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The protozoan parasite Entamoeba histolytica is the microbial agent of amoebiasis – an infection that is endemic worldwide and is associated with high morbidity and mortality rates. As the disease develops, virulent E. histolytica deplete the mucus layer, interact with the intestinal epithelium, and then degrade the colonic mucosa and disrupt the extracellular matrix (ECM). Our research demonstrated that virulent parasites with an invasive phenotype display rapid, highly specific changes in their transcriptome (notably for essential factors involved in carbohydrate metabolism and the processing of glycosylated residues). Moreover, combined activation of parasite and host lytic enzymes leads to the destruction of the intestinal parenchyma. Together, these enzymes degrade the mucus layer and the ECM, and trigger the inflammatory response essential to the development of amoebiasis.

Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis. Entamoeba histolytica est un parasite extracellulaire des tissus provoquant des colites et occasionnellement des abcès du foie chez l’homme. Les produits de sécrétion dérivés d’E. histolytica (SPs) contiennent de grandes quantités de cystéine-protéases (CPs), l’un des principaux facteurs de virulence amibiens. Bien que les mastocytes tissulaires jouent un rôle important dans la réponse inflammatoire de la muqueuse à ce pathogène, on ne sait pas si les SPs induisent l’activation des mastocytes. Dans cette étude, lorsque des mastocytes humains (cellules HMC-1) ont été stimulés avec des SPs recueillis à partir d’amibes pathogènes de type sauvage, l’expression et la production de l’interleukine IL-8 ont été significativement augmentées par rapport à des cellules incubées avec du milieu seul. L’inhibition de l’activité des CPs dans les SPs avec la chaleur ou avec E64, un inhibiteur de CP, a entraîné une réduction significative de la production d’IL-8. En outre, les SPs obtenus à partir d’amibes surexprimant l’inhibiteur de protéases à cystéine (ICP) à faible activité de CP ont montré des effets stimulants plus faibles sur la production d’IL-8 que le contrôle de type sauvage. La pré-incubation des cellules HMC-1 avec des anticorps contre le récepteur 2 activé par la protéase humaine (PAR2) n’a pas affecté la production d’IL-8 induite par SPs. Ces résultats suggèrent que les cystéine-protéases des produits de sécrétion dérivés d’E. histolytica stimulent les mastocytes pour produire de l’IL-8 par l’intermédiaire d’un mécanisme indépendant de PAR2, ce qui contribue à la réponse inflammatoire tissulaire médiée par IL-8 au cours de la phase précoce de l’amibiase humaine.

Entamoeba histolytica , the causative agent of fatal diarrhoeal disease in children in the developing world, is shown here to kill human cells by biting off and ingesting pieces of cells, in a process reminiscent of the trogocytosis seen between immune cells; ingestion of bites is required for killing and this mechanism is used both in tissue culture and during invasion of intestinal explants. Entamoeba take a bite of intestine Entamoeba histolytica , the causative agent of fatal diarrhoeal disease in children in the developing world, was so named for its ability to destroy host tissues, although the mechanism underlying this effect was unclear. Here Katherine Ralston et al . describe how these amoeba kill intestinal epithelial cells by biting off and ingesting pieces of cell, in a process reminiscent of the trogocytosis seen between immune cells. Ingestion of the bites is required for killing, and the mechanism operates both in tissue culture and during invasion of intestinal explants. The authors suggest that intercellular exchange via trogocytosis may be more evolutionarily ancient and widespread than was assumed. This work also highlights amoebic trogocytosis as a potential target for new drugs to treat amoebiasis — a major neglected disease. Entamoeba histolytica is the causative agent of amoebiasis, a potentially fatal diarrhoeal disease in the developing world. The parasite was named “ histolytica ” for its ability to destroy host tissues, which is probably driven by direct killing of human cells. The mechanism of human cell killing has been unclear, although the accepted model was that the parasites use secreted toxic effectors to kill cells before ingestion 1 . Here we report the discovery that amoebae kill by ingesting distinct pieces of living human cells, resulting in intracellular calcium elevation and eventual cell death. After cell killing, amoebae detach and cease ingestion. Ingestion of human cell fragments is required for cell killing, and also contributes to invasion of intestinal tissue. The internalization of fragments of living human cells is reminiscent of trogocytosis (from Greek trogo , nibble) observed between immune cells 2 , 3 , 4 , 5 , 6 , but amoebic trogocytosis differs because it results in death. The ingestion of live cell material and the rejection of corpses illuminate a stark contrast to the established model of dead cell clearance in multicellular organisms 7 . These findings change the model for tissue destruction in amoebiasis and suggest an ancient origin of trogocytosis as a form of intercellular exchange.

Tetraspanins (TSPANs) are a family of highly conserved proteins present in a wide variety of eukaryotes. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica . Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native-PAGE analysis showed that these TSPANs form several complexes of 120–250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases, while repression of tspan4 led to a four-fold increase in the activity of cysteine proteases in crude extracellular vesicles (EVs) fraction. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites.

Entamoeba histolytica , a protozoan parasite, is the causative agent of amoebiasis, which is a significant global health concern. The virulence mechanisms underlying its pathogenicity are multifaceted and complex. However, endocytic processes and motility are well accepted virulence determinants. As previously reported, an AGCK family kinase, EhAGCK1 to be involved in trogocytosis exclusively while another one from same family named EhAGCK2 participates in all actin dependent endocytic processes. As the kinase dead mutants of EhAGCK1 showed significant defect in destruction of live host cells and also the localisation pattern of same is distinguishable from EhAGCK2. From observations so far, it appears that former initiates a distinguishable signaling cascade. In this work, we have demonstrated distinct biochemical properties of kinases involved in related yet distinguishable endocytic processes for the first time. Our biochemical characterization highlights distinct ion dependency of EhAGCK1 along with substrate specificity. We also show upstream activator of these kinases, 3-phosphoinositide dependent kinase 1 (PDK1) activity and its role in activating the kinase activity. The kinases exhibit property of autophosphorylation, and which may regulate the kinase activity subsequently. Summarily, these studies show that EhAGCK1 and EhAGCK2 show distinct biochemical properties which further confirm their unique role in related endocytic processes of trogocytosis and phagocytosis.

The parasitic protozoan Entamoeba histolytica secretes extracellular vesicles (EVs), but so far little is known about their function in the interaction with the host immune system. Infection with E. histolytica trophozoites can lead to formation of amebic liver abscesses (ALAs), in which pro-inflammatory immune responses of Ly6C hi monocytes contribute to liver damage. Men exhibit a more severe pathology as the result of higher monocyte recruitment and a stronger immune response. To investigate the role of EVs and pathogenicity in the host immune response, we studied the effect of EVs secreted by low pathogenic Eh A1 and highly pathogenic Eh B2 amebae on monocytes. Size and quantity of isolated EVs from both clones were similar. However, they differed in their proteome and miRNA cargo, providing insight into factors potentially involved in amebic pathogenicity. In addition, EVs were enriched in proteins with signaling peptides compared with the total protein content of trophozoites. Exposure to EVs from both clones induced monocyte activation and a pro-inflammatory immune response as evidenced by increased surface presentation of the activation marker CD38 and upregulated gene expression of key signaling pathways (including NF-κB, IL-17 and TNF signaling). The release of pro-inflammatory cytokines was increased in EV-stimulated monocytes and more so in male- than in female-derived cells. While Eh A1 EV stimulation caused elevated myeloperoxidase (MPO) release by both monocytes and neutrophils, Eh B2 EV stimulation did not, indicating the protective role of MPO during amebiasis. Collectively, our results suggest that parasite-released EVs contribute to the male-biased immunopathology mediated by pro-inflammatory monocytes during ALA formation.

Amoebiasis: a well-tuned genome The genome sequence of the pathogen Entamoeba histolytica is reported this week. E. histolytica causes amoebiasis, the second most deadly protozoan disease after malaria. The genome contains adaptations shared with other anaerobic pathogens such as Trichomonas and Giardia . And there is evidence that the genome has been shaped by many gene transfers from bacteria, which may suggest possible targets for drugs against these organisms. The identification of a large number of sensing and signalling proteins challenges the idea that E. histolytica is a simple organism: in fact it is finely attuned to its environment. Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries 1 . Here we present the genome of E. histolytica , which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis . These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica 's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Amebiasis is caused by the protozoan parasite Entamoeba histolytica (Eh), a potentially fatal disease occurring mainly in developing countries. How Eh interacts with innate host factors in the gut is poorly understood. Eh resides and feed in/on the outer colonic mucus layer and thus share an ecological niche with indigenous microbiota. As gut microbiota regulates innate immune responses, in this study we characterized the cooperative roles that microbiota and the mucus layer play in Eh-induced pro-inflammatory responses in the colon. To study this, we used antibiotics treated and non-treated specific pathogen free Muc2-/- and Muc2+/+ littermates and germ-free mice inoculated with Eh in colonic loops as a short infection model. In antibiotic treated Muc2-/- and Muc2+/+ littermates, Eh elicited robust mucus and water secretions, enhanced pro-inflammatory cytokines and chemokine expression with elevated MPO activity and higher pathology scores as compared to the modest response observed in non-antibiotic treated littermates. Host responses were microbiota specific as mucus secretion and pro-inflammatory responses were attenuated following homologous fecal microbial transplants in antibiotic-treated Muc2+/+ quantified by secretion of 3H-glucosamine newly synthesized mucin, Muc2 mucin immunostaining and immunohistochemistry. Eh-elicited pro-inflammatory responses and suppressed goblet cell transcription factor Math1 as revealed by in vivo imaging of Eh-colonic loops in Math1GFP mice, and in vitro using Eh-stimulated LS174T human colonic goblet cells. Eh in colonic loops increased bacterial translocation of bioluminescent E. coli and indigenous bacteria quantified by FISH and quantitative PCR. In germ-free animals, Eh-induced mucus/water secretory responses, but acute pro-inflammatory responses and MPO activity were severely impaired, allowing the parasite to bind to and disrupt mucosal epithelial cells. These findings have identified key roles for intestinal microbiota and mucus in regulating innate host defenses against Eh, and implicate dysbiosis as a risk factor for amebiasis that leads to exacerbated immune responses to cause life-threatening disease.

Entamoeba histolytica is the pathogenic amoeba responsible for amoebiasis, an infectious disease targeting human tissues. Amoebiasis arises when virulent trophozoites start to destroy the muco-epithelial barrier by first crossing the mucus, then killing host cells, triggering inflammation and subsequently causing dysentery. The main goal of this study was to analyse pathophysiology and gene expression changes related to virulent (i.e. HM1:IMSS) and non-virulent (i.e. Rahman) strains when they are in contact with the human colon. Transcriptome comparisons between the two strains, both in culture conditions and upon contact with human colon explants, provide a global view of gene expression changes that might contribute to the observed phenotypic differences. The most remarkable feature of the virulent phenotype resides in the up-regulation of genes implicated in carbohydrate metabolism and processing of glycosylated residues. Consequently, inhibition of gene expression by RNA interference of a glycoside hydrolase (β-amylase absent from humans) abolishes mucus depletion and tissue invasion by HM1:IMSS. In summary, our data suggest a potential role of carbohydrate metabolism in colon invasion by virulent E. histolytica.

Amoebiasis is the second leading cause of death from parasitic disease worldwide. The causative protozoan parasite, Entamoeba histolytica, is a potent pathogen. Secreting proteinases that dissolve host tissues, killing host cells on contact, and engulfing red blood cells, E histolytica trophozoites invade the intestinal mucosa, causing amoebic colitis. In some cases amoebas breach the mucosal barrier and travel through the portal circulation to the liver, where they cause abscesses consisting of a few E histolytica trophozoites surrounding dead and dying hepatocytes and liquefied cellular debris. Amoebic liver abscesses grow inexorably and, at one time, were almost always fatal, but now even large abscesses can be cured by one dose of antibiotic. Evidence that what we thought was a single species based on morphology is, in fact, two genetically distinct species—now termed Entamoeba histolytica (the pathogen) and Entamoeba dispar (a commensal)—has turned conventional wisdom about the epidemiology and diagnosis of amoebiasis upside down. New models of disease have linked E histolytica induction of intestinal inflammation and hepatocyte programmed cell death to the pathogenesis of amoebic colitis and amoebic liver abscess.