E. coli DH5α-inoculated animals. In closed colonic loops from mice, intense inflammation was observed with E. histolytica-EPEC manifested by downregulation of Math1 mRNA with a corresponding increase in the expression of Muc2 mucin and proinflammatory cytokine genes il-6, il-12, and mcp-1 These results demonstrate that E. histolytica/EPEC interaction enhanced the expression and production of key molecules associated with E. histolytica virulence, critical in pathogenesis and progression of disease.

Pathology Associated With an Outbreak of Entamoebiasis in Wild Cane Toads (Rhinella marina) in Tropical Australia.

Infection due to Entamoeba spp. is known to cause serious disease in primates (Entamoeba histolytica) and snakes (Entamoeba invadens), but there are no detailed descriptions of the pathology associated with Entamoeba spp. infection in amphibians. In 2014, an outbreak of entamoebiasis associated with a novel species of Entamoeba induced clinical illness and poor body condition in free-ranging cane toads in Australia's Northern Territory. Here, we describe the gross pathology, histology, and clinical pathology linked to the outbreak. The study compared 25 toads with invasive entamoebiasis, defined as histologically visible amoebas within tissue, and 12 toads without invasive entamoebiasis. Grossly, affected toads had mild to marked congestion of colonic serosal vasculature, with variable thickening of the intestinal wall and serosanguineous to hemorrhagic colonic content. Histologically, invasive entamoebiasis manifested primarily as moderate to severe, variably hyperplastic to ulcerative colitis. The small intestine was affected in 10 of 25 toads, and 5 of 25 toads also had gastric lesions. Amoebas consistent in morphology with Entamoeba sp. were commonly intermingled with mucosal epithelium, frequently along the basement membrane, with deeper invasion into the superficial lamina propria in only 5 toads. Toads with invasive entamoebiasis had neutrophilia, monocytosis, and lymphopenia, and thus elevated neutrophil to lymphocyte ratios, suggestive of an inflammatory and/or stress leukogram.

Development of a Novel Ex-vivo 3D Model to Screen Amoebicidal Activity on Infected Tissue.

Amoebiasis is a parasitic disease that causes thousands of deaths every year, its adverse effects and resistance to conventional treatments have led to the search of new treatment options, as well as the development of novel screening methods. In this work, we implemented a 3D model of intestine and liver slices from hamsters that were infected ex vivo with virulent E. histolytica trophozoites. Results show preserved histology in both uninfected tissues as well as ulcerations, destruction of the epithelial cells, and inflammatory reaction in intestine slices and formation of micro abscesses, and the presence of amoebae in the sinusoidal spaces and in the interior of central veins in liver slices. The three chemically synthetized compounds T-001, T-011, and T-016, which act as amoebicides in vitro, were active in both infected tissues, as they decreased the number of trophozoites, and provoked death by disintegration of the amoeba, similar to metronidazole. However, compound T-011 induced signs of cytotoxicity to liver slices. Our results suggest that ex vivo cultures of precision-cut intestinal and liver slices represent a reliable 3D approach to evaluate novel amoebicidal compounds, and to simultaneously detect their toxicity, while reducing the number of experimental animals commonly required by other model systems.

Coinfection with Entamoeba polecki and Brachyspira hyodysenteriae in a pig with severe diarrhea.

Enteric disease in pigs is usually of multifactorial etiology, including infectious and non-infectious factors. In many cases of endemic diarrhea in weaner-to-finisher pigs, the combination of 2 or more microorganisms leads to aggravation of intestinal lesions and, consequently, clinical signs. We autopsied a 4-mo-old fattening pig with diarrhea and diagnosed severe fibrinonecrotizing typhlocolitis. Numerous spiral-shaped bacteria and amoeba-like PAS-positive protozoa were observed in the cecal and colonic mucosa and submucosa. Brachyspira hyodysenteriae was detected by PCR from colonic content. By in situ hybridization, large numbers of Entamoeba polecki were found within the lamina propria and submucosa; moderate numbers of Blastocystis sp. and scattered trichomonads were present in intestinal content. In addition, Entamoeba polecki, Balantidium spp., Blastocystis sp., and Trichomonas sp. were also detected by PCR.

Amoebic colitis presenting with hypo-albuminaemia in an eight-month-old breastfed girl.

Entamoeba histolytica is a protozoan parasite that affects a large proportion of the world's population and causes amoebic dysentery and extra-intestinal disease. Many individuals remain asymptomatic during colonisation; in 10% of individuals, the parasite breaks through the mucosal barrier and leads to invasive disease. An eight-month-old girl who was evaluated for hypo-albuminaemia and was diagnosed with amoebic colitis is reported. To the best of our knowledge, this is the first report of hypo-albuminaemia owing to amoebic colitis in any age group.

Clinical and microscopic predictors of Entamoeba histolytica intestinal infection in travelers and migrants diagnosed with Entamoeba histolytica/dispar infection.

BACKGROUND: Amebiasis is a protozoal infection caused by Entamoeba histolytica, while the morphologically indistinguishable E. dispar is considered as non-pathogenic. Polymerase chain reaction (PCR) assays are necessary to differentiate both species. The most common clinical presentations of E. histolytica disease are amebic colitis and amebic liver abscess, but asymptomatic infection is also possible. We assessed the frequency and pattern of clinical symptoms and microscopic features in travelers/migrants associated with E. histolytica intestinal infection and compared them to those found in individuals with E. dispar infection. METHODS: We conducted a retrospective study at the travel clinic of the Institute of Tropical Medicine, Antwerp, Belgium on travelers/migrants found from 2006 to 2016 positive for Entamoeba histolytica/dispar through antigen detection and/or through microscopy confirmed by PCR. All files of individuals with a positive PCR for E. histolytica (= cases) and a random selection of an equal number of Entamoeba dispar carriers (= controls) were reviewed. We calculated the sensitivity, specificity and likelihood ratios (LRs) of clinical symptoms (blood in stool, mucus in stool, watery diarrhea, abdominal cramps, fever or any of these 5 symptoms) and of microscopic features (presence of trophozoites in direct and in sodium acetate-acetic acid-formalin (SAF)-fixed stool smears) to discriminate between E. histolytica and E. dispar infection. RESULTS: Of all stool samples positive for Entamoeba histolytica/dispar for which PCR was performed (n = 810), 30 (3.7%) were true E. histolytica infections, of which 39% were asymptomatic. Sensitivity, specificity and positive LRs were 30%, 100% and 300 (p 0.007) for presence of blood in stool; 22%, 100% and 222 (p 0.03) for mucus in stool; 44%, 90% and 4.7 (p 0.009) for cramps and 14%, 97% and 4.8 (p = 0.02) for trophozoites in direct smears. For watery diarrhea, fever and for trophozoites in SAF fixated smears results were non-significant. CONCLUSIONS: E. histolytica infection was demonstrated in a small proportion of travelers/migrants with evidence of Entamoeba histolytica/dispar infection. In this group, history of blood and mucus in stool and cramps had good to strong confirming power (LR+) for actual E. histolytica infection. Trophozoites were also predictive for true E. histolytica infection but in direct smears only.

Characteristics of inflammatory reactions during development of liver abscess in hamsters inoculated with Entamoeba nuttalli.

BACKGROUND: Entamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica. METHODOLOGY/PRINCIPAL FINDINGS: Trophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1ß) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro. CONCLUSIONS/SIGNIFICANCE: The results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species.

Entamoeba Histolytica: Updates in Clinical Manifestation, Pathogenesis, and Vaccine Development.

Entamoeba histolytica is the responsible parasite of amoebiasis and remains one of the top three parasitic causes of mortality worldwide. With increased travel and emigration to developed countries, infection is becoming more common in nonendemic areas. Although the majority of individuals infected with E. histolytica remain asymptomatic, some present with amoebic colitis and disseminated disease. As more is learned about its pathogenesis and the host's immune response, the potential for developing a vaccine holds promise. This narrative review outlines the current knowledge regarding E. histolytica and E. dispar and insight in the development of a vaccine.

Entamoeba histolytica EhCP112 Dislocates and Degrades Claudin-1 and Claudin-2 at Tight Junctions of the Intestinal Epithelium.

During intestinal invasion, Entamoeba histolytica opens tight junctions (TJs) reflected by transepithelial electrical resistance (TEER) dropping. To explore the molecular mechanisms underlying this, we studied in vitro and in vivo the damage produced by the recombinant E. histolytica cysteine protease (rEhCP112) on TJ functions and proteins. rEhCP112 reduced TEER in Caco-2 cells in a dose- and time-dependent manner; and EhCP112-overexpressing trophozoites provoked major epithelial injury compared to control trophozoites. rEhCP112 penetrated through the intercellular space, and consequently the ion flux increased and the TJs fence function was disturbed. However, macromolecular flux was not altered. Functional in vitro assays revealed specific association of rEhCP112 with claudin-1 and claudin-2, that are both involved in regulating ion flux and fence function. Of note, rEhCP112 did not interact with occludin that is responsible for regulating macromolecular flux. Moreover, rEhCP112 degraded and delocalized claudin-1, thus affecting interepithelial adhesion. Concomitantly, expression of the leaky claudin-2 at TJ, first increased and then it was degraded. In vivo, rEhCP112 increased intestinal epithelial permeability in the mouse colon, likely due to apical erosion and claudin-1 and claudin-2 degradation. In conclusion, we provide evidence that EhCP112 causes epithelial dysfunction by specifically altering claudins at TJ. Thus, EhCP112 could be a potential target for therapeutic approaches against amoebiasis.

Adherens junctions and desmosomes are damaged by Entamoeba histolytica: Participation of EhCPADH complex and EhCP112 protease.

Entamoeba histolytica trophozoites adhere to epithelium at the cell-cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded ß-cat, E-cad, Dsp l/ll, and Dsg-2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with ß-cat, E-cad, Dsp l/ll, and Dsg-2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin-coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with ß-cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E-cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.

MUC2 Mucin and Butyrate Contribute to the Synthesis of the Antimicrobial Peptide Cathelicidin in Response to Entamoeba histolytica- and Dextran Sodium Sulfate-Induced Colitis.

Embedded in the colonic mucus are cathelicidins, small cationic peptides secreted by colonic epithelial cells. Humans and mice have one cathelicidin-related antimicrobial peptide (CRAMP) each, LL-37/hCAP-18 and Cramp, respectively, with related structure and functions. Altered production of MUC2 mucin and antimicrobial peptides is characteristic of intestinal amebiasis. The interactions between MUC2 mucin and cathelicidins in conferring innate immunity against Entamoeba histolytica are not well characterized. In this study, we quantified whether MUC2 expression and release could regulate the expression and secretion of cathelicidin LL-37 in colonic epithelial cells and in the colon. The synthesis of LL-37 was enhanced with butyrate (a product of bacterial fermentation) and interleukin-1ß (IL-1ß) (a proinflammatory cytokine in colitis) in the presence of exogenously added purified MUC2. The LL-37 responses to butyrate and IL-1ß were higher in high-MUC2-producing cells than in lentivirus short hairpin RNA (shRNA) MUC2-silenced cells. Activation of cyclic adenylyl cyclase (AMP) and mitogen-activated protein kinase (MAPK) signaling pathways was necessary for the simultaneous expression of MUC2 and cathelicidins. In Muc2 mucin-deficient (Muc2-/-) mice, murine cathelicidin (Cramp) was significantly reduced compared to that in Muc2+/- and Muc2+/+ littermates. E. histolytica-induced acute inflammation in colonic loops stimulated high levels of cathelicidin in Muc2+/+ but not in Muc2-/- littermates. In dextran sodium sulfate (DSS)-induced colitis in Muc2+/+ mice, which depletes the mucus barrier and goblet cell mucin, Cramp expression was significantly enhanced during restitution. These studies demonstrate regulatory mechanisms between MUC2 and cathelicidins in the colonic mucosa where an intact mucus barrier is essential for expression and secretion of cathelicidins in response to E. histolytica- and DSS-induced colitis.