Comparative Genomic Hybridizations ofEntamoebaStrains Reveal Unique Genetic Fingerprints That Correlate with Virulence

Abstract

Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in ϳ10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.Entamoeba histolytica causes amebic colitis and liver abscesses. Worldwide, 50 million people have invasive E. histolytica, and 100,000 die each year, making it the second most common cause of parasitic death in humans (71). In Dhaka, Bangladesh, where diarrheal diseases are the leading cause of death in children less than 6 years old, ϳ50% of children have serological evidence of exposure to E. histolytica by age 5 (34, 35). Infected children suffer from significant morbidity with malnourishment and growth delays. A number of interesting epidemiologic trends have been identified for amebic disease. The data, although limited, indicate that a minority (ϳ10%) of individuals who become infected with E. histolytica progress to clinically overt disease; others remain asymptomatically colonized (29). In individuals with invasive disease, occurrence of amebic liver abscess (ALA) is 5 to 50 times less common than diarrhea (65). Geographically variable disease predilections have been observed, and invasive disease predominantly affects men (1, 2, 11, 61).The extent of genetic diversity among E. histolytica clinical isolates is unclear. Studies have analyzed a small number of highly repetitive and polymorphic genetic loci by techniques, such as randomly amplified polymorphic DNA (RAPD), RNA arbitrarily primed PCR, and restriction fragment length polymorphism (RFLP), to conclude that there is significant genetic diversity (7,18,23,31,33,64,72,73). Contradictory data were obtained by two studies; in one study, the lectin gene was sequenced, and in the other study, the intergenic region between the superoxide dismutase gene and the actin 3 gene was analyzed (9, 30). These studies found minimal genetic diversi...

Unusually Low Levels of Genetic Variation among Giardia lamblia Isolates

“…It is still unclear whether this is due to host or parasite effects or hostparasite interactions (reviewed in reference 42). For instance, studies of virulent and avirulent strains of E. histolytica have shown that the least virulent strain exhibited the most divergence, thus potentially linking genotypic variants to disease outcomes (45). Efforts to relate genotypic markers with various aspects of the infection could lead to promising means for an effective fight against the disease.…”

Laboratory Diagnostic Techniques for Entamoeba Species

“…A microarray-based genotyping assay (comparative genomic hybridization) technique was later developed using sequenced genomic DNA clones from E. histolytica (HM-1:IMSS). This was the first genome-wide analysis of Entamoeba strains, and it revealed that this technology can be used to distinguish E. histolytica from E. dispar, to identify genes restricted to virulent strains, and to find potential genotypic-phenotypic associations (164).…”

Identification of an Entamoeba histolytica Serine-, Threonine-, and Isoleucine-Rich Protein with Roles in Adhesion and Cytotoxicity

“…E. histolytica Rahman was recently shown to have decreased levels of cytotoxicity, decreased ability to phagocytose erythrocytes, decreased ability to elicit colitis in human colonic xenografts, and decreased resistance to oxidative stress (19). Interestingly, relatively few genetic differences were noted between E. histolytica HM-1:IMSS and E. histolytica Rahman based on comparative genomic hybridization analysis (45). However, a number of genes solely expressed in virulent strains and species of Entamoeba have recently been identified (18,24,33 (24).…”