Entamoeba histolytica cell surface calreticulin binds human c1q and functions in amebic phagocytosis of host cells

Phagocytosis of host cells is characteristic of tissue invasion by the intestinal ameba Entamoeba histolytica, which causes amebic dysentery and liver abscesses. Entamoeba histolytica induces host cell apoptosis and uses ligands, including C1q, on apoptotic cells to engulf them. Two mass spectrometry analyses identified calreticulin in amebic phagosome preparations, and, in addition to its function as an endoplasmic reticulum chaperone, calreticulin is believed to be the macrophage receptor for C1q. The purpose of this study was to determine if calreticulin functions as an E. histolytica C1q receptor during phagocytosis of host cells. Calreticulin was localized to the surface of E. histolytica during interaction with both Jurkat lymphocytes and erythrocytes and was present in over 75% of phagocytic cups during amebic erythrophagocytosis. Presence of calreticulin on the cell surface was further demonstrated using a method that selectively biotinylated cell surface proteins and by flow cytometry using trophozoites overexpressing epitope-tagged calreticulin. Regulated overexpression of calreticulin increased E. histolytica's ability to phagocytose apoptotic lymphocytes and calcium ionophore-treated erythrocytes but had no effect on amebic adherence to or destruction of cell monolayers or surface expression of the GalNAc lectin and serine-rich E. histolytica protein (SREHP) receptors. Finally, E. histolytica calreticulin bound specifically to apoptotic lymphocytes and to human C1q. Collectively, these data implicate cell surface calreticulin as a receptor for C1q during E. histolytica phagocytosis of host cells.     

Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites.     

Cytokine Genes are Down-Regulated When Attachment of Entamoeba histolytica to HT-29 Colon Epithelial Cells is Prevented

Entamoeba histolytica can cause invasive disease by disruption of the intestinal barriers and subsequent lysis of the intestinal cells. Adherence to and contact dependent killing of host cells requires the galactose inhibitable lectin. To elucidate the mechanism whereby E. histolytica influences host defence, the authors assessed the change of proinflammatory cytokine genes expressed by colon epithelial cells in response to co-culture with E. histolytica trophozoites and carbohydrates, including galactose, N -acetyl-galactosamine or N -acetyl-lactosamine, which prevented E. histolytica from attaching to epithelial cells. After HT-29 human colon epithelial cells were co-cultured with E. histolytica trophozoites in the presence or absence of carbohydrates (0.1–100 m m ), RNA was extracted from the epithelial cells by an acid guanidinium thiocyanate–phenol–chloroform method. Cytokine gene expression was assessed by quantitative RT-PCR using a synthetic internal standard, and proteins were determined by ELISA. IL-8 mRNA expressed by HT-29 cells in response to E. histolytica trophozoites was downregulated in the presence of galactose, N -acetyl-galactosamine or N -acetyl-lactosamine (0.1–100 m m ), and this was paralleled by decreased IL-8 protein secretion. GM-CSF and IL-1α/β mRNAs were also downregulated in those cells in the presence of these agents. These results suggest that the expression of proinflammatory cytokine genes could be inhibited by preventing E. histolytica from attaching to the host's colon epithelial cells.     

In vitro effect of human recombinant tumor necrosis factor on Entamoeba histolytica trophozoites

The effect of tumor necrosis factor (TNF) on E. histolytica trophozoites was examined by using three virulent (IP: 0682:1, HM-1: IMSS, 200: NIH) and one nonvirulent (DKB) strain of E. histolytica. Various concentrations of recombinant TNF were added to E. histolytica trophozoites and the total parasite numbers and their viability were periodically assessed by microscopic observation and trypan blue staining after incubation at 37 degrees C in a nonhumidified chamber. In this study, concentrations of 10(1)-10(6) units of TNF were used. Over a concentration range of 10(4)-10(6) units, the number of trophozoites was significantly lowered in the amoebic cultures containing TNF as compared to untreated controls. It was also found that the effect of TNF was dependent on the densities of both virulent and non-virulent strains of E. histolytica trophozoites in axenic conditions. TNF has no significant affect on the growth of amoebae at the lower starting number of amoebae. The amoebae cultured at the higher density were growth-inhibited significantly in comparison with the control groups. When the growth of the virulent and nonvirulent strains of amoebae was compared in TNF treated culture, it was found that TNF has an inhibitory effect on both the virulent and nonvirulent strans of E. histolytica.     

Sexual dimorphism in the control of amebic liver abscess in a mouse model of disease

Amebic liver abscess (ALA) is the most common extraintestinal manifestation of human infection by the enteric protozoan parasite Entamoeba histolytica. In contrast to intestinal infection, ALA greatly predominates in males but is rare in females. Since humans are the only relevant host for E. histolytica, experimental studies concerning this sexual dimorphism have been hampered by the lack of a suitable animal model. By serial liver passage of cultured E. histolytica trophozoites in gerbils and mice, we generated amebae which reproducibly induce ALA in C57BL/6 mice. Interestingly, all animals developed ALA, but the time courses of abscess formation differed significantly between the genders. Female mice were able to clear the infection within 3 days, whereas in male mice the parasite could be recovered for at least 14 days. Accordingly, male mice showed a prolonged time of recovery from ALA. Immunohistology of abscesses revealed that polymorphonuclear leukocytes and macrophages were the dominant infiltrates, but in addition, gamma,delta-T cells, NK cells, and natural killer T (NKT) cells were also present at early times during abscess development, whereas conventional alpha,beta-T cells appeared later, when female mice had already cleared the parasite. Interestingly, male and female mice differed in early cytokine production in response to ameba infection. Enzyme-linked immunospot assays performed with spleen cells of infected animals revealed significantly higher numbers of interleukin-4-producing cells in male mice but significantly higher numbers of gamma interferon (IFN-gamma)-producing cells in female mice. Early IFN-gamma production and the presence of functional NKT cells were found to be important for the control of hepatic amebiasis as application of an IFN-gamma-neutralizing monoclonal antibody or the use of NKT knockout mice (Valpha14iNKT, Jalpha 18(-/-)) dramatically increased the size of ALA in female mice. In addition, E. histolytica trophozoites could be reisolated from liver abscesses of Jalpha18(-/-) mice on day 7 postinfection, when wild-type mice had already cleared the parasite. These data suggest that the sexual dimorphism in the control of ALA is due to gender-specific differences in early cytokine production mediated at least in part by NKT cells in response to E. histolytica infection of the liver.     

Genes induced by a high-oxygen environment in Entamoeba histolytica

Entamoeba histolytica, although a microaerophilic protozoan parasite, encounters a high-oxygen environment, during invasive amoebiasis. The parasite requires specific regulation of certain proteins to maintain its physiological functions to survive in the more oxygenated condition. Our endeavor was to know how does amoeba adapt itself in a high-oxygen environment. Reactive oxygen species (ROS) was found to accumulate in an increasing concentration within the stressed trophozoites in a time-dependent manner. Increased cytopathic activity was detected at 2h in high-oxygen-exposed E. histolytica lysate compared to lysate of normal E. histolytica trophozoites by Ussing chamber assay. The differential display and semi-quantitative polymerase chain reaction showed overexpression in the mRNA levels of thiol-dependent peroxidase (Eh29), superoxide dismutase (SOD), EhCP5, G protein, HSP70, and peptidylprolyl isomerase at different time periods of oxidative stressed trophozoites compared to normally cultured E. histolytica. Analyses of the up-regulated genes that are associated with stress response, viz., signal transduction, tissue destruction, and oxidative stress management, including enhanced expression of a 29-kDa Eh29, suggest that this organism has several protective mechanisms to deal with oxidative stress during invasion.     

Cellular bases of experimental amebic liver abscess formation

The complete sequence of morphologic events during amebic liver abscess formation in the hamster has been studied, from the lodgement of amebas in the hepatic sinusoids to the development of extensive liver necrosis. Following intraportal inoculation of live amebas, the early stages of the lesion (from 1 to 12 hours) were characterized by acute cellular infiltration composed of an increasingly large number of polymorphonuclear leukocytes, which surrounded centrally located trophozoites. Histiocytes and lysed leukocytes were situated on the periphery of the lesions. Hepatocytes close to the early lesions showed degenerative changes which led to necrosis; however, direct contact of liver cells with amebas was very rarely observed. At later stages, the extent of necrosis increased, macrophages and epithelioid cells replaced most leukocytes, and well-organized granulomas developed. Extensive necrosis associated with fused granulomas was present by Day 7. The results suggest that Entamoeba histolytica trophozoites do not produce amebic liver abscesses in hamsters through direct lysis of hepatocytes. Rather, tissue destruction is the result of the accumulation and subsequent lysis of leukocytes and macrophages surrounding the amebas.     

Inflammatory reaction in experimental hepatic amebiasis. An ultrastructural study.

One of the hallmarks of tissue necrosis produced by the human protozoan parasite Entamoeba histolytica, the causative agent of human amebiasis, appeared to be the lack of inflammatory reaction to the invading trophozoites. Recent evidence suggests, however, that inflammatory cells do appear during early stages of amebic destructive lesions and that they contribute to the establishment of foci of tissue necrosis in intestinal and liver lesions. The present analysis of the fine-structural changes that take place during early stages of amebic liver abscesses induced in hamsters after the intraportal inoculation of axenic amebas has shown that large numbers of polymorphonuclear leukocytes (PMNs) are recruited around invading amebas. These leukocytes lyse as a consequence of contact-mediated damage induced by the trophozoites. Amebas were also capable of ingesting apparently intact PMNs. Macrophages and eosinophils were also recruited at the foci of inflammation. At all times examined, trophozoites of Entamoeba histolytica survived in spite of being in close contact with PMNs or degranulating eosinophils. The ultrastructural observations have also shown the lack of direct contact between amebas and liver parenchymal cells during the initial stages of the focal liver necrosis induced by the parasite, therefore supporting the view that hepatic damage may be effected indirectly through lysis of inflammatory cells. The results also provide a basis for the understanding of the induction of experimental protective immunity against invasive amebiasis, a process which seems to be mostly dependent on cellular mechanisms.     

Effect of splenectomy on the size of amoebic liver abscesses and metastatic foci in hamsters.

The role of the spleen in hepatic amoebiasis in hamsters was studied. In hamsters receiving an intrahepatic inoculation of 10(5) trophozoites of axenic Entamoeba histolytica at 7 or 14 days postsplenectomy, the mean weight of metastatic foci increased significantly when compared with sham-splenectomized or intact controls. In contrast, when both splenectomy and intrahepatic inoculation with amoebae were carried out at the same time, there was not only a significant increase in the mean weight of metastatic foci but also in the liver abscess. It is suggested that the spleen is important for host defense against E. histolytica infection, especially in the reduction in the degree of metastatic spread from the primary site.     

Sequential histopathology of cavitary liver abscess. Formation induced by axenically grown Entamoeba histolytica

Multiple hamster liver passage of Entamoeba histolytica trophozoites with intervening recovery into axenic culture caused increased virulence as measured by increase in the size of the lesion produced. Lesions produced by amebae that had not been liver-passaged did not persist; however, multiply liver-passaged substrains produced large, fluid-filled abscesses one month to six weeks after inoculation. Six days after inoculation, lesions consisted of multiple granulomas, lymphocytes, and E histolytica trophozoites. Large, fluid-filled abscesses produced by liver-passaged substrains lacked the granulomatous appearance of the earlier lesions. The abscesses had a fibrous wall, with E histolytica trophozoites at the inner aspect. To our knowledge, the evolution of early granulomatous lesions into a cavitary abscess with features closely resembling those of human amebic abscess has not been reported previously in the experimental disease in the hamster.     

Roles of cell adhesion and cytoskeleton activity in Entamoeba histolytica pathogenesis: a delicate balance

The protozoan parasite Entamoeba histolytica colonizes the human large bowel. Invasion of the intestinal epithelium causes amoebic colitis and opens the route for amoebic liver abscesses. The parasite relies on its dynamic actomyosin cytoskeleton and on surface adhesion molecules for dissemination in the human tissues. Here we show that the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin clusters in focal structures localized in the region of E. histolytica that contacts monolayers of enterocytes. Disruption of myosin II activity impairs the formation of these structures and renders the trophozoites avirulent for liver abscess development. Production of the cytoplasmic domain of the E. histolytica Gal/GalNAc lectin in engineered trophozoites causes reduced adhesion to enterocytes. Intraportal delivery of these parasites to the liver leads to the formation of a large number of small abscesses with disorganized morphology that are localized in the vicinity of blood vessels. The data support a model for invasion in which parasite motility is essential for establishment of infectious foci, while the adhesion to host cells modulates the distribution of trophozoites in the liver and their capacity to migrate in the hepatic tissue.     

An Entamoeba sp. strain isolated from rhesus monkey is virulent but genetically different from Entamoeba histolytica

An Entamoeba sp. strain, P19-061405, was isolated from a rhesus monkey in Nepal and characterized genetically. The strain was initially identified as Entamoeba histolytica using PCR amplification of peroxiredoxin genes. However, sequence analysis of the 18S rRNA gene showed a 0.8% difference when compared to the reference E. histolytica HM-1:IMSS human strain. Differences were also observed in the 5.8S rRNA gene and the internal transcribed spacer (ITS) regions 1 and 2, and analysis of the serine-rich protein gene from the monkey strain showed unique codon usages compared to E. histolytica isolated from humans. The amino acid sequences of two hexokinases and two glucose phosphate isomerases also differed from those of E. histolytica. Isoenzyme analyses of these enzymes in the monkey strain showed different electrophoretic mobility patterns compared with E. histolytica isolates. Analysis of peroxiredoxin genes indicated the presence of at least seven different types of protein, none of which were identical to proteins in E. histolytica. When the trophozoites from the monkey strain were inoculated into the livers of hamsters, formation of amebic abscesses was observed 7 days after the injection. These results demonstrate that the strain is genetically different from E. histolytica and is virulent. Revival of the name Entamoeba nuttalli is proposed for the organism.     

Expression of amoebapores is required for full expression of Entamoeba histolytica virulence in amebic liver abscess but is not necessary for the induction of inflammation or tissue damage in amebic colitis

Entamoeba histolytica trophozoites produce amoebapores, a family of small amphipathic peptides capable of insertion into bacterial or eukaryotic membranes and causing cellular lysis. Recently, E. histolytica trophozoites that are totally deficient in the production of amoebapore-A were created through a gene silencing mechanism (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). Here we tested the virulence of amoebapore A(-) trophozoites in models of the two major forms of amebic disease: amebic liver abscess and amebic colitis. We demonstrate that amoebapore expression is required for full virulence in the SCID mouse model of amebic liver abscess, but E. histolytica trophozoites that do not express amoebapore-A can still cause inflammation and tissue damage in infected human colonic xenografts. These data are consistent with the concept that tissue damage may proceed by different mechanisms in amebic liver abscess compared to amebic colitis.     

Susceptibility of Entamoeba histolytica to oxidants.

The effect of hydrogen peroxide and hypochlorite on culture forms of Entamoeba histolytica trophozoites was examined by using two strains of E. histolytica, virulent (IP:0682:1) and nonvirulent (DKB). The amoebae were incubated with various concentrations of hydrogen peroxide and hypochlorite, and their viability was determined at different times after incubation. When the viability of the virulent and nonvirulent strains was compared to different oxidant strengths, it became apparent that the virulent strain was less susceptible than the nonvirulent one to the cytotoxic effect of hydrogen peroxide and hypochlorite. Our studies further showed that the toxic effect was both time and dose dependent. To confirm that the killing of amoebae in this system was associated with the presence of hydrogen peroxide, amoebae were incubated with hydrogen peroxide and catalase. Catalase reduced the killing effect of hydrogen peroxide to the control level. These data confirmed previous observations of the susceptibility of amoebic trophozoites to hydrogen peroxide and also demonstrated susceptibility to hypochlorite.     

The cell cycle of Entamoeba invadens during vegetative growth and differentiation

The cell division cycle of Entamoeba invadens was studied during vegetative growth of trophozoites and during their differentiation into cysts. During vegetative growth of trophozoites, it was observed that DNA synthesis typically continued after one genome content had been duplicated. During encystation, DNA synthesis was arrested after 4n genome content had been synthesised. Using multi-parameter flow cytometry, the light scattering properties of cysts and trophozoites were studied. The cytoplasmic granularity, reflected by the side scatter of light, was proportional to DNA content of trophozoites, whereas cysts with similar DNA contents showed heterogeneity in their cytoplasmic granularity. Dynamic changes in the intracellular calcium pools were observed during differentiation of trophozoites to cysts. Comparison of E. invadens and Entamoeba histolytica cell cycles suggest that both organisms may have similar regulatory processes during cell division and differentiation. Since E. histolytica cannot be induced to encyst in axenic culture, analysis of the E. invadens cell cycle during encystation may be useful for identifying homologous processes in E.histolytica.     

Localization of myosin heavy chain A in the human pathogen Entamoeba histolytica.

To recognize myosin II in trophozoites of the human pathogen Entamoeba histolytica, a specific antimyosin polyclonal serum was raised against a fusion protein consisting of a 146-amino-acid fragment of the myosin II heavy chain A of E. histolytica (MhcA) fused with beta-galactosidase. The hybrid protein was encoded by a chimera gene formed by a DNA fragment, from the mhcA gene, amplified by polymerase chain reaction and fused with the lacZ gene of Escherichia coli. Polymerase chain reaction-amplified DNA is located within the region encoding the tail domain of myosin. This antibody recognized a 250-kDa protein in extracts of E. histolytica trophozoites. Confocal microscope analysis of antibody-labelled trophozoites indicated that MhcA localizes at the posterior pole of locomoting cells and concentrates within the uroid. These results might indicate that MhcA is involved in movement and in the uroid formation which help amoebas to escape the host immune response. These data are the first evidence indicating that myosin exists in E. histolytica. In addition, two other peptides were found in myosin-enriched extracts of amoebas, indicating that other myosins may be present in this parasite.     

Development of inflammation and augmented chemotactic responsiveness of murine peritoneal macrophages following treatment with Entamoeba histolytica trophozoites

The accumulation of inflammatory cells in the peritoneal cavity of C57BL/6 mice was examined following intraperitoneal injection of Entamoeba histolytica trophozoites. Two different strains of E. histolytica were used: a virulent strain (IP:0682:1) and a non-virulent strain (DKB). Injection of 10(6) trophozoites of either strain resulted in significant increases in the numbers of total peritoneal cells, macrophages and polymorphonuclear cells as compared to either saline-injected control mice or mice injected with 10-fold lower doses of trophozoites. The in vitro chemotactic response of macrophages from amoebae-induced exudates was also examined. Macrophages from mice treated with strain IP:0682:1 or DKB strain trophozoites were more responsive to complement-derived chemotactic factors than macrophages from saline-injected mice. This increase was significant on day 2 and persisted at enhanced levels until day 20 when the experiment was terminated. In addition, it was found that trophozoites activated normal mouse serum resulting in the production of serum-derived chemotactic activity.