Toll-like Receptor Signaling Activation by Entamoeba histolytica Induces Beta Defensin 2 in Human Colonic Epithelial Cells: Its Possible Role as an Element of the Innate Immune Response

Background

Entamoeba histolytica, a protozoan parasite of humans, produces dysenteric diarrhea, intestinal mucosa damage and extraintestinal infection. It has been proposed that the intestinal microbiota composition could be an important regulatory factor of amebic virulence and tissue invasion, particularly if pathogenic bacteria are present. Recent in vitro studies have shown that Entamoeba histolytica trophozoites induced human colonic CaCo2 cells to synthesize TLR-2 and TLR-4 and proinflammatory cytokines after binding to the amebic Gal/GalNac lectin carbohydrate recognition domain. The magnitude of the inflammatory response induced by trophozoites and the subsequent cell damage were synergized when cells had previously been exposed to pathogenic bacteria.

Methodology/Principal Findings

We show here that E. histolytica activation of the classic TLR pathway in CaCo2 cells is required to induce β defensin-2 (HBD2) mRNA expression and production of a 5-kDa cationic peptide with similar properties to the antimicrobial HBD2 expressed by CaCo2 cells exposed to enterotoxigenic Escherichia coli. The induced peptide showed capacity to permeabilize membranes of bacteria and live trophozoites. This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody.

Conclusions/Significance

Entamoeba histolytica trophozoites bind to human intestinal cells and induce expression of HBD2; an antimicrobial molecule with capacity to destroy pathogenic bacteria and trophozoites. HDB2''s possible role as a modulator of the course of intestinal infections, particularly in mixed ameba/bacteria infections, is discussed.     

The interplay between Entamoeba and enteropathogenic bacteria modulates epithelial cell damage

Background

Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined.

Methodology

Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified.

Principal Findings

E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica.

Conclusions

Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage.

Significance

The in vitro system presented here provides evidence that the Entamoeba/enteropathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon.     

The Antiretroviral Lectin Cyanovirin-N Targets Well-Known and Novel Targets on the Surface of Entamoeba histolytica Trophozoites

Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man₅GlcNAc₂). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools.Entamoeba histolytica causes amebic dysentery and liver abscess in the developing world (10, 20, 29). We are interested in E. histolytica glycoproteins containing Asn-linked glycans (N-glycans) for numerous reasons. E. histolytica makes an N-glycan precursor that contains 7 sugars (Man₅GlcNAc₂-PP-dolichol) rather than 14 sugars (Glc₃Man₉GlcNAc₂-PP-dolichol) made by most animals, plants, and fungi (21, 31, 44). E. histolytica N-glycans are used for quality control of glycoprotein folding in the endoplasmic reticulum (ER) lumen, and there is positive selection for sites of N-linked glycosylation in secreted and membrane proteins of E. histolytica (5, 11, 53).Unprocessed Man₅GlcNAc₂, by far the most abundant E. histolytica N-glycan, is present on the plasma membrane and vesicular membranes (31). The antiretroviral lectin cyanovirin-N, which is specific for α-1,2-linked mannose present on unprocessed N-glycans, binds E. histolytica N-glycans and forms aggregates or caps on the surface of E. histolytica trophozoites (1, 25, 31, 44, 45). E. histolytica glycoproteins are also capped by the plant lectin concanavalin A (ConA), which has a broader carbohydrate specificity (mannose and glucose) than cyanovirin-N (3, 16, 18, 19). Heavy subunits of the Gal/GalNAc lectin, the most important E. histolytica vaccine candidate, have 7 to 10 potential sites for N-linked glycosylation (32, 39, 43). Inhibition of N-glycan synthesis results in Gal/GalNAc lectins that are unable to bind to sugars on host epithelial cells.Carbohydrates appear to be an important target on the surface of E. histolytica as anti-proteophosphoglycan (PPG) monoclonal antibodies bind to O-phosphodiester-linked glycans and protect animal models from amebic infection (6, 33, 35, 40, 48). Lectin affinity columns are a powerful method for enriching unique parasite glycoproteins that may be identified by mass spectrometry (MS) of tryptic fragments (17, 55). For example, we recently used the plant lectin wheat germ agglutinin to dramatically enrich glycoproteins with short N-glycans of Giardia (42).The goal of the present studies was to explore further the interaction of the antiretroviral lectin cyanovirin-N with E. histolytica trophozoites in vitro. Questions asked included the following: Are E. histolytica glycoproteins with N-glycans replenished on the plasma membrane after capping with cyanovirin-N? What is the effect of cyanovirin-N capping on other amebic virulence factors and/or vaccine candidates (e.g., the Gal/GalNAc lectin and PPG)? Is capping by cyanovirin-N mediated by actin, as described for capping by the Gal/GalNAc lectin and ConA? What is the effect of the cyanovirin-N on amebic phagocytosis of mucin-coated beads, a surrogate assay for virulence? Which trophozoite glycoproteins are potential targets of cyanovirin-N (identified by mass spectrometry of lectin-enriched E. histolytica proteins)? Are any of them potential vaccine candidates?     

Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.     

Vaccination with Leishmania infantum Acidic Ribosomal P0 but Not with Nucleosomal Histones Proteins Controls Leishmania infantum Infection in Hamsters

Background

Several intracellular Leishmania antigens have been identified in order to find a potential vaccine capable of conferring long lasting protection against Leishmania infection. Histones and Acid Ribosomal proteins are already known to induce an effective immune response and have successfully been tested in the cutaneous leishmaniasis mouse model. Here, we investigate the protective ability of L. infantum nucleosomal histones (HIS) and ribosomal acidic protein P0 (LiP0) against L. infantum infection in the hamster model of visceral leishmaniasis using two different strategies: homologous (plasmid DNA only) or heterologous immunization (plasmid DNA plus recombinant protein and adjuvant).

Methodology/Principal Findings

Immunization with both antigens using the heterologous strategy presented a high antibody production level while the homologous strategy immunized group showed predominantly a cellular immune response with parasite load reduction. The pcDNA-LiP0 immunized group showed increased expression ratio of IFN-γ/IL-10 and IFN-γ/TGF-β in the lymph nodes before challenge. Two months after infection hamsters immunized with the empty plasmid presented a pro-inflammatory immune response in the early stages of infection with increased expression ratio of IFN-γ/IL-10 and IFN-γ/TGF-β, whereas hamsters immunized with pcDNA-HIS presented an increase only in the ratio IFN-γ/ TGF-β. On the other hand, hamsters immunized with LiP0 did not present any increase in the IFN-γ/TGF-β and IFN-γ/IL-10 ratio independently of the immunization strategy used. Conversely, five months after infection, hamsters immunized with HIS maintained a pro-inflammatory immune response (ratio IFN-γ/ IL-10) while pcDNA-LiP0 immunized hamsters continued showing a balanced cytokine profile of pro and anti-inflammatory cytokines. Moreover we observed a significant reduction in parasite load in the spleen, liver and lymph node in this group compared with controls.

Conclusions/Significance

Our results suggest that vaccination with L. infantum LiP0 antigen administered in a DNA formulation could be considered a potential component in a vaccine formulation against visceral leishmaniasis.     

Molecular Epidemiology of Amoebiasis: A Cross-Sectional Study among North East Indian Population

Background

Epidemiological studies carried out using culture or microscopy in most of the amoebiasis endemic developing countries, yielded confusing results since none of these could differentiate the pathogenic Entamoeba histolytica from the non-pathogenic Entamoeba dispar and Entamoeba moshkovskii. The Northeastern part of India is a hot spot of infection since the climatic conditions are most conducive for the infection and so far no systemic study has been carried out in this region.

Methodology/Principal Findings

Following a cross-sectional study designed during the period 2011–2014, a total of 1260 fecal samples collected from the Northeast Indian population were subjected to microscopy, fecal culture and a sensitive and specific DNA dot blot screening assay developed in our laboratory targeting the Entamoeba spp. Further species discrimination using PCR assay performed in microscopy, culture and DNA dot blot screening positive samples showed E. histolytica an overall prevalence rate of 11.1%, 8.0% and 13.7% respectively. In addition, infection rates of nonpathogenic E. dispar and E. moshkovskii were 11.8% (95% CI = 10.2, 13.8) and 7.8% (95% CI = 6.4, 9.4) respectively. The spatial distributions of infection were 18.2% (107/588) of Assam, 11.7% (23/197) of Manipur, 10.2% (21/207) of Meghalaya, and 8.2% (22/268) of Tripura states. Association study of the disease with demographic features suggested poor living condition (OR = 3.21; 95% CI = 1.83, 5.63), previous history of infection in family member (OR = 3.18; 95% CI = 2.09, 4.82) and unhygienic toilet facility (OR = 1.79; 95% CI = 1.28, 2.49) as significant risk factors for amoebiasis. Children in age group <15 yr, participants having lower levels of education, and daily laborers exhibited a higher infection rate.

Conclusions/Significance

Despite the importance of molecular diagnosis of amoebiasis, molecular epidemiological data based on a large sample size from endemic countries are rarely reported in the literature. Improved and faster method of diagnosis employed here to dissect out the pathogenic from the nonpathogenic species would help the clinicians to prescribe the appropriate anti-amoebic drug.     

Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess

Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses.     

Entamoeba histolytica: Expression and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain

We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG’s anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh.     

A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

Investigations of human parasitic diseases depend on the availability of appropriate in vivo animal models and ex vivo experimental systems, and are particularly difficult for pathogens whose exclusive natural hosts are humans, such as Entamoeba histolytica, the protozoan parasite responsible for amoebiasis. This common infectious human disease affects the intestine and liver. In the liver sinusoids E. histolytica crosses the endothelium and penetrates into the parenchyma, with the concomitant initiation of inflammatory foci and subsequent abscess formation. Studying factors responsible for human liver infection is hampered by the complexity of the hepatic environment and by the restrictions inherent to the use of human samples. Therefore, we built a human 3D-liver in vitro model composed of cultured liver sinusoidal endothelial cells and hepatocytes in a 3D collagen-I matrix sandwich. We determined the presence of important hepatic markers and demonstrated that the cell layers function as a biological barrier. E. histolytica invasion was assessed using wild-type strains and amoebae with altered virulence or different adhesive properties. We showed for the first time the dependence of endothelium crossing upon amoebic Gal/GalNAc lectin. The 3D-liver model enabled the molecular analysis of human cell responses, suggesting for the first time a crucial role of human galectins in parasite adhesion to the endothelial cells, which was confirmed by siRNA knockdown of galectin-1. Levels of several pro-inflammatory cytokines, including galectin-1 and -3, were highly increased upon contact of E. histolytica with the 3D-liver model. The presence of galectin-1 and -3 in the extracellular medium stimulated pro-inflammatory cytokine release, suggesting a further role for human galectins in the onset of the hepatic inflammatory response. These new findings are relevant for a better understanding of human liver infection by E. histolytica.     

Protection against murine intestinal amoebiasis induced by oral immunization with the 29 kDa antigen of Entamoeba histolytica and cholera toxin

Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29 kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29-CTxB fusion retained the critical ability to bind ganglioside GM₁, as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29-CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis.     

Natural Killer T Cells Activated by a Lipopeptidophosphoglycan from Entamoeba histolytica Are Critically Important To Control Amebic Liver Abscess

The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jα18^-/- mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using α-galactosylceramide (α-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d^−/− mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-γ but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28∶0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-γ production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to α-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess.     

Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica

Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC₅₀ was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca²⁺ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.     

Expression of Inducible Nitric Oxide Synthase mRNA and Nitric Oxide Production During the Development of Liver Abscess in Hamster Inoculated with Entamoeba histolytica

The present study analyzed iNOS and eNOS mRNA expression and NO production during development of hepatic abscess caused by Entamoeba histolytica trophozoites. One 374-bp sequence, which displayed 88% identity to mammalian iNOS protein, was isolated from LPS-stimulated peritoneal hamster macrophages. A separate 365-bp cDNA sequence showed 99% identity with eNOS protein. iNOS mRNA was detected in hamsters during formation of amoebic liver abscesses, but not in control hamsters. eNOS mRNA expression was not modified. Serum nitrite concentration in hamsters infected with E. histolytica was 33 ± 6 μM, in control hamsters was 20 ± 3 μM. The study shows that iNOS mRNA expression and NO production are induced by E. histolytica trophozoites during amoebic liver abscess formation. However, in spite of iNOS mRNA expression and NO production, E. histolytica trophozoites induced liver abscess formation in hamster.     

Effect of the sesquiterpene lactone incomptine A in the energy metabolism of Entamoeba histolytica

Entamoeba histolytica is the causative agent of human amoebiasis, which mainly affects developing countries. Although several drugs are effective against E. histolytica trophozoites, the control of amoebiasis requires the development of new and better alternative therapies. Medicinal plants have been the source of new molecules with remarkable antiprotozoal activity. Incomptine A isolated from Decachaeta incompta leaves, is a sesquiterpene lactone of the heliangolide type which has the major in vitro activity against E. histolytica trophozoites. However the molecular mechanisms involved in its antiprotozoal activity are still unknown. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that 21 E. histolytica proteins were differentially expressed in response to incomptine A treatment. Notably, three glycolytic enzymes, namely enolase, pyruvate:ferredoxin oxidoreductase and fructose-1,6-biphosphate aldolase, were down-regulated. Moreover, ultrastructural analysis of trophozoites through electronic microscopy showed an increased number of glycogen granules. Taken together, our data suggested that incomptine A could affect E. histolytica growth through alteration of its energy metabolism.     

Differential expression of surface glycoconjugates on Entamoeba histolytica and Entamoeba dispar

The human large intestine can harbor two morphologically similar amoebae; the invasive Entamoeba histolytica and the non-invasive Entamoeba dispar. Whereas E. histolytica can produce intestinal and extra-intestinal lesions, E. dispar is present in non-symptomatic carriers. Although biochemical, genetic and proteomic studies have identified clear differences between these Entamoebae, it has become clear that several molecules, once assumed to be involved in tissue destruction, exist in both the virulent and the avirulent species. As surface molecules may play a role in invasion and could therefore determine which amoebae are invasive, we analyzed the glycoconjugate composition of E. histolytica and E. dispar using lectins. There was a significant difference between E. histolytica and E. dispar in the expression of glycoconjugates containing d-mannose and N-acetyl-α-d-galactosamine residues, but not between virulent and avirulent strains of E. histolytica. N-glycoconjugates with terminal α (1–3)-linked mannose residues participate in the adhesion and subsequent cytotoxicity of E. histolytica to cultured hamster hepatocytes. One of them probably is the Gal/GalNAc lectin.     

SNAP-Tag Technology Optimized for Use in Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite responsible for invasive intestinal and extraintestinal amebiasis. The pathology of amebiasis is still poorly understood, which can be largely attributed to lack of molecular tools. Here we present the optimization of SNAP-tag technology via codon optimization specific for E. histolytica. The resultant SNAP protein is highly expressed in amebic trophozoites, and shows proper localization when tagged with an endoplasmic reticulum retention signal. We further demonstrate the capabilities of this system using super resolution microscopy, done for the first time in E. histolytica.     

Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis

Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30 μg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings.     

Molecular Analysis of the Gal/GalNAc Adhesin of Entamoeba histolytica1

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactosc/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E. coli, retained at least some of its native conformation.     

Initiation of inflammation and cell death during liver abscess formation by Entamoeba histolytica depends on activity of the galactose/N-acetyl-D-galactosamine lectin

The parasite Entamoeba histolytica colonizes the human intestine causing amoebic colitis and disseminates through the vascular route to form liver abscesses. The Gal/GalNAc lectin is an adhesion protein complex which sustains tissue invasion by E. histolytica. Disruption of the Gal/GalNAc lectin function in engineered parasites (HGL-2 trophozoites) changed the pathophysiology of hamster liver abscess formation. HGL-2 trophozoites produced numerous small inflammatory foci located in the vicinity of blood vessels. The low penetration of HGL-2 trophozoites into hepatic tissue was shown to be associated with weak attraction of neutrophils and macrophages to the infiltrated areas and absence of pro-inflammatory tumour necrosis factor, in contrast to wild type or control vector infections. The low host inflammatory response in HGL-2 infections correlated with a delay in apoptosis of hepatic cells, whereas apoptosis of endothelial cells was not detected. Triggering of apoptosis in both host cell types most likely has a central role in modulating inflammation, a major landmark in hepatic amoebiasis. These data highlight the key role of the Gal/GalNAc lectin in initiation of E. histolytica hepatic infection.     

Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterised. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analysed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant-negative mutants. While TMK39 co-localised with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amoebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilised for non-redundant functions by the parasite.