Entamoeba histolytica and Entamoeba dispar, mechanisms of adherence and implications for virulence

grantor: University of TorontoIn 1925, Emile Brumpt had proposed the existence of two morphologically identical species, 'Entamoeba histolytica' (a human pathogen) and 'Entamoeba dispar' (a non-pathogenic human commensal). Recent molecular and genetic evidence has supported the two-species concept. The pathogen relies on the galactose/'N'-Acetyl-D-galactosamine lectin for adherence that leads to contact-dependent cytolysis of host target cells, a key step in amebic virulence. Work presented here explores the structure, function, and expression of the lectin from 'E. histolytica' and 'E. dispar'. We hypothesized that molecular differences between the pathogenic and non-pathogenic lectin homologues would be instructive in understanding the differential role of each in colonization ('E. dispar') versus invasion ('E. histolytica'). To achieve this, a gene encoding the lectin in 'E. dispar' ('dhgl2 ') was cloned and sequenced. Homologous lectin message and protein levels in 'E. histolytica' and 'E. dispar' under axenic culture conditions were compared. A heterologous expression system (in COS 7 cells) was constructed to test anti-lectin monoclonal antibody reactivity for 'E. histolytica' and 'E. dispar' lectin homologues. Lectin protein, which was generated in COS7 cells, was used for enzyme-linked immunosorbent assays in which antisera from patients infected with pathogen or non-pathogen were tested. Further experiments were also performed in order to localize the carbohydrate recognition domain(s) of the amebic lectin using a newly developed 'in vitro' assay. Taken together, evidence is presented supporting the view that 'E. histolytica' and 'E. dispar' possess highly conserved lectin molecules that are differentially expressed in pathogen versus non-pathogen. Additional experiments were performed to isolate and characterize a ß2 integrin-like molecule expressed on the amebic surface. Data supporting the identification of a distinct ß2 integrin on the surface of 'E. histolytica' is presented. A putative role for this molecule in amebic virulence is discussed. Finally, epidemiological questions regarding 'E. histolytica' and ' E. dispar' infections were revisited, now relying on stool antigen immunoassays. These immunoassays, which are able to distinguish 'E. histolytica' from 'E. dispar', demonstrated that the overwhelming majority of infections in a non-endemic setting are caused by the non-pathogen. These results have broad clinical implications since specific molecular diagnosis would obviate the need for unnecessary chemotherapy of ' E. dispar', infections, with its attendant costs, risk of side effects, and danger of drug resistance.Ph.D