Multilocus sequence typing system (MLST) reveals a significant association of Entamoeba histolytica genetic patterns with disease outcome

The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needed to be explored. Proper identification and genetic characterization of Entamoeba histolytica clinical isolates is an effective tool for exploring this relation. Along with conventional polymorphic marker (Chitinase), tRNA linked short tandem repeat (STR) loci has been employed as multilocus genotyping tool due to its better resolution and evolutionary significance. Some common as well as exclusive repeat patterns showing significant relation with disease outcome have been identified. Phylogenetic analysis revealed that repeat patterns exclusively found in asymptomatic and amoebic liver abscess derived isolates are placed in a common lineage and has similar association pattern with the disease outcome. Assumption can be made that isolates of E. histolytica remaining asymptomatic is genetically closer siblings of those causing liver abscess rather than the diarrheal isolates.     

The MAK16 Gene of Entamoeba histolytica and Its Identification in Isolates from Patients

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher''s exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient''s symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.     

Characterization of a novel Entamoeba histolytica strain from Burkina Faso, Africa, possessing a unique hexokinase-2 gene

An Entamoeba histolytica strain (BF-841 cl1) that originated from Burkina Faso, Africa presented with novel, polymorphic genotypes of the serine-rich E. histolytica protein and the anodic hexokinase-2 (HXK-2) isoenzyme band, which showed less electrophoretic mobility than that of an E. histolytica reference strain [HM-1:IMSS cl6 (zymodeme (Z)-II)] by starch gel electrophoresis and isoelectric focusing (IEF). The HXK-2 gene of BF-841 cl1 had amino acid variations at four positions compared to the sequence of HM-1: IMSS cl6. These variations were absent from the sequences of four other E. histolytica strains with different zymodemes [KU27 (Z-II), SAW1627 (Z-IIα-), SAW755CR clB (Z-XIV), and KU2 (Z-XIX)]. The results of IEF showed no difference in the substrate specificity of HXK (HXK-1 and HXK-2) between BF-841 cl1 and the three reference E. histolytica strains (HM-1:IMSS cl6, SAW755 clB, and KU27). It was also confirmed that BF-841 cl1 was able to form liver abscesses in Syrian hamsters.     

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.     

TNFα-Mediated Liver Destruction by Kupffer Cells and Ly6Chi Monocytes during Entamoeba histolytica Infection

Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis.     

Multilocus sequence typing (MLST) of Entamoeba histolytica identifies kerp2 as a genetic marker associated with disease outcomes

Amoebiasis caused by protozoan parasite Entamoeba histolytica has diverse infection outcomes. The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needs to be explored. Genome information of infecting strains from endemic areas throughout the world is essential to explore this relation. Comparative genetics between E. histolytica populations from different disease outcomes have been studied to identify potential genetic markers having single nucleotide polymorphisms (SNPs) significantly associated with specific clinical outcome. Coding and non-coding regions have significantly different rates of polymorphism. Non-synonymous base substitutions were significantly more frequent than synonymous within coding loci. Both synonymous and non-synonymous SNPs within lysine- and glutamic acid rich protein 2 (kerp2) locus were significantly associated with disease outcomes. An incomplete linkage disequilibrium (LD) value with potential recombination events and significant population differentiation (F_ST) value have also been identified at kerp2 locus within the study population. Presence of disease specific SNPs, potential recombination events, and significant F_ST value at kerp2 locus indicate that kerp2 gene and its gene product are under constant selection pressure exerted by host on parasite and could also be a potential determinant of disease outcome of E. histolytica infection. Furthermore, E. histolytica isolated from asymptomatic carriers are phylogenetically closer to those causing liver abscess in human and exhibit potential inter-population recombination among them. Individuals with persistent asymptomatic E. histolytica infection may be under high risk of developing amoebic liver abscess formation in future and detailed investigation of asymptomatic individuals from endemic areas should be always required.     

Entamoeba histolytica: Virulence enhancement of isoenzyme-stable parasites

Pathogenic Entamoeba histolytica isolated from patients with clinical amoebiasis can be differentiated from nonpathogenic E. histolytica obtained from asymptomatic carriers on the basis of the electrophoretic pattern of their isoenzymes. Virulence of different strains of axenically grown trophozoites of Entamoeba histolytica, as determined by various laboratory tests, such as damage to tissue culture monolayers, or their ability to cause an hepatic abscess in a hamster, are known to vary considerably. Reassociation of trophozoites of strain HK-9 with certain Escherichia coli strains for short periods of time markedly augmented their virulence, as tested by the above-mentioned methods. The bacterial association, however, did not cause any change in the electrophoretic pattern of amoebic isoenzymes (zymodeme).     

Identification of Significant Variation in the Composition of Lipophosphoglycan-like Molecules of E. histolytica and E. dispar1

The lipophosphoglycan-like (LPG-like) molecules of E. histolytica virulent strains are clearly distinct from those of the avirulent E. histolytica and E. dispar strains. Abundant ‘LPG’ levels are apparently limited to virulent strains, while lipophosphopeptidoglycans (‘LPPG's) are common to both virulent and avirulent strains of E. histolytica and E. dispar. It is therefore conceivable that ‘LPPG’ performs a function that is essential to survival within the host, while the ‘LPG’ performs a more specific function related to virulence.     

The Jacob2 Lectin of the Entamoeba histolytica Cyst Wall Binds Chitin and Is Polymorphic

Background

The infectious and diagnostic form of Entamoeba histolytica (Eh), cause of amebic dysentery and liver abscess, is the quadranucleate cyst. The cyst wall of Entamoeba invadens (Ei), a model for Eh, is composed of chitin fibrils and three sets of chitin-binding lectins that cross-link chitin fibrils (multivalent Jacob lectins), self-aggregate (Jessie lectins), and remodel chitin (chitinase). The goal here was to determine how well the Ei model applies to Entamoeba cysts from humans.

Methods/Results

An Eh Jacob lectin (EhJacob2) has three predicted chitin-binding domains surrounding a large, Ser-rich spacer. Recombinant EhJacob2 made in transfected Eh trophozoites binds to particulate chitin. Sequences of PCR products using primers flanking the highly polymorphic spacer of EhJacob2 may be used to distinguish Entamoeba isolates. Antibodies to the EhJacob2, EhJessie3, and chitinase each recognize cyst walls of clinical isolates of Entamoeba. While numerous sera from patients with amebic intestinal infections and liver abscess recognize recombinant EhJacob1 and EhJessie3 lectins, few of these sera recognize recombinant EhJacob2.

Conclusions/Significance

The EhJacob2 lectin binds chitin and is polymorphic, and Jacob2, Jessie3, and chitinase are present in cyst walls of clinical isolates of Entamoeba. These results suggest there are substantial similarities between cysts of the human pathogen (Eh) and the in vitro model (Ei), even though there are quantitative and qualitative differences in their chitin-binding lectins.     

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model

Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.     

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.     

Proton-transporting and calcium ion-transporting ATPases of Entamoeba histolytica

Entamoeba histolytica is unique among human protozoan parasites in its ability to phagocytose bacteria and red blood cells and to destroy host epithelial cells via a contact-mediated cytolysis. Antagonists of vacuolar acidification and calcium ion-transport inhibit amebic lysis of epithelial cells in vitro. In this review, John Samuelson, Nnecka Azikiwe and Pei-Shen Shen describe the primary structures of E. histolytica V-type proton-transporting ATPase (V-ATPase) and P-type calcium-transporting ATPase, which probably mediate amebic vacuolar acidification and calcium ion sequestration, respectively. The function of the amebic V-ATPase is discussed with regard to pinocytosis, bacterial killing and host cell lysis. Phylogenetic trees incorporating the sequences of the proteolipid and catalytic peptides of the amebic V-ATPase are described. The amebic P-type calcium-transporting ATPase is compared to those of the red blood cell plasma membrane and yeast vacuolar membrane. Finally, the potential of the V-ATPase proteolipid and P-type calcium ion-transporting ATPase as targets for anti-amebic antibodies or for bacteria loaded with recombinant toxins is explored.     

ROP18 and ROP5 alleles combinations are related with virulence of T. gondii isolates from Argentina

The allelic combination of ROP18/ROP5 genes of Toxoplasma gondii has been shown to be highly predictive of mouse virulence in canonical isolates and strains. The aims of this study were to analyze the alleles present in the ROP18/ROP5 genes from T. gondii isolates obtained in Argentina, to associate the results with the virulence registered in mouse model, and to compare with other isolates and reference strains using a phylogenetic network. Fourteen T. gondii isolates from Argentina were analyzed by nPCR-RFLP for ROP18/ROP5. Phylogenetic network analysis was inferred using the ToxoDB genotypes and the ROPs molecular markers. All isolates and reference strains were categorized as lethal or non-lethal. As results, combinations 2/2, 3/3 and 4/3 for ROP18/ROP5 were detected in 12 isolates, whereas only alleles 1 and 2 of ROP5 were detected in 2 isolates. The majority of the isolates had a mouse virulence matching to that predicted by the ROP18/ROP5 allele combination. The 3 isolates that differed from the expected virulence presented non-clonal genotypes. ROPs incorporation increased the accuracy of the phylogenetic network relations among the T. gondii samples, prevailing the clustering according to regions. Our results indicate a predominance of type 3 allele in both ROP18 and ROP5 markers and an association of allelic profiles 3/3 and 4/3 of non-clonal genotypes from Argentina, both with virulent and avirulent profiles in mice.     

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?     

Different Clinical Outcomes of Entamoeba histolytica in Malaysia: Does Genetic Diversity Exist?

The present study was conducted to investigate the clinical outcomes of Entamoeba histolytica infection in symptomatic and asymptomatic Orang Asli (aborigine) communities in Malaysia. Examination was performed on 500 stool samples obtained from Orang Asli communities in 3 different states using formalin-ether concentration, trichrome staining, and single-round PCR techniques. Out of 500 stool samples, single infection of E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii was identified in 3.2%, 13.4%, and 1%, respectively. In addition, 10 samples had mixed infections with E. histolytica and E. dispar. Six samples containing E. dispar were also positive for E. moshkovskii, and only 2 samples had E. histolytica in association with E. dispar and E. moshkovskii. Seventeen E. histolytica-positive samples were from symptomatic subjects, whereas the remaining 11 samples came from asymptomatic subjects. These findings suggest a predominant distribution of pathogenic potential of E. histolytica strains in this community. Therefore, further studies on genotyping of E. histolytica is required, to find out association between E. histolytica genotype and the outcome of the infection.     

Isolation and Molecular Characterization of Entamoeba nuttalli Strains Showing Novel Isoenzyme Patterns from Wild Toque Macaques in Sri Lanka

We have proposed the revival of the name Entamoeba nuttalli for a virulent ameba strain, P19‐061405, from a rhesus macaque and located it phylogenetically between E. histolytica and E. dispar. As E. nuttalli was originally described for an ameba found in a toque macaque in Sri Lanka, the prevalence and characteristics of Entamoeba species in wild toque macaques were examined. PCR analysis of 227 stool samples from six locations showed positive rates for E. nuttalli, E. dispar, and E. histolytica of 18.5%, 0.4%, and 0%, respectively. Fifteen E. nuttalli strains were cultured successfully from five locations. The 18S ribosomal RNA gene showed only three nucleotide differences in comparison with P19‐061405 strain. In isoenzyme analysis, the pattern of hexokinase in Sri Lankan strains was different from that of P19‐061405 strains and the difference was confirmed by analysis of the genes. Hepatic inoculation of one of the Sri Lankan E. nuttalli strains in hamsters resulted in amebic abscess formation and body weight loss. These results demonstrate that E. nuttalli is prevalent in wild toque macaques and that several characteristics of the strains are unique. We conclude that use of the name E. nuttalli is appropriate for the new Entamoeba species found in nonhuman primates.     

Pathogenic and Non-pathogenic Entamoeba: Pore Formation and Hemolytic Activity1

Pore-forming activity in planar lipid bilayers and liposomes of extracts from differentially pathogenic Entamoeba and the-capacity of trophozoites and subcellular fractions to lyse human red blood cells (hrbc) were investigated. In all amebas studied, the two activities paralleled each other. They were high in E. histolytica irrespective of the virulence of the particular strain, but low in non-pathogenic E. histolytica-like amebas of human origin as well as in E. invadens, which is pathogenic for reptiles, and in E. moshkovskii isolated from sewage. We conclude that the capacities to insert pores and to lyse are not sufficient for virulence although they may be necessary. The subcellular distribution of the hemolytic activity of E. histolytica and its sensitivity to a variety of inhibitors and activators differ from those of other known amebic cytotoxic activities including pore formation. Therefore, there may be an additional constituent of E. histolytica involved in the cytotoxicity of the parasite.     

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.