Remarkable genetic polymorphism among Entamoeba histolytica isolates from a limited geographic area

Abstract

In order to understand genetic polymorphisms among Entamoeba histolytica strains in a limited geographic area and among restricted social populations, we studied nucleotide polymorphism in DNA regions that do not encode proteins (locus 1-2 and locus 5-6) and in genes coding for chitinase and for serine-rich E. histolytica protein. Thirty E. histolytica isolates from domestically infected Japanese amebiasis patients (male homosexuals and residents in institutions for the mentally handicapped) and four reference strains were examined. PCR revealed remarkable polymorphisms in both the number and size of the PCR fragments containing these loci. Polymorphisms in lengths, types, and numbers of internal repeat units were observed in locus 1-2 and the repeat-containing region of serine-rich E. histolytica protein among the Japanese isolates. In contrast, polymorphism at locus 5-6 was observed almost exclusively in the number of repeats of a 16-nucleotide unit. The repeat-containing region of chitinase appeared to be the least polymorphic among the four loci with a single dominant genotype representing 66% (20 out of 30) of all of the isolates. Isolates obtained from male homosexuals showed a more complex genetic polymorphism than those from residents in institutions. Considering all four polymorphic loci together, all 19 Japanese isolates from male homosexuals were distinct. In contrast, all isolates obtained from mass-infection cases at a single institution had an identical genotype, suggesting that these cases were caused by a single E. histolytica strain. No significant correlation was found between genotypes and zymodemes or between genotypes and clinical presentations, e.g., colitis or liver abscess. Certain genotypes were observed with higher frequencies in male homosexuals or residents of institutions. These data indicate that genotyping of the E. histolytica isolates by using these four polymorphic loci could serve as a tool to fingerprint individual isolates. We propose that genotyping of ameba isolates should help to determine geographic origins of isolates and routes of transmission.

FIG. 1.

Agarose gel electrophoresis of locus 1-2 (A), locus 5-6 (B), chitinase (C), and SREHP (D) from representative E. histolytica isolates. Only results for representative isolates that belong to each genotype (shown in parentheses) are shown. The individual genotype is designated for each polymorphic DNA fragment. Bands with asterisks are irrelevant PCR fragments (verified by sequencing) observed only in KU1, KU5, KH5, KH9, and KH15, for unknown reasons.

FIG. 2.

Schematic representation of polymorphism in locus 1-2 among the Japanese isolates and reference strains. The numbers shown correspond to nucleotide numbers of locus 1-2 of HM-1:IMSS cl9 (GenBank/EMBL/DDBJ accession number AF276055). Sequences prior to nucleotide 30 are not shown because these nucleotides overlap the PCR primer. Gaps are introduced to optimize alignments. Conserved regions are highlighted with gray rectangles. Names of reference strains previously reported are underlined, and those analyzed in this study are double underlined. Previously unidentified repeats units are also included in this and following figures. A T-to-C nucleotide substitution is indicated by an arrow. An asterisk next to an isolate designation indicates that more information can be found in reference .

FIG. 3.

Schematic representation of polymorphism in locus 5-6 among the Japanese isolates and reference strains. The numbers shown correspond to nucleotide numbers of locus 5-6 of HM-1:IMSS cl9 (GenBank/EMBL/DDBJ accession number AF276060). Sequences prior to nucleotide 52 are not shown for the reason described in the legend to Fig. 2. The first GTATGTTTCTAT and the second GATTTTAT repeats described in text are marked with a thick arrow and an arrowhead, respectively. Variant forms of A5 and C, in which two and three nucleotides were replaced (indicated by thin arrows), are designated A5v and Cv, respectively. An asterisk next to an isolate designation indicates that more information can be found in reference .

FIG. 4.

Schematic representation of polymorphism in the repeat-containing region of the chitinase gene among the Japanese isolates and reference strains. The numbers shown in this figure correspond to nucleotide numbers of chitinase of HM-1:IMSS (GenBank/EMBL/DDBJ accession number U78319). Nucleotide and deduced amino acid sequences of heptapeptide repeats are also shown. An asterisk next to an isolate designation indicates that more information can be found in reference .

FIG. 5.

Schematic representation of polymorphism in the repeat-containing region of the SREHP gene among the Japanese isolates and reference strains. The numbers shown in this figure correspond to nucleotide numbers of SREHP of HM-1:IMSS (GenBank/EMBL/DDBJ accession number M80910). Nucleotide and deduced amino acid sequences of tetra-, octa-, and nonapeptide repeats are also shown. An asterisk next to an isolate designation indicates that more information can be found in reference .