Localization of Phosphatidylinositol 4,5-Bisphosphate to Lipid Rafts and Uroids in the Human Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica is an intestinal protozoan parasite and is the causative agent of amoebiasis. During invasive infection, highly motile amoebae destroy the colonic epithelium, enter the blood circulation, and disseminate to other organs such as liver, causing liver abscess. Motility is a key factor in E. histolytica pathogenesis, and this process relies on a dynamic actomyosin cytoskeleton. In other systems, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is known to regulate a wide variety of cellular functions, including signal transduction, actin remodeling, and cell motility. Little is known about the role of PI(4,5)P₂ in E. histolytica pathogenicity. In this study, we demonstrate that PI(4,5)P₂ is localized to cholesterol-rich microdomains, lipid rafts, and the actin-rich fractions of the E. histolytica membrane. Microscopy revealed that the trailing edge of polarized trophozoites, uroids, are highly enriched in lipid rafts and their constituent lipid, PI(4,5)P₂. Polarization and enrichment of uroids and rafts with PI(4,5)P₂ were enhanced upon treatment of E. histolytica cells with cholesterol. Exposure to cholesterol also increased intracellular calcium, which is a downstream effector of PI(4,5)P₂, with a concomitant increase in motility. Together, our data suggest that in E. histolytica, PI(4,5)P₂ may signal from lipid rafts and cholesterol may play a role in triggering PI(4,5)P₂-mediated signaling to enhance the motility of this pathogen.     

The dynamic interdependence of amebiasis, innate immunity, and undernutrition

Entamoeba histolytica, the protozoan parasite that causes amebic dysentery, greatly contributes to disease burden in the developing world. Efforts to exhaustively characterize the pathogenesis of amebiasis have increased our understanding of the dynamic host–parasite interaction and the process by which E. histolytica trophozoites transition from gut commensals to invaders of the intestinal epithelium. Mouse models of disease continue to be instrumental in this area. At the same time, large-scale studies in human populations have identified genetic and environmental factors that influence susceptibility to amebiasis. Nutritional status has long been known to globally influence immune function. So it is not surprising that undernutrition has emerged as a critical risk factor. A better understanding of how nutritional status affects immunity to E. histolytica will have dramatic implications in the development of novel treatments. Future work should continue to characterize the fascinating host–parasite arms race that occurs at each stage of infection.     

Antiamoebic properties of the actinomycete metabolites echinomycin A and tirandamycin A

Entamoeba histolytica infects 50 million people per year, causing 100,000 deaths worldwide. The primary treatment for amoebiasis is metronidazole. However, increased pathogen resistance combined with the drug’s toxic side effects encourages a search for alternative therapeutic agents. Secondary metabolites from marine bacteria are a promising resource for antiprotozoan drug discovery. In this study, extracts from a collection of marine-derived actinomycetes were screened for antiamoebic properties, and the activities of antibiotics echinomycin A and tirandamycin A are shown. Both antibiotics inhibited the in vitro growth of a E. histolytica laboratory strain (HM-1:IMSS) and a clinical isolate (Colombia, Col) at 30- to 60-μM concentrations. EIC₅₀ (estimated inhibitory concentration) values were comparable for both antibiotics (44.3–46.3 μM) against the E. histolytica clinical isolate.     

Effect of silica on resistance of mice to Entamoeba histolytica infection.

The role of macrophages in intestinal amoebiasis in mice has been investigated. The effect of injuring host macrophages on the course of infection was examined by using strains selected as being either genetically susceptible (C3H/HeJ, C57BL/6) or genetically resistant (A/J) to amoebiasis. Mice were treated with an intravenous injection of silica particles 1 day before infection with 2.5 X 10(5) or 5 X 10(5) polyxenic trophozoites of Entamoeba histolytica. The animals were killed at various times after infection, and the parasite burden in the cecum was measured. Silica treatment dramatically increased the growth of parasites in the susceptible mice. The same trend was evident, although less marked, in the resistant mice. The effect of silica treatment in experimental amoebiasis was much more pronounced in animals inoculated with 5 X 10(5) amoebae than in those with 2.5 X 10(5) amoebae. These data suggest that macrophages play an important role in host defense against amoebiasis in mice.     

Attenuated recombinant Yersinia as live oral vaccine carrier to protect against amoebiasis

Various attenuated Yersinia enterocolitica strains expressing different sections of the Entamoeba histolytica surface lectin via the type III protein secretion system (T3SS) were assessed for their use to orally vaccinate rodents against invasive amoebiasis. The T3SS was found to efficiently express and secrete or translocate subfragments as well as the entire heavy subunit of the lectin. Oral vaccination with recombinant Yersinia conferred significant protection against amoebic liver abscess formation when the antigen was expressed as a fusion molecule with the translocation domain of Yersinia outer protein E. However, effectiveness of vaccination was dependent on gender and the rodent species used. Protection was mediated primarily by cellular immune mechanisms as it was independent from the antibody titre against the amoeba lectin but correlated with an antigen-specific Th1-cytokine response. The results suggest that Gram-negative bacteria expressing E. histolytica antigens via T3SS may constitute a suitable oral vaccine carrier against amoebiasis and that an effective IFN-γ response is required for protection against invasive amoebiasis.     

Comparison of two immunoassays for detection of Entamoeba histolytica

Effective diagnostic tools are essential in order to combat disease caused by the parasite Entamoeba histolytica. In this study, we compared the commercially available Ridascreen Entamoeba test (R-Biopharm) and the E. histolytica II test (Techlab), and we found that the E. histolytica II test detects E. histolytica infections more accurately.     

Caerulomycin,an antifungal antibiotic with marked in vitro and in vivo activity againstEntamoeba histolytica

The anti-amoebic action of the bipyridyl antibiotic caerulomycin was assessed in vitro and in vivo using various strains ofEntamoeba histolytica from polyxenic, axenic and monoxenic cultures. Minimum inhibition concentrations of caerulomycin (metronidazole) were 7.5 (5), 15.6 (1.95) and 60 (2.5) g/ml against polyxenic, axenic and monoxenic cultures ofE. histolytica, respectively. The ED₅₀ values ascertained in golden hamsters (extraintestinal amoebiasis) and rats (intestinal amoebiasis) after the oral route were 136 and 199 mg/kg (×4), respectively. Metronidazole proved to be approximately four times more active against tissue forms ofE. histolytica than caerulomycin [ED₅₀ of metronidazole: <40 mg/kg (×4)]. The antibiotic was slightly superior to metronidazole in its action on lumen forms ofE. histolytica [ED₅₀ of metronidazole: 233 mg/kg (×4)]. The antibiotic was in some cases toxic to hamsters and rats within the therapeutic range.     

Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.     

Participation of the serine-rich Entamoeba histolytica protein in amebic phagocytosis of apoptotic host cells

Entamoeba histolytica is an intestinal ameba that causes dysentery and liver abscesses. Cytotoxicity and phagocytosis of host cells characterize invasive E. histolytica infection. Prior to phagocytosis of host cells, E. histolytica induces apoptotic host cell death, using a mechanism that requires contact via an amebic galactose-specific lectin. However, lectin inhibition only partially blocks phagocytosis of already dead cells, implicating at least one additional receptor in phagocytosis. To identify receptors for engulfment of apoptotic cells, monoclonal antibodies against E. histolytica membrane antigens were screened for inhibition of phagocytosis. Of 43 antibodies screened, one blocked lectin-independent uptake of apoptotic cells, with >90% inhibition at a dose of 20 μg/ml (P < 0.0003 versus control). The same antibody also inhibited adherence to apoptotic lymphocytes and, to a lesser extent, adherence to and killing of viable lymphocytes. The antigen recognized by the inhibitory antibody was purified by affinity chromatography and identified by liquid chromatography-mass spectrometry as the serine-rich E. histolytica protein (SREHP). Consistent with this, the inhibitory antibody bound to recombinant SREHP present in bacterial lysates on immunoblots. The SREHP is an abundant immunogenic surface protein of unclear function. The results of this unbiased antibody screen strongly implicate the SREHP as a participant in E. histolytica phagocytosis and suggest that it may play an important role in adherence to apoptotic cells.     

Serum IgE levels in amoebiasis

Elevated levels of serum immunoglobulin E (IgE) are found in many parasitic diseases. In order to determine the influence of Entamoeba histolytica on IgE levels of clinically symptomatic patients with intestinal amoebiasis, a controlled study was performed. There was no difference between the serum IgE values from Brazilian patients with amoebiasis and those from age-, race- and sex-matched normal Brazilian controls. Based also on our previous similar studies on giardiasis and Chagas’ disease, we conclude that protozoan infections do not elevate serum IgE levels.     

Amoebic colitis

Amoebic colitis is caused by the parasite Entamoeba histolytica (E. histolytica). The condition is prevalent in countries with poor sanitation but is increasing within developed countries secondary to global travel and immigration. We report a case of amoebic colitis diagnosed in a patient with a clinical diagnosis of Crohn's disease and with no travel history. Colonic biopsies showed focal active colitis with ulcer slough; deeper levels looking for granulomas fortuitously revealed the amoebae. The diagnosis was confirmed by positive serology and symptomatic improvement after a course of eradication treatment. Pathologists should look for amoebae in biopsies for inflammatory bowel disease (IBD), as endoscopic and histological appearances of IBD and amoebic colitis can be similar but importantly immunosuppressive therapy is contraindicated in amoebic colitis.     

A radioimmunoprecipitation polyethylene glycol assay for circulating Entamoeba histolytica antigens

An assay capable of detecting circulating Entamoeba histolytica antigens in amoebiasis is described. This assay utilised a radiolabelled affinity purified rabbit anti-E. histolytica antibody that had been depleted of antibodies that cross-react with human serum proteins, and a polyethylene glycol precipitation step.     

Prostaglandin E2 produced by Entamoeba histolytica binds to EP4 receptors and stimulates interleukin-8 production in human colonic cells

Entamoeba histolytica pathogenesis in the colon occurs in a stepwise fashion. It begins with colonization of the mucin layer, which is followed by stimulation of a proinflammatory response that causes nonspecific tissue damage that may facilitate parasite invasion of the underlying colonic mucosa. Unfortunately, the parasite and/or host factors that stimulate a proinflammatory response in the gut are poorly understood. In this study, we found that live E. histolytica or secretory or proteins (SP) and soluble ameba components (SAP) can markedly increase interleukin-8 (IL-8) mRNA expression and protein production in colonic epithelial cells. The IL-8-stimulating molecule produced by live amebae was identified as prostaglandin E₂ (PGE₂) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE₂ and eliminated IL-8 production induced by live parasites or ameba components. Moreover, using specific prostaglandin EP2 and EP4 receptor agonists and antagonists, we found that PGE₂ binds exclusively through EP4 receptors in colonic epithelial cells to stimulate IL-8 production. Silencing of EP4 receptors with EP4 small interfering RNA completely eliminated SP- and SAP-induced IL-8 production. These studies identified bioactive PGE₂ as a one of the major virulence factors produced by E. histolytica that can stimulate the potent neutrophil chemokine and activator IL-8, which can trigger an acute host inflammatory response. Thus, the induction of IL-8 production in response to E. histolytica-derived PGE₂ may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.     

Toll-like receptor 9-dependent macrophage activation by Entamoeba histolytica DNA

Activation of the innate immune system by bacterial DNA and DNA of other invertebrates represents a pathogen recognition mechanism. In this study we investigated macrophage responses to DNA from the intestinal protozoan parasite Entamoeba histolytica. E. histolytica genomic DNA was purified from log-phase trophozoites and tested with the mouse macrophage cell line RAW 264.7. RAW cells treated with E. histolytica DNA demonstrated an increase in levels of tumor necrosis factor alpha (TNF-α) mRNA and protein production. TNF-α production was blocked by pretreatment with chloroquine or monensin. In fact, an NF-κB luciferase reporter assay in HEK cells transfected with human TLR9 demonstrated that E. histolytica DNA signaled through Toll-like receptor 9 (TLR9) in a manner similar to that seen with CpG-ODN. Immunofluorescence assays confirmed NF-κB activation in RAW cells, as seen by nuclear translocation of the p65 subunit. Western blot analysis demonstrated mitogen-activated protein kinase activation by E. histolytica DNA. E. histolytica DNA effects were abolished in MYD88^−/− mouse-derived macrophages. In the context of disease, immunization with E. histolytica DNA protected gerbils from an E. histolytica challenge infection. Taken together, these results demonstrate that E. histolytica DNA is recognized by TLR9 to activate macrophages and may provide an innate defense mechanism characterized by the induction of the inflammatory mediator TNF-α.     

Plasmodium and mononuclear phagocytes

Plasmodium, the causative agent of malaria, initially multiplies inside liver cells and then in successive cycles inside erythrocytes, causing the symptoms of the disease. In this review, we discuss interactions between the extracellular and intracellular forms of the Plasmodium parasite and innate immune cells in the mammalian host, with a special emphasis on mononuclear phagocytes. We overview here what is known about the innate immune cells that interact with parasites, mechanisms used by the parasite to evade them, and the protective or detrimental contribution of these interactions on parasite progression through its life cycle and pathology in the host.     

Comparison of PCR, Isoenzyme Analysis, and Antigen Detection for Diagnosis of Entamoeba histolytica Infection

The diagnosis of amebiasis by microscopic identification of the parasite in stool is insensitive and unable to distinguish the invasive parasite Entamoeba histolytica from the commensal parasite E. dispar. In this study, we have tested a PCR technique for the detection of E. histolytica and compared it with isoenzyme analysis and the TechLab E. histolytica-specific antigen detection test. The nested-PCR test we used is based on amplification of the small subunit rRNA gene of E. histolytica and E. dispar followed by restriction digest analysis of the PCR product. Single stool samples were obtained from 98 patients from Dhaka, Bangladesh, with diarrhea: 88 patients diagnosed by microscopy and/or culture with E. histolytica and/or E. dispar infection and 10 patients without infection. Isoenzyme analysis identified 53 of the infections as E. histolytica and 28 as E. dispar. PCR and isoenzyme identification of E. histolytica agreed in 96% (51 of 53) of amebic cultures. PCR for E. histolytica was negative in all 10 samples that were negative for E. histolytica by isoenzyme and antigen detection. PCR and antigen detection had comparable sensitivities when performed directly on fresh stool specimens, identifying 87% (46 of 53) and 85% (45 of 53), respectively, of E. histolytica infections identified by isoenzyme analysis. The correlation of results by antigen detection and PCR for identification of E. histolytica in stool was 93% (45 of 48 cases). Mixed infections with E. histolytica and E. dispar were detected by PCR in 14% (12 of 88) of cases. In conclusion, all three techniques for specific identification of E. histolytica in fresh stool showed excellent correlation. Only the TechLab E. histolytica antigen detection test was both rapid and technically simple.