Molecular epidemiology of Entamoeba spp.: evidence of a bottleneck (Demographic sweep) and transcontinental spread of diploid parasites

Abstract

Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico and India. Entamoeba dispar is morphologically identical but is not associated with disease. Here we determined the ploidy of E. histolytica and developed PCR-based methods for distinguishing field isolates of E. histolytica or E. dispar. Fluorescence in situ hybridization showed that E. histolytica trophozoites are diploid for five "single-copy" probes tested. Intergenic sequences between superoxide dismutase and actin 3 genes of clinical isolates of E. histolytica from the New and Old Worlds were identical, as were those of E. dispar. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast, E. histolytica and E. dispar genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic. chitinase alleles suggested that the early axenized strains of E. histolytica, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similar E. dispar parasites can be identified in both the New and Old Worlds. Ser-rich protein alleles, which suggested the presence of the HM-1 strain in Mexico City, included some E. histolytica genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection at chitinase and Ser-rich protein loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of E. histolytica and E. dispar.

FIG. 1

Fluorescence micrograph of a colchicine-treated E. histolytica trophozoite stained with propidium iodide. Condensed chromosomes number 28, which is twice the number of chromosomes (14) identified on pulsed-field gels.

FIG. 2

Confocal micrographs of FISH of E. histolytica with single-copy genes encoding chitinase (A), pyruvate:ferredoxin oxidoreductase (B), nicotinamide nucleotide transhydrogenase (C), plasma membrane calcium-transporting ATPase (D), and cysteine proteinase 5 (E), which bind twice (yellow) to each nucleus. In contrast, FISH with p-glycoprotein genes (F), which are in multiple copies, shows numerous spots.

FIG. 3

Alignment of the intergenic sequences between superoxide dismutase and actin 3 genes of E. histolytica and E. dispar. Periods indicate identity of E. dispar with E. histolytica, and dashes indicate gaps. Unshaded boxes indicate superoxide dismutase and actin gene coding regions, and the dotted box indicates the TATA-like sequence upstream of the start codon of actin 3, while arrows indicate locations of PCR primers. The E. histolytica sequence was identical to that reported previously with GenBank accession no. X70852 (40).

FIG. 4

Patterns of E. histolytica and E. dispar chitinase repeats. (A) Building blocks of chitinase repeats. Each 21-nucleotide sequence, which encodes a unique heptapeptide repeat, is given a number. The numbers are marked to indicate silent nucleotide changes, which are underlined. (B) Patterns of chitinase repeats. Shown are the chitinase PCR products from clinical isolates of E. histolytica (Eh) and E. dispar (Ed) in Mexico City (HI), San Diego (SD), and Calcutta (K). Each PCR product is coded using the numbers in panel A, while gaps are indicated by dashes.

FIG. 5

Patterns of E. histolytica and E. dispar Ser-rich protein repeats. (A) Building blocks of Ser-rich protein repeats. Each 24-nucleotide sequence, which encodes a unique octapeptide repeat, or 12-nucleotide sequence, which encodes a unique tetrapeptide repeat, is given a number. The numbers are marked to indicate silent nucleotide changes, which are underlined. (B) Patterns of Ser-rich protein repeats. Shown are Ser-rich protein PCR products from clinical isolates of E. histolytica (Eh) and E. dispar (Ed) in Mexico City (HI) and Calcutta (K). Each PCR product is coded using the numbers in panel A, while gaps are indicated by dashes.