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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.

Amebiasis, caused by Entamoeba histolytica , is a global health concern, affecting millions and causing significant mortality, particularly in areas with poor sanitation. Although recent studies have examined E. histolytica ’s interaction with human intestinal microbes, the impact of bacterial presence on the parasite’s motility, mechanical forces, and their potential role in altering invasiveness have not been fully elucidated. In this study, we utilized a micropillar-array system combined with live imaging to investigate the effects of enteropathogenic Escherichia coli on E. histolytica ’s motility characteristics, F-actin spatial localization, and traction force exerted on fibronectin-coated substrates. Our findings indicate that co-incubation with live enteropathogenic E. coli significantly enhances the motility of E. histolytica , as evidenced by superdiffusive movement—characterized by increased directionality and speed—resulting in broader dispersal and more extensive tissue/cell damage. This increased motility is accompanied by a reduction in F-actin-dependent traction forces and podosome-like structures on fibronectin-coated substrates, but with increased F-actin localization in the upper part of the cytoplasm. These findings highlight the role of physical interactions and cellular behaviors in modulating the parasite’s virulence, providing new insights into the mechanistic basis of its pathogenicity.

Oxidative stress is one of the strongest toxic factors in nature: it can harm or even kill cells. Cellular means of subverting the toxicity of oxidative stress are important for the success of infectious diseases. Many types of bacterium inhabit the intestine, where they can encounter pathogens. During oxidative stress, we analyzed the interplay between an intestinal parasite (the pathogenic amoeba Entamoeba histolytica - the agent of amoebiasis) and enteric bacteria (microbiome residents, pathogens and probiotics). We found that live enteric bacteria protected E. histolytica against oxidative stress. By high-throughput RNA sequencing, two amoebic regulatory modes were observed with enteric bacteria but not with probiotics. The first controls essential elements of homeostasis, and the second the levels of factors required for amoeba survival. Characteristic genes of both modes have been acquired by the amoebic genome through lateral transfer from the bacterial kingdom (e.g. glycolytic enzymes and leucine-rich proteins). Members of the leucine-rich are homologous to proteins from anti-bacterial innate immune such as Toll-like receptors. The factors identified here suggest that despite its old age in evolutionary terms, the protozoan E. histolytica displays key characteristics of higher eukaryotes’ innate immune systems indicating that components of innate immunity existed in the common ancestor of plants and animals.

Macropinocytosis is an evolutionarily conserved endocytic process that plays a vital role in internalizing extracellular fluids and particles in cells. This non-selective endocytic pathway is crucial for various physiological functions such as nutrient uptake, sensing, signaling, antigen presentation, and cell migration. While macropinocytosis has been extensively studied in macrophages and cancer cells, the molecular mechanisms of macropinocytosis in pathogens are less understood. It has been known that Entamoeba histolytica , the causative agent of amebiasis, exploits macropinocytosis for survival and pathogenesis. Since macropinocytosis is initiated by actin polymerization, leading to the formation of membrane ruffles and the subsequent trapping of solutes in macropinosomes, actin cytoskeleton regulation is crucial. Thus, this study focuses on unraveling the role of well-conserved actin cytoskeleton regulators, Rho small GTPase family proteins, in macropinocytosis in E . histolytica . Through gene silencing of highly transcribed Ehrho / Ehrac genes and following flow cytometry analysis, we identified that silencing EhracM enhances dextran macropinocytosis and affects cellular migration persistence. Live imaging and interactome analysis unveiled the cytosolic and vesicular localization of EhRacM, along with its interaction with signaling and membrane traffic-related proteins, shedding light on EhRacM’s multiple roles. Our findings provide insights into the specific regulatory mechanisms of macropinocytosis among endocytic pathways in E . histolytica , highlighting the significance of EhRacM in both macropinocytosis and cellular migration.

Background Entamoeba histolytica , an enteric pathogen, causes disease by adhering to and destroying the host tissues. The interaction between the parasite and host tissue enables the rewiring of gene expression and global membrane trafficking in the parasite. A fine balance between endocytosis and exocytosis of cargoes/receptors is required to establish infection in the host. Multivesicular bodies (MVBs) act as sorting platforms, delivering cargoes/receptors to lysosomes for degradation or secreting their contents through plasma membrane fusion. Some small GTPases are known to control MVB biogenesis in various organisms. However, the functional contribution of Rab GTPases in MVB biogenesis is poorly studied in E. histolytica . Results Here, we identified a novel atypical protein RabD2, with an N-terminal glutamic acid-lysine rich motif and a C-terminal conserved Rab domain. Our biochemical and cell biological assays provide evidence that RabD2 self-associates, and this interaction is controlled by the N-terminal EK-rich motif and the GTPase activity mutants. RabD2 localizes on the surface of MVBs and controls their biogenesis. In line with these findings, overexpression of RabD2 upregulates ubiquitination levels, directing the decreased abundance of the heavy chain of GalNAc lectin, ultimately leading to decreased adherence of E. histolytica trophozoites to host cells. Conclusions Thus, amoebic RabD2 is a new class of Rab protein that forms high-ordered self-association variants and controls adhesion to the host cells, which impacts the pathogenesis of E. histolytica through the biogenesis of MVBs.

Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson's medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples.

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.

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infant's intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasite Entamoeba histolytica, as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) and N-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site of E. histolytica infection. The objective of the present study was to investigate whether HMO reduce E. histolytica attachment and cytotoxicity. Our in vitro results show that physiological concentrations of isolated, pooled HMO detach E. histolytica by more than 80 %. In addition, HMO rescue E. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80 % of the HT-29 cells, while HMO with fucose α1–2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolished E. histolytica attachment and cytotoxicity at 8 mg/ml. Although our results need to be confirmed in vivo, they may provide one explanation for why breast-fed infants are at lower risk of E. histolytica infections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents.

Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica.

Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5β1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5β1 integrin and NLRP3 into the intercellular junction, where α5β1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5β1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5β1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5β1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5β1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection.