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Electron Microscope Studies of Experimental Entamoeba Histolytica Infection in the Guinea Pig: I. Penetration of the Intestinal Epithelium by Trophozoites
Abstract
Germ-free guinea pigs were inoculated intracecally with Entamoeba histolytica and the enteric flora derived from a human patient with acute amebic colitis. Aanimals were killed at post-inoculation intervals of 7 to 12 days. The mode of penetration of cecal epithelium by the ameba was examined by light and electron microscopy. The following sequence was reconstructed from numerous individual observations. When the amebae were in moderately close proximity to the brush, border, the microvilli became shortened, irregular, and sometimes disappeared. Dense material was observed between the amebae and microvilli. When the ameba was very close to the epithelium the apical portion of epithelial cytoplasm projected into the lumen contacting the organism, thus becoming detached from adjoining cells. This produced spaces between epithelial cells through which amebae invaded interepithelial spaces. Initially the ameba penetrated the interglandular epithelium. Later, it penetrated equally the glandular and interglandular epithelium barrier. There were marked alterations of cytoplasmic components of epithelial cells. Polymorphonuclear leukocytes migrated into the epithelium filling these spaces; these often showed a variety of degenerative processes. Amebae, utilizing their pseudopodia, moved further through the intercellular spaces and reached the lamina propria.
... Mutants of E. histolytica strain HM-1 which are deficient in both proteinase expression and cytopathic effect have been identified (45). The in vitro cytopathic effect correlates with the early pathology of invasion in animal models in which the intestinal epithelial cells separate before making direct contact with trophozoites, presumably from disruption of the extracellular matrix (118). ...
... One of the main drawbacks of experimental amebiasis is the lack of an animal model that closely mimics human disease, in which liver abscesses develop after intestinal infection. Early stages of amebic invasion have been observed when trophozoites were injected into the ceca of gerbils (18,107) or guinea pigs (118). Two of the most widely used models of amebic liver abscess utilize direct inoculation of trophozoites into the livers of baby gerbils or hamsters (69), (17). ...
Amebiasis is a major cause of morbidity and mortality throughout the tropical world. Entamoeba histolytica is now recognized as a separate species from the morphologically identical E. dispar, which cannot invade. Cysteine proteinases are a key virulence factor of E. histolytica and play a role in intestinal invasion by degrading the extracellular matrix and circumventing the host immune response through cleavage of secretory immunoglobulin A (sIgA), IgG, and activation of complement. Cysteine proteinases are encoded by at least seven genes, several of which are found in E. histolytica but not E. dispar. A number of new animal models, including the formation of liver abscesses in SCID mice and intestinal infection in human intestinal xenografts, have proven useful to confirm the critical role of cysteine proteinases in invasion. Detailed structural analysis of cysteine proteinases should provide further insights into their biochemical function and may facilitate the design of specific inhibitors which could be used as potential chemotherapeutic agents in the future.
... Virulent E. histolytica trophozoites invade the mucosa of the large intestine and from there they may colonize and damage extraintestinal organs (P~rezTamayo 1986). Early intestinal invasion events include the adhesion of trophozoites to epithelial cells, the lysis and dislocation of such cells, the degradation of the basement membrane, and the penetration of amebae to the lamina propria (Prathap and Gilman 1970; Takeuchi and Phillips 1975). Although the initial interaction of trophozoites with the colon mucosa probably is crucial for invasion, an analysis of this process is impossible in humans and extremely difficult in experimental animal models due to their variability and to the nearly impossible task of localizing the earliest microscopic lesions produced by intracecal inoculation of amebae (Ghadirian and Konshvan 1984; Anaya-Vel/tzquez et al. 1985). ...
... The structural lesions described herein are similar to those previously seen in human biopsies and in experimental intestinal amebiasis. For instance, in human biopsies Prathap and Gilman (1970) found mucosal edema , epithelial cell shortening, and interglandular lesions, whereas in guinea pigs Takeuchi and Phillips (1975) observed a shortening and disappearance of microvilli, a loss of tight junctions, and interglandular epithelial cell damage. On the other hand, Knight and co-workers (1974) showed that during the early interaction of amebae with RK13 monolayer cells, electron microscopic damage was not detectable in the plasma membrane but occurred in the cytoplasm, consisting of vacuolation and a loss of mitochondrial crests. ...
Although in acute intestinal amoebiasis the events occurring during the initial interaction of Entamoeba histolytica trophozoites with the mucosa of the large intestine probably determine the invasion by the parasites, an appropriate experimental model to study them does not exist. To develop such a model we used rabbit colonic segments (0.28 cm2) mounted in Ussing-type chambers. These preparations had electrophysiological properties (potential difference, short-circuit current, and electrical resistance) similar in magnitude and duration to those reported for stripped colonic mucosa. Lysates from 50,000 Entamoeba invadens PZ strain trophozoites (virulent to reptiles) added to the mucosal side of preparations had no effect, whereas lysates from the same number of E. histolytica HM1 strain trophozoites (virulent to humans) caused an immediate decay of electrophysiological properties. E. histolytica lysates induced the formation of translucent vacuoles at the bases of epithelial cells and separation between the epithelium and the lamina propria. The specificity and speed of the electrophysiologic effects of E. histolytica lysates and their correlation with the microscopic lesions suggest that this model may be useful to analyze the initial pathogenic events of intestinal amoebiasis.
... Cysteine proteases are a characteristic feature of parasitic protozoa (e.g., Entamoeba histolytica and Cryptosporidium spp.) that have been shown to facilitate invasion of host tissue, as well as immune evasion [135]. For instance, Entamoeba histolytica utilizes cysteine proteases for degradation of colonic mucins and extracellular matrix components that lead to separation of epithelial cells, consequently breaching the epithelial barrier and enabling the protozoa to invade the host tissue [136,137]. Additionally, cysteine proteases are potent modulators of the host immune defense via direct degradation of immunoglobulin A (IgA), IgG, and IL-18, as well as attenuation of protective Th1-type responses (Figure 1) [135]. Besides Blastocystis ST7, no evidence of a similar mechanism has been found in other protozoa species thus far. ...
The human gastrointestinal microbiota contains a diverse consortium of microbes, including bacteria, protozoa, viruses, and fungi. Through millennia of co-evolution, the host–microbiota interactions have shaped the immune system to both tolerate and maintain the symbiotic relationship with commensal microbiota, while exerting protective responses against invading pathogens. Microbiome research is dominated by studies describing the impact of prokaryotic bacteria on gut immunity with a limited understanding of their relationship with other integral microbiota constituents. However, converging evidence shows that eukaryotic organisms, such as commensal protozoa, can play an important role in modulating intestinal immune responses as well as influencing the overall health of the host. The presence of several protozoa species has recently been shown to be a common occurrence in healthy populations worldwide, suggesting that many of these are commensals rather than invading pathogens. This review aims to discuss the most recent, conflicting findings regarding the role of intestinal protozoa in gut homeostasis, interactions between intestinal protozoa and the bacterial microbiota, as well as potential immunological consequences of protozoa colonization.
... Intestinal epithelial cells are the first cell types to come into contact with E. histolytica (Fig. 2). It has been known for a while that before making cell contact, trophozoites secrete immunomodulatory proteins that stimulate epithelial cells, resulting in cytokine production and subsequent inflammatory cell infiltration [17,18]. A variety of cells, including intestinal epithelial cells, produce interleukin-8 (IL-8), a potent neutrophil chemoattractant cytokine. ...
This review outlines some of the recent advances that have furthered our understanding of the processes that lead to the tissue damage caused by E. histolytica
... Intestinal epithelial cells are the first cell types to come into contact with E. histolytica (Fig. 2). It has been known for a while that before making cell contact, trophozoites secrete immunomodulatory proteins that stimulate epithelial cells, resulting in cytokine production and subsequent inflammatory cell infiltration [17,18]. A variety of cells, including intestinal epithelial cells, produce interleukin-8 (IL-8), a potent neutrophil chemoattractant cytokine. ...
Purpose of Review
Entamoeba histolytica is a protozoan parasite that causes amebiasis, which remains a significant cause of morbidity and mortality worldwide. E. histolytica causes tissue destruction which leads to clinical disease. This review outlines some of the recent advances that have furthered our understanding of the processes that lead to the tissue damage caused by E. histolytica.
Recent Findings
Recent studies have identified new mechanisms involved in E. histolytica–induced tissue damage. These include (i) new form of contact-dependent killing called trogocytosis; (ii) parasite-produced cytokine, macrophage migration inhibitory factor, that contributes to inflammation; (iii) exploitation of host immune response to promote invasion; and (iv) the contribution of the gut microbiome to clinical disease.
Summary
Targeting these mechanisms that result in tissue injury should be a focus of future research for the development of improved preventive and therapeutic strategies for amebiasis.
... The importance of amebic adherence has been emphasized morphologic with in vivo and in vitro studies which demonstrated amebas adherent to guinea pig intestinal mucosa prior to penetration (30,31). The hemagglutinating lectin isolated by Kobiler and Mirelman (8) from E. histolytica may not be involved in the adherence of whole viable amebas to target cells, as chitotriose (at -50 times the 50% lectin inhibitory concentration [8]) did not decrease amebic adherence to CHO cells. ...
The enteric pathogen, Entamoeba histolytica, appears to cause disease by adhering to and then destroying mucosal barriers. Using an in vitro method of studying the interaction of E. histolytica with target cells (Chinese hamster ovary [CHO] and human erythrocytes [RBC]), we examined the mechanism of amebic adherence and its role in lysis of target cells. Killing and phagocytosis of target cells by amebas ceases at 4°C, allowing observation of adherence. Amebas adhere to CHO cells at 4°C, 78.9% formed rosettes (amebas with ≥3 adherent CHO cells each) at 2 h. At 37°C, cytochalasins B and D inhibit adherence of amebas to CHO cells (P < 0.0005). Amebas adhere to and kill CHO cells in media with <0.1 μM calcium and magnesium plus 10 mM EDTA, indicating that divalent cations are not required in the medium. Adherence of amebas to human RBC was not ABO blood group specific and showed greater adherence to human than bovine or sheep RBC (P < 0.005). Neither Fc nor complement receptors were found on amebas by standard rosette studies. The amebic adherence receptor is not trypsin (0.125%) sensitive nor inhibited by trypan blue (1 mM). N-acetyl-d-galactosamine (GALNAc) inhibited the adherence of amebas to CHO cells and human RBC (0.1 g/100 ml or 4.5 mM GALNAc, P < 0.005) by binding to a receptor on the amebic surface. GALNAc abolishes amebic cytolysis of target CHO cells (determined by 111Indium oxine release from CHO cells, P < 0.001) but not amebic phagocytosis of CHO cells. By suspending ameba-CHO cells rosettes in dextran, we found that GALNAc (1%) reversibly inhibits amebic adherence (P < 0.0005) and that cytochalasins decrease amebic killing of adherent CHO cells (P < 0.025).
These findings indicate that the adherence of E. histolytica to target cells requires microfilament function, is via a specific amebic receptor that has affinity for GALNAc, and is required to lyse cells. Inhibition of the adherence of E. histolytica may alter the pathogenicity of this organism.
... E. histolytica invades tissue and causes clinical disease following ingestion of the infectious cyst form of the parasite from fecally contaminated food or water [2,9,34,35,42]. Excystation of the amebic trophozoites occurs in the intestinal lumen. Trophozoites adhere to the colonic mucus and epithelial cells through interaction of a Gal/GalNAc-specific lectin [29,31]. ...
Entamoeba histolytica, which causes amebic colitis and liver abscess, is considered a major enteric pathogen in residents and travelers to developing countries where the disease is endemic. Interaction of this protozoan parasite with the intestine is mediated through the binding of the trophozoite stage to intestinal mucin and epithelium via a galactose and N-acetyl-d-galactosamine (Gal/GalNAc) lectin comprised of a disulfide linked heavy (ca. 180kDa) and light chain (ca. 35kDa) and a noncovalently bound intermediate subunit (ca. 150kDa). Our efforts to develop a vaccine against this pathogen have focused on an internal 578 amino acid fragment, designated LecA, located within the cysteine-rich region of the heavy chain subunit because: (i) it is a major target of adherence-blocking antibodies of seropositive individuals and (ii) vaccination with his-tagged LecA provides protection in animal models. We developed a purification process for preparing highly purified non-tagged LecA using a codon-optimized gene expressed in Escherichia coli. The process consisted of: (i) cell lysis, collection and washing of inclusion bodies; (ii) solubilization and refolding of denatured LecA; and (iii) a polishing gel filtration step. The purified fragment existed primarily as a random coil with β-sheet structure, contained low endotoxin and nucleic acid, was highly immunoreactive, and elicited antibodies that recognized native lectin and that inhibited in vitro adherence of trophozoites to CHO cells. Immunization of CBA mice with LecA resulted in significant protection against cecal colitis. Our procedure yields sufficient amounts of highly purified LecA for future studies on stability, immunogenicity, and protection with protein-adjuvant formulations.
... Invasive disease is characterized by ulcerations of the intestinal wall and in some cases by invasion through the wall and dissemination to the liver, resulting in the clinical syndromes of amebic dysentery and liver abscess, respectively (8). Ultrastructural studies of experimental E. histolytica infection revealed the degeneration of epithelial cells adjacent to invading trophozoites and the penetration of trophozoites into the lamina propria through the basement membrane (16). The interaction of amebic factors with components of the extracellular matrix potentially plays an important role in the penetration of trophozoites through the intestinal mucosa. ...
The protozoan parasite Entamoeba histolytica is the caus- ative agent of human amebiasis. Invasive disease is character- ized by ulcerations of the intestinal wall and in some cases by invasion through the wall and dissemination to the liver, re- sulting in the clinical syndromes of amebic dysentery and liver abscess, respectively (8). Ultrastructural studies of experimen- talE. histolyticainfection revealed the degeneration of epithe- lial cells adjacent to invading trophozoites and the penetration of trophozoites into the lamina propria through the basement membrane (16). The interaction of amebic factors with com- ponents of the extracellular matrix potentially plays an impor- tant role in the penetration of trophozoites through the intes- tinal mucosa. In order to identify E. histolytica proteins that interact with components of the extracellular matrix, amebic lysates were fractionated over laminin-Sepharose,fibronectin- Sepharose, and collagen-Sepharose. We report here that E. histolytica27-kDa cysteine proteinases exhibit striking binding specificities for immobilized laminin over immobilized fi- bronectin or collagen. Furthermore, the coinjection of laminin but notfibronectin or collagen withE. histolyticatrophozoites greatly reduces liver abscess formation in severe combined immunodeficient (SCID) mice.
... Cell fractionation studies indicate that although present in the cytosol, they are enriched in the plasma and internal membranes [47,48]. CPs have been implicated in the in vitro cytopathology of cell monolayers [49,50], which correlates with the observed separation of colonic epithelial cells before invasion [51]. Other correlates with invasion include the ability of CPs to degrade extracellular matrix components such as laminin, collagen types I and IV, fibronectin [22,52], and colonic mucin [53,54]. ...
Entamoeba histolytica is able to invade human tissues by means of several molecules and biological properties related to the virulence. Pathogenic amebas use three major virulence factors, Gal/GalNAc lectin, amebapore and proteases, for lyse, phagocytose, kill and destroy a variety of cells and tissues in the host. Responses of the parasite to host components such as mucins and bacterial flora influence the behavior of pathogenic amebas altering their expression of virulence factors. The relative virulence of different strains of E. histolytica has been shown to vary as a consequence of changes in conditions of in vitro cultivation which implies substantial changes in basic metabolic aspects and factors directly and indirectly related to amebic virulence. Comparison of E. histolytica strains with different virulence phenotypes and under different conditions of growth will help to identify new virulence factor candidates and define the interplay between virulence factors and invasive phenotype. Virulence attenuate mutants of E. histolytica are useful also to uncover novel virulence determinants. The comparison of biological properties and virulence factors between E. histolytica and E. dispar, a non-pathogenic species, has been a useful approach to investigate the key factors involved in the experimental presentation of amebiasis and its complex regulation. The molecular mechanisms that regulate these variations in virulence are not yet known. Their elucidation will help us to better understand the gene expression plasticity that enables the effective adaptation of the ameba to changes in growth culture conditions and host factors.
The Entamoeba histolytica 27-kDa cysteine proteinases exhibit striking binding specificities for immobilized laminin over other components of the extracellular matrix, such as collagen and fibronectin. Inactivation of the proteinase with the active-site inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane abolishes laminin binding by the enzyme, and conversely, laminin inhibits cleavage of a fluorogenic dipeptide substrate of the amebic cysteine proteinase, suggesting that the substrate binding pocket of the enzyme is involved in the binding of laminin. The addition of laminin but not fibronectin or collagen to E. histolytica trophozoites significantly reduces amebic liver abscess formation in severe combined immunodeficient mice, further supporting the hypothesis that E. histolytica cysteine proteinases play an important role in amebic pathogenesis. The specific interaction of amebic proteinases with laminin may be exploited in designing new inhibitors of these enzymes.
The present study was designed to develop and characterize animal models for the assay of enterotoxic activity in extracts of Entamoeba histolytica trophozoites. Marked water and electrolyte secretion occurred in both in vivo rabbit ileal loops and rat colon loops exposed to clarified sonic fluids of E. histolytica strain HM-1 trophozoites (10(6)/ml) when the animals were first administered indomethacin (0.1 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg) dose of indomethacin. Higher doses (greater than or equal to 1 mg/kg) of indomethacin inhibited extract-induced secretion. No enterotoxic activity was detected with or without indomethacin, using extracts from the nonpathogenic E. histolytica-like Laredo strain, even at 10-fold-higher cell concentrations. The HM-1 enterotoxic activity was heat labile. Prior exposure of the loop lumen to fetuin (100 micrograms/ml) blocked the secretory response to subsequent HM-1 extract exposure, but postexposure of the loop to fetuin did not block secretion that had already been established by the amoeba extract. No histological changes were seen associated with the amoeba extract-induced secretion. The data suggest that E. histolytica HM-1 strain elaborates an enterotoxic activity capable of causing consistent secretion in the mammalian intestine that has had its mucosal cytoprotection impaired by indomethacin.
Entamoeba histolytica is the cause of high levels of morbidity and mortality throughout the developing world, having been ranked as the third most important parasitic agent of disease after malaria and schistosomiasis. In 1981, it was estimated that up to 500 million people are infected annually [265]. The majority of infections are asymptomatic and apparently non-pathogenic. In most of these cases, spontaneous eradication of the parasite is thought to occur within several months [28, 153]. Some 10%, however, go on to develop invasive amoebiasis [201] in which symptoms range from mild and transient diarrhoea to fulminating colitis characterized by the passage of bloody stools. In some cases dissemination via the bloodstream may result in the production of extraintestinal abscesses in other organs, principally the liver. Invasive amoebiasis is held to account for around 75 000 deaths per year [79].
Protozoa are unicellular animals. The single cell which carries out all the functions comprises a cytoplasm which contains one or more nuclei. The nucleus consists of a membrane with a network of fine filaments inside it; on this membrane and on the enclosed network are arranged granules of chromatin. Inside the nucleus is a mass, usually central in position and roughly circular in shape, known as the karyosome.
Introduction Genetic Diversity Link between Genotype and Virulence Virulence Mechanisms Summary, Limitations, and Opportunities for Future Work
1.1. In vitro cultures of Entamoeba histolytica strains HK-9 and NIH-200 contained considerable amounts of the acid hydrolase β-N-acetylglucosaminidase. The results indicate that this enzyme is produced by the amoebae and secreted into the growth medium.2.2. The enzyme was purified 1000–2000 fold and preliminarily characterized. In cultures from NIH-200 two β-N-acetylglucosaminidases with distinct pls, pH optima and kinetics were demonstrated. From cultures of HK-9 only one enzyme was isolated and it seems that it was identical with one of the NIH-200 enzymes. The molecular weight of all three enzymes was 125,000 ± 10,000.3.3. As it is known that hydrolases can be involved in inflammatory conditions it is suggested that released β-N-acetylglucosaminidases participate in the pathogenesis of amoebiasis.
Summary The development of histolytic lesions of acute amebic colitis was studied by light (LM) and electron (EM) microscopy and by
histochemistry (HC) in the germ-free guinea pig inoculated intracecally with amebae and the enteric flora derived from patients
with acute amebic colitis. Between 7 and 12 days after inoculation the animals had developed focal amebic ulcerations ranging
from microscopic in size to large ulcers. The ulcerated areas were covered with gray to gray-red exudate and were surrounded
by edematous and hyperemic areas. The smallest ulcerations involved only the epithelium and the immediate underlying lamina
propria, while the more extensive ulcerations with histolysis involved the whole thickness of the mucosa. The lytic areas
consisted of amorphous material, which was intensely and diffusely alkaline phosphatase positive, while the intraepithelial
enzymes were completely lost. By EM this material was identified as cellular debris from degenerating PMN, epithelial and
other host cells and as fibrin. Although intact PMN’s were present adjacent to the histolytic lesions, PMN’s within the lytic
area showed active degranulation and severe alteration. The vascular structures were extensively altered and showed lysing
endothelial cells and thrombosis. The areas surrounding the ulcers were edematous, hyperemic and infiltrated by numerous PMN’s.
The crypts were elongated and the epithelium cuboidal and showing degenerative changes when in contact or in close vicinity
to amebae. Goblet cells were depleted. The activity of intracellular enzymes of epithelial cells was depressed, while the
alkaline phosphatase activity in the PMN was greatly increased. The influx of PMN’s in the lesions and their massive degeneration
in the lytic areas, may well explain contradictory reports of leukotaxis into amebic lesion as well as of absence of PMN’s
in amebic colonic exudate.
These observations suggest that lysis of colonic mucosa in amebic infection is due in part to tissue ischemia caused by vascular
thrombosis, and in part, to the lytic action of degenerating PMN’s, possibly through the discharge of their lysosomal enzymes
into the intercellular spaces.
Trophozoites ofEntamoeba histolytica HM-1werecultivated axenically inTYI-Smedium.Theamoebae were thentransferred intothismediumlacking serum (TYI)andinoculated intoinvivocolonloopsofadult Sprague-Dawley rats. Thetrophozoites were rapidly absorbed bythemucus, andfewwere foundfreeinthe luminal fluid by1h.By4h,theamoebaebegantoreappear inthelumen,aggregated insloughed mucus blanket fragments. Thecolon wasexamined histologically andbyscanning electron microscopy. There was no evidence ofinvasion or evenbrush-border attachment bythetrophozoites within 4h.InTYI,trophozoite motility was low.Exposure tothecolonic lumenenvironment for 5 mininthis mediumsignificantly increased motility. However, asthetrophozoites becameabsorbed tomucus fragments, their observed motility virtually ceased despite some morphological evidence ofpseudopod extension. Erythrophagocytosis was notsignificantly affected byeither exposing trophozoites toTYIwashings ofthecolonic lumen, or bythemore complete medium,TYI-S,inwhichtheamoebae were significantly more motile. Two majormucus glycoprotein oligosaccharide end-group sugars,L-fucose andN-acetyl-neuraminic acid, were tested fortheir effects on
acid(NANA)fromN-acetylneuramin-lactose (aL2->3 or a2-6)or mucinintheir medium. Theneuraminidase was found tobemembraneassociated, with more than50%ofthe yield beingrecovered intheplasma membranefraction. Theneuraminidase specific activity oftheplasma membranefraction was sixtimesthatofinternal membranefraction enzyme. TheoptimumpH and temperature forthis enzyme were6.7and37°C, respectively. Neuraminidase activity was inhibited byethylene glycol-bis(,I-aminoethyl ether)-N,N,N',N'-tetraacetic acid, andtheoptimumCa2+concentration was 2mM. Themicrofilament disruptor cytochalasin D (30,ug/ml) inhibited motility andneuraminidase activity ofintact Entamoeba trophozoites. Thecytochalasin D-induced loss ofsurface neuraminidase activity was explained in partbya redistribution ofenzyme witha loss ofplasma membrane enzyme andan increase inintracellular membrane enzyme. A qualitatively similar cytochalasin D effect was observed withtwoothermembrane- associated enzymes,calcium-regulated ATPaseandacidphosphatase. Membrane-associated
Using a closed-circuit television system, Entamoeba histolytica strain IP-106 was observed to penetrate through a meniscus separating a medium–nitrogen gas interface. Both the forces exerted on the amoeba by the surface tension of the liquid medium and the resultant forces applied by the amoeba, at various stages during penetration, were calculated. During penetration the force applied increased to a maximum of 3.3 × 10−6 N. However it was found that the shape of the amoeba allowed it to penetrate with a constant pressure. Intercellular movement due to active pseudopodial motion may play an important role in the process of penetration.
As the fungus Coelomomyces punctatus develops in the coelomic cavity of the mosquito Anopheles quadrimaculatus, the conformation of the plasma membrane and extracellular coat of the fungus changes markedly. The vegetative stage was surrounded by a granular and fibrillar extracellular coat which reacted positively in the silver methenamine procedure for the localization of polysaccharides. Numerous simple, branched or contorted cytoplasmic protuberances covered the irregularly shaped hyphal bodies. The surface of the hyphal body adjacent to the fat body of the mosquito had occasional involutions of the plasma membrane sheathed by cisternae of endoplasmic reticulum. In contrast with these hyphal bodies, cytoplasmic protuberances were spaced at wide intervals along filamentous hyphae. Aborting thalli were contorted and deeply lobed. The plasma membrane was smooth, and cytoplasmic protuberances were absent on other hyphae and hyphal bodies, particularly at advanced stages of infection. Instead unattached vesicles, morphologically similar to the protuberances found on some thalli, were embedded in granular material clustered around the smooth plasma membrane of these thalli. Mosquito hemocytes appeared to engulf these vesicles and granular material. As the vegetative stage was transformed into the reproductive stage, a newly formed, compact extracellular layer surrounded the sporangial initial. Later, a darkly staining wall appeared around the resting sporangium. Cisternae of endoplasmic reticulum consistently subtended thin areas in this pitted wall.
Attachment to and Ingestion of Bacteria by Trophozoites of E. HistolyticaInteractions Between Trophozoites and Epithelial CellsAugmentation of E. Histolytica Virulence on Association With BacteriaCytopathic Effects of Cell-Free Extracts of E. HistolyticuConcluding RemarksReferencesDiscussionCell Biology of AmoebiasisReferences
Organic requirements for attachment to glass, elongation, and motility of Entamoeba histolytica, have been determined. the trophozoite, which has been grown axenically only in highly complex media with reduced oxygen tensions, remains rounded and detached when placed in a Tris-HCl buffered solution containing NaCI, KCI, MgCI2, and CaCI2. A maintenance medium in which the amebae could attach to glass, elongate, and remain motile and viable for 12 to 24 h was devised with the addition of cysteine, ascorbic acid, bovine serum albumin, and the vitamin solution of medium NCTC #107. Tris-HCI was the most effective buffer tested and the optimal pH was 6.9 to 7.0. Survival, but not attachment, of the amebae was decreased at osmolalities ranging between 110 and 180 milliosmoles/kg, whereas both functions were decreased above ∼260 milliosmoles/kg. Bovine serum albumin, the most effective of the proteins tested, and the vitamin solution helped maintain attachment of some ameba strains, but were not required by other strains. the requirements for cysteine and ascorbic acid were absolute and highly specific. During incubation in the maintenance medium, cell volumes decreased. Sensitivity of the organisms to agglutination by concanavalin A, wheat germ agglutinin, soybean agglutinin and fucose binding protein remained unchanged.
Early cellular and vascular changes in response to invasion of lamina propria byEntamoeba histolytica were studied sequentially, at the ultrastructural level, in germfree guinea pigs inoculated intracecally with amebae and
enteric flora derived from patients with acute amebic colitis. Approximately one week post-inoculation the animals developed
acute colitis with mucosal invasion by trophic amebae. Although epithelial cells at the sites of amebic invasion showed progressive
cytoplasmic changes and desquamation resulting in microerosions, most mesenchymal elements in the lamina propria appeared
normal without cytopathic changes even when in direct contact with invading amebae. Only the polymorpho-nuclear leukocytes
(PMN) apposed or topographically close to amebae exhibited degenerative changes which were characterized by condensation of
nucleoplasm and cytoplasm, extracellular release of cytoplasmic components including granules, and, finally, lysis of cell
membranes. Capillaries and venules in the lamina propria showed a variety of changes such as swelling and gap formation at
the intercellular endothelial junctions and more rarely at the fenestrae. Blood vessels physically close to amebae showed
formation of endothelial cytoplasmic blebs which pinched off into the vascular or extravascular space. Platelet and fibrin
thromboses were common in the more severely damaged capillaries and venules. Fragments or clumps of fibrin-like material were
found also in the extracellular spaces.
Amebic invasion of the lamina propria, then, is accompanied by continued epithelial shedding, PMN degeneration, and changes
in both capillaries and venules consisting of endothelial damage and occlusive thrombosis. The vascular changes appeared to
be closely related to PMN degeneration resulting from interaction of PMN with invading amebae.
Explants and swabs from the pericardium and mantle of three strains of Biomphalaria glabrata, two of them resistant to infection with Schistosoma mansoni, have yielded small amoebae, 3–5μm in diameter, in culture. These amoebae have been grown axenically through > 50 passages to date. The amoebae form cysts in dense cultures. When mixed with S. mansoni mother sporocysts in vitro, the amoebae adhere to and kill the trematodes within several hours. For 1–2 days thereafter, the amoebae proliferate rapidly at a generation time of about 5 hr, then return to normal growth. Sonically disrupted sporocysts also induce proliferation. Live sporocysts do not attract the amoebae or emit soluble substances which influence amoebal growth. Amoebae also adhered to and killed S. mansoni daughter sporocysts and cells derived from B. glabrata embryos; however, they did not harm S. mansoni cercariae or rediae of other trematode species. The proportion of mantle explants yielding amoebae was significantly higher (P<0.05) in one of the resistant snail strains than in the susceptible strain; however, whether amoebae contribute to snail resistance is unknown. Exposure of snails to S. mansoni miracidia did not influence the proportion of snails yielding amoebae.
Attachment of Entamoeba histolytica and of Giardia lamblia trophozoites to glass was monitored during the culture cycle. Attachment of each parasite was greatest during the exponential phase of axenic growth. The effects of l-cysteine upon the kinetics of attachment of trophozoites to glass were determined quantitatively. Attachment in complex growth media required cysteine, even under N2, atmosphere. With cysteine, the rates of attachment were greatest for the first 2 hr, then continued more slowly. The numbers of attached trophozoites decreased immediately upon exposure to medium without cysteine. The role of cysteine in protecting trophozoites of both species from the lethal effects of oxygen was assessed using clonal growth in agar or agarose medium to determine viability following exposure to varying oxygen tensions in liquid medium. Cysteine was required for viability of trophozoites. Without cysteine, decreasing the oxygen tension prolonged survival. Under increased oxygen tension, cysteine delayed the onset of exponential killing. Although it has no thiol reducing group, l-cystine similarly protected E. histolytica.
The present study was designed to develop and characterize animal models for the assay of enterotoxic activity in extracts of Entamoeba histolytica trophozoites. Marked water and electrolyte secretion occurred in both in vivo rabbit ileal loops and rat colon loops exposed to clarified sonic fluids of E. histolytica strain HM-1 trophozoites (10(6)/ml) when the animals were first administered indomethacin (0.1 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg) dose of indomethacin. Higher doses (greater than or equal to 1 mg/kg) of indomethacin inhibited extract-induced secretion. No enterotoxic activity was detected with or without indomethacin, using extracts from the nonpathogenic E. histolytica-like Laredo strain, even at 10-fold-higher cell concentrations. The HM-1 enterotoxic activity was heat labile. Prior exposure of the loop lumen to fetuin (100 micrograms/ml) blocked the secretory response to subsequent HM-1 extract exposure, but postexposure of the loop to fetuin did not block secretion that had already been established by the amoeba extract. No histological changes were seen associated with the amoeba extract-induced secretion. The data suggest that E. histolytica HM-1 strain elaborates an enterotoxic activity capable of causing consistent secretion in the mammalian intestine that has had its mucosal cytoprotection impaired by indomethacin.
Using fluorescent antibody techniques (FA) and light microscopy (LM) and electron microscopy (EM), this paper describes the morphological features of the ileum in the DK1 mouse orally challenged with adenovirus K87. At the peak of infection, virus is easily identified by FA in the epithelium of the villi and crypts of the ileum. LM shows that fluorescent cells have large, bizarre, uniformly basophilic nuclei containing deoxyribonucleic acid, as indicated by histochemical tests. EM further identifies these nuclei as belonging to columnar, goblet, or Paneth cells, all epithelial cells facing the lumen with a microvillus border. The basophilic material in the nuclei consists of virus particles 75 nm in diameter arranged in crystalline arrays. When found in the cell cytoplasm, the virions do not form arrays but are scattered or form irregular aggregates, which may or may not be enclosed by single membranes. Infected columnar cells show mild cytopathic effects with no cell degeneration and necrosis, whereas the goblet and Paneth cells appear normal and maintain synthetic and secretory functions. All infected cells, however, share an abnormally accelerated extrusion rate, with columnar and goblet cells often being shed from the side rather than from the tip of the villi. The Paneth cells, which do not migrate out of the crypts, show a higher than normal rate of extrusion in the crypt lumen.
Although the events occurring during the initial interaction of E. histolytica trophozoites with the mucosa of the large intestine probably determine the invasion by the parasites, an appropriate experimental model does not exist. To develop such a model we used full-thickness rabbit colon preparations (0.28 cm2) mounted in Ussing-type chambers. Untreated preparations had electrophysiological properties (potential difference, short-circuit current and electrical resistance) similar in magnitude and duration to those reported for stripped colonic mucosa. Exposure to E. histolytica trophozoite lysates for up to 80 min produced dose-dependent lesions in the colon, consisting of: (a) increased decay rates for potential difference, short-circuit current and transmural resistance, and (b) mucosal lesions involving vacuolation at the bases and shortening of epithelial cells, loss of intercellular junctions, destruction of microvilli, and necrosis of interglandular epithelial zones. The specificity and speed of the electrophysiologic effects and their correlation with the microscopic lesions suggest that this new model will help to understand the initial pathogenic events of intestinal amebiasis.
We studied human T-lymphocyte responses to the purified Entamoeba histolytica galactose-inhibitable adherence protein. Individuals having serum anti-adherence protein antibodies possess peripheral blood lymphocytes which demonstrate antigen-specific responses to the purified adherence protein (10 micrograms/ml) and whole soluble amebic antigen (100 micrograms/ml). This was determined by incorporation of [3H]thymidine (53,080 and 73,114 dpm, respectively) and by increased production of interleukin-2 and gamma interferon (42.0 and 67.5 U/ml, respectively) (P less than 0.05 for each in comparison with values for control lymphocyte responses). Lymphocytes from antiamebic antibody-positive subjects develop in vitro amebicidal activity only when incubated for 5 days with the purified adherence protein (P = 0.02). In conclusion, the E. histolytica galactose-inhibitable adherence protein elicits an in vitro amebicidal cell-mediated immune response, further supporting the potential for the use of this protein in a subunit amebiasis vaccine.
The Entamoeba histolytica galactose-binding lectin is a surface glycoprotein composed of 170- and 35-kDa subunits. Inhibition of this lectin with galactose or anti-170 kDa subunit polyclonal antibody blocks amebic adherence to target cells and colonic mucin glycoproteins. We describe the properties of 10 mAb with specificity for the 170-kDa subunit. Based on competitive binding studies, six nonoverlapping antigenic determinants on the lectin were identified. The effect of the mAb on adherence of amebic trophozoites to both Chinese hamster ovary (CHO) cells and human colonic mucins was measured. Antilectin antibodies directed against epitopes 1 and 2 enhanced adherence, with the number of amebae having at least three adherent CHO cells increasing with the addition of epitope 1 mAb from 26 +/- 9 to 88 +/- 2% and the binding of colonic mucins increasing from 34 +/- 1 to 164 +/- 3 pg/10(5) amebae. Antibody-enhanced adherence remained 90 to 100% galactose inhibitable, occurred at 4 degrees C and was not Fc mediated. Univalent Fab fragments of epitope 1 mAb augmented mucin binding by 238% and CHO cell adherence by 338%. The binding of purified lectin to CHO cells was increased from 1.1 +/- 0.1 to 2.4 +/- 0.3 ng/10(3) CHO cells by mAb directed to epitope 1, demonstrating that enhanced adherence was due to direct activation of the lectin. mAb to epitope 3 bound to the lectin only upon its solubilization from the membrane and had no effect on adherence. Adherence to CHO cells and mucins was inhibited from 50 to 75% by mAb to epitopes 4 and 5; epitope 6 mAb inhibited amebic adherence to CHO cells but not mucins. The pooled sera from 10 patients with amebic liver abscess blocked the binding to the 170-kDa subunit of mAb directed to all six epitopes. Striking individual variations in the effects of immune sera on adherence were observed. Although the sera of all 44 South African patients with amebic liver abscess had high titer anti-lectin antibodies, 16 patients' sera significantly (more than 3 SEM) enhanced adherence whereas 25 patients' sera significantly inhibited adherence. Antilectin antibodies exert profound functional effects on the interaction of E. histolytica with target cells and human colonic mucins. Exploration of the clinical consequences of adherence-enhancing and inhibitory antibody responses may give insight into the role of antilectin antibodies in immunity to invasive amebiasis.
The term lectin (from Latin = to pick out, to choose) was coined by Boyd and Shapeleigh (1954) to denote a heterogeneous class of (glyco)proteins, primarily of plant origin, possessing different biological activities such as hemagglutination (phytohemagglutinins), blastic transformation of lymphocytes (mitogens), and toxic effects on vertebrate cells (toxins). These proteins vary markedly in their physicochemical properties; for instance while the potato lectin has a carbohydrate content as high as 50%, other lectins like concanavalin A or the peanut agglutinin, are devoid of carbohydrates. The molecular weights of lectins vary from 11000 for the blood group B specific lectin from Streptomyces species to 335 000 for the lectin from the horseshoe crab. Despite their wide range of physicochemical properties and biological activities, lectins share a common feature which is responsible for their various biological, biochemical and immunochemical activities: Lectins bind with high affinity and specificity to mono- and oligosaccharides of complex carbohydrates in solutions, cell surfaces, subcellular organelles, and tissue sections.
Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HM1 strain of E. histolytica and measure vesicle pH (5.1 +/- 0.2 by spectrofluorimetry). Concentrations of NH4Cl (1.0-10.0 mM) sufficient to increase vesicle pH to greater than or equal to 5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of 3H-thymidine previously incorporated into CHO cell monolayers, and by the release of 111indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 microM). In contrast, NH4Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4 degrees C and by the binding and ingestion of 3H-leucine-labeled bacteria. In the presence of NH4Cl and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 micrograms/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH4Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 micrograms for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.
Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.
The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.
Effect of Entamoeba histolytica proteinase/toxin (Ehp/t) on the luminol-dependent chemiluminescence (CL) in stimulated human polymorphonuclear leukocytes (PMN) was studied. The role of superoxide (SO) and hydroxyl (OH) anions in the Ehp/t-associated enhancement/inhibition of CL was also studied using specific scavengers and a biological response modifier, muramyldipeptide (MDP). Ehp/t was isolated from axenic trophozoites of the HM-1:IMSS strain of virulent strain of E. histolytica. Proteinase activity was assayed on a synthetic substrate, Z-arg-arg-AFC and cytotoxicity was tested on HeLa cell monolayers. PMN isolated from blood were incubated with Ehp/t prior to stimulation by phorbol myristateacetate (PMA, 2 micrograms/ml), serum-treated zymosan (2.5 mg/ml) and glucan (2 mg/ml). CL was monitored in an LKB (Wallac) Luminometer. Ehp/t was found to depress up to 90% of CL induced by PMA, glucan and zymosan. Such a depression was Ehp/t concentration-dependent. A 150 micrograms/ml concentration of Ehp/t, obtained from a 0.015-1.5 mg/ml concentration range tested at different incubation times and temperatures, was used in most of our experiments. Incubation time and temperature optima were 15 min and 37 degrees C, respectively. Ehp/t partially inhibited the CL associated with SO and OH. MDP, in the presence of Ehp/t, enhanced CL response in human PMN to about 67% with reference to normal CL without inhibitor. PMN were confirmed to play a vital role in amebic tissue invasion mechanisms.
The action of the major protease from the parasitic protozoon Entamoeba histolytica, a cysteine protease of Mr 27,000-29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated alpha 2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.
We studied the adherence of [3H]thymidine-labeled axenic Entamoeba histolytica (strain HM1-IMSS) to in vitro preparations of rat and human colonic mucosa. Studies were performed with fixed or unfixed rat colonic mucosa, unfixed rat mucosa exposed to trypsin, unfixed rat submucosa, and fixed human colonic mucosa. Twenty percent of the amebae adhered to fixed rat colonic mucosa; adherence was specifically inhibited by N-acetyl-D-galactosamine (GalNAc), galactose, and asialofetuin. The adherence of amebae to fixed human colonic mucosa was also GalNAc inhibitable. Greater adherence was found with unfixed rat colonic mucosa (40.9%) and was not GalNAc inhibitable unless the tissue was first exposed to trypsin. However, GalNAc did inhibit the adherence of amebae to unfixed rat submucosa. Glutaraldehyde fixation of amebae inactivates known amebic adhesion proteins; there was a markedly decreased adherence of fixed amebae to trypsin-exposed mucosa or fixed rat colonic mucosa. However, fixed or viable amebae had equal levels of adherence to unfixed rat colonic mucosa, suggesting the presence of a host adhesion protein that binds to receptors on amebae. Human (10%) and rabbit (5%) immune sera reduced the adherence of viable amebae to fixed rat colonic mucosa. We concluded that the GalNAc-inhibitable adhesion protein on the surface of E. histolytica trophozoites mediated adherence to fixed rat mucosa, fixed human colonic mucosa, trypsin-exposed unfixed rat mucosa, and unfixed rat submucosa. The surface of unfixed rat colonic mucosa contained a glutaraldehyde- and trypsin-sensitive host adhesion protein, perhaps in the overlying mucus blanket, which bound viable or fixed E. histolytica trophozoites.
Trophozoites of Entamoeba histolytica HM-1 were cultivated axenically in TYI-S medium. The amoebae were then transferred into this medium lacking serum (TYI) and inoculated into in vivo colon loops of adult Sprague-Dawley rats. The trophozoites were rapidly absorbed by the mucus, and few were found free in the luminal fluid by 1 h. By 4 h, the amoebae began to reappear in the lumen, aggregated in sloughed mucus blanket fragments. The colon was examined histologically and by scanning electron microscopy. There was no evidence of invasion or even brush-border attachment by the trophozoites within 4 h. In TYI, trophozoite motility was low. Exposure to the colonic lumen environment for 5 min in this medium significantly increased motility. However, as the trophozoites became absorbed to mucus fragments, their observed motility virtually ceased despite some morphological evidence of pseudopod extension. Erythrophagocytosis was not significantly affected by either exposing trophozoites to TYI washings of the colonic lumen, or by the more complete medium, TYI-S, in which the amoebae were significantly more motile. Two major mucus glycoprotein oligosaccharide end-group sugars, L-fucose and N-acetyl-neuraminic acid, were tested for their effects on trophozoite motility in both TYI and TYI-S. L-Fucose reduced motility; the sialic acid increased motility. It is concluded that the intestinal lumen contains several compartments, including the luminal fluid and the mucus blanket, and that Entamoeba trophozoites exist in a highly motile state in the former and a low motility state in the latter. The mucus blanket provided a significant barrier to trophozoite access to intestinal epithelium target tissue.
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