Entamoeba histolytica: identification of a distinct beta2 integrin-like molecule with a potential role in cellular adherence

Entamoeba histolytica infection causes dysentery, intestinal colitis, and hepatic abscess in an estimated 50 million people worldwide. Attachment of E. histolytica trophozoites to intestinal epithelium and vascular endothelium during liver metastasis results in an inflammatory process. We report the identification of a distinct amebic beta2 integrin (CD18)-like molecule which affords adherence to TNF-alpha-activated endothelial cells. Data from flow cytometry and indirect immunofluorescence assays suggest the amebic beta2 integrin was localized to focal adhesion plates and was present in both E. histolytica and Entamoeba dispar. The amebic beta2 integrin appeared to be distinct from the amebic Gal/GalNAc lectin based on recombinant expression, amebic colocalization, and ELISA studies. Trophozoite adherence to endothelial cells expressing ICAM-1 (CD54) following activation with TNF-alpha or ICAM-1-transfected CHO cells was specifically inhibited with anti-CD18 or anti-CD54 MAbs. In summary, evidence in support of a distinct beta2 integrin-like molecule participating in amebic adherence to TNF-alpha-activated endothelial cells expressing ICAM-1 is presented. The presence of integrin-dependent binding may allow trophozoites to opportunistically adhere to activated intestinal epithelium or vascular endothelium expressing ICAM-1 during amebic colitis or hepatic abscess.     

Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family

Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)). Expression of the carboxy-truncation of PATMK(delta932) also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.     

Entamoeba histolytica: the serine-rich gene polymorphism-based genetic variability of clinical isolates from Georgia

It is generally accepted that a majority of individuals infected by Entamoeba histolytica do not develop symptomatic disease. However, the parasite and the host factors contributing to the development of the disease, remain undetermined. It is also unclear why certain individuals develop extra-intestinal amebiasis without exhibiting apparent intestinal symptoms. An outbreak of amebic liver abscess in Tbilisi, Georgia in 1998-1999 suggested that the causative E. histolytica strain had an unusual propensity for extra-intestinal spread. To correlate the genetic differences with pathogenic potential of the parasite, we have examined the SREHP gene polymorphisms among Georgian E. histolytica isolates. Comparison of polymorphic patterns revealed the presence of several different genotypes of E. histolytica, thus preventing an association of a single genotype with hepatic disease, but supporting the previous finding of extensive genetic diversity among E. histolytica isolates from the same geographic origin.     

Tissue invasion by Entamoeba histolytica: evidence of genetic selection and/or DNA reorganization events in organ tropism

Entamoeba histolytica infection may have various clinical manifestations. Nine out of ten E. histolytica infections remain asymptomatic, while the remainder become invasive and cause disease. The most common form of invasive infection is amebic diarrhea and colitis, whereas the most common extra-intestinal disease is amebic liver abscess. The underlying reasons for the different outcomes are unclear, but a recent study has shown that the parasite genotype is a contributor. To investigate this link further we have examined the genotypes of E. histolytica in stool- and liver abscess-derived samples from the same patients. Analysis of all 18 paired samples (16 from Bangladesh, one from the United States of America, and one from Italy) revealed that the intestinal and liver abscess amebae are genetically distinct. The results suggest either that E. histolytica subpopulations in the same infection show varying organ tropism, or that a DNA reorganization event takes place prior to or during metastasis from intestine to liver.     

Cellular and molecular mechanisms that underlie Entamoeba histolytica pathogenesis: prospects for intervention

The protozoan parasite Entamoeba histolytica is the causative agent of amoebic dysentery. It is prevalent in developing countries that cannot prevent its fecal-oral spread and ranks second in worldwide causes of morbidity by parasitic infection. Improvements in sanitation would help curb disease spread. However, a lack of significant progress in this area has resulted in the need for a better understanding of the molecular and cellular biology of pathogenesis in order to design novel methods of disease treatment and prevention. Recent insight into the cellular mechanisms regulating virulence of E. histolytica has indicated that processes such as endocytosis, secretion, host cell adhesion and encystation play major roles in the infectious process. This review focuses on components of the molecular machinery that govern these cellular processes and their role in virulence, and discusses how an understanding of this might reveal opportunities to interfere with E. histolytica infection.     

Pathogenic and non-pathogenic Entamoeba: pore formation and hemolytic activity

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.     

The lysine- and glutamic acid-rich protein KERP1 plays a role in Entamoeba histolytica liver abscess pathogenesis

The parasite Entamoeba histolytica colonizes the large bowel where it may persist as an asymptomatic luminal gut infection, which changes to virulence. Parasite invasion of the intestine leads to dysentery and spreads to the liver, where amoebae form abscesses. We took advantage of changes in virulence that occurs after long-term in vitro culture of E. histolytica strains. Using microarrays, we concluded that virulence correlates with upregulation of key genes involved in stress response, including molecular chaperones, ssp1 and peroxiredoxin; as well as the induction of unknown genes encoding lysine-rich proteins. Seven of these were retained with respect to their lysine content higher than 25%. Among them, we found KERP1, formerly identified as associated to parasite surface and involved in the parasite adherence to host cells. Experimentally induced liver abscesses, using molecular beacons and protein analysis, allowed us to draw a parallel between the intricate upregulation of kerp1 gene expression during abscess development and the increased abundance of KERP1 in virulent trophozoites. Following its characterization as a marker for the progression of infection, KERP1 was also seen to be a virulence marker as trophozoites affected in kerp1 expression by an antisense strategy were unable to form liver abscesses.     

A Detoxifying Oxygen Reductase in the Anaerobic Protozoan Entamoeba histolytica

We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ～5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.     

Genomic and proteomic approaches highlight phagocytosis of living and apoptotic human cells by the parasite Entamoeba histolytica

Phagocytosis plays a major role during the invasive process of the human intestine by the pathogenic amoeba E. histolytica. This parasite is the etiologic agent causing amoebic dysentery, a worldwide disease causing 50 million of clinical cases leading to about 100,000 deaths annually. The invasive process is characterized by a local acute inflammation and the destruction of the intestinal tissue at the invasion site. The recent sequencing of the E. histolytica genome has opened the way to large-scale approaches to study parasite virulence such as processes involved in human cell phagocytosis. In particular, two different studies have recently described the phagosome proteome, providing new insights into the process of phagocytosis by this pathogenic protozoan. It has been previously described that E. histolytica induces apoptosis and phagocytosis of the human target cells. Induction of apoptosis by the trophozoites is thought to be involved in the close regulation of the inflammatory response occurring during infection. Little is known about the molecular mechanisms responsible for induction of apoptosis or in the recognition of apoptotic cells by E. histolytica. In this review, we comment on the recent data we obtained after isolation of the early phagosomes and the identification of its associated proteins. We focus on the surface molecules potentially involved in human cell recognition. In particular, we propose several parasite molecules, potentially involved in the induction of apoptosis and/or the phagocytosis of human apoptotic cells.     

The role of extracellular cysteine proteinases in pathogenesis of Entamoeba histolytica invasion

The extracellular cysteine proteinases of Entamoeba histolytica have been implicated as important virulence factors in the pathogenesis of amebiasis and play a key role in tissue invasion and disruption of host defenses. These proteinases have attracted considerable interest as targets for novel therapeutic agents and as vaccine candidates. Here, Xuchu Que and Sharon Reed highlight some of the more recent findings, focusing in particular on functional and structural features of the extracellular cysteine proteinases of E. histolytica.     

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.     

Molecular Changes in Entamoeba histolytica in Response to Bacteria1

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.     

Pathophysiology of amoebiasis

Few organisms are more aptly named than Entamoeba histolytica, an intestinal protozoan parasite that can lyse and destroy human tissue. Within the past four years, new models of E. histolytica infection have begun to illuminate how amoebic trophozoites cause intestinal disease and liver abscess, and have expanded our understanding of the remarkable killing ability of this parasite. Here, I summarize recent work on the interactions between E. histolytica and human intestine, and between E. histolytica and hepatocytes, and discuss what these studies tell us about the role of inflammation and programmed cell death in the pathogenesis of amoebiasis.