Isolation and characterization of polymorphic DNA from Entamoeba histolytica

Abstract

An important gap in our understanding of the epidemiology of amebiasis is what determines the outcome of Entamoeba histolytica infections. To investigate the possible existence of invasive and noninvasive strains as one factor, the ability to differentiate individual isolates of E. histolytica is necessary. Two new loci containing internal repeats, locus 1-2 and locus 5-6, have been isolated. Each contains a single repeat block with two types of related direct repeats arranged in tandem. Southern blot analysis suggests that both loci are multicopy and may themselves be arranged in tandem arrays. Three other previously reported, internally repetitive loci containing at least two repeat blocks each with one or more related repeat units were also investigated. PCR was used to study polymorphism at each of these loci, which was detected to various degrees in each case. Variation was seen in the total number of bands obtained per isolate and their sizes. Nucleotide sequence comparison of loci 1-2 and 5-6 in five axenic isolates revealed differences in the number of repeat units, which correlated with the observed PCR product size variation, and in repeat sequence. Use of multiple loci collectively allowed differentiation of a majority of the 13 isolates studied, and we believe that these loci have the potential to be used as polymorphic molecular markers for investigating the epidemiology of E. histolytica and the potential existence of genetically distinct invasive and noninvasive strains.

FIG. 1

Locus 1-2. (A) Nucleotide sequence. The main block of internal tandem repeats is in boldface. Underlined regions indicate one of the two types of repeat units. (B) Schematic representation. The two types of internal tandem repeats and their arrangement with respect to each other are shown. Tandem duplications in the flanking regions are not shown. The positions of the amplification primers are indicated.

FIG. 2

Locus 5-6. (A) Nucleotide sequence. The main block of internal tandem repeats is in boldface. Underlined regions indicate one of the two types of repeat units. (B) Schematic representation. The two types of internal tandem repeats and their arrangement with respect to each other is shown. Tandem duplications in the flanking regions are not shown. The positions of the amplification primers are indicated. Two primer pairs were designed for locus 5-6 (Table 2). Amplification products generated by primers R5 and R6 were cloned, sequenced, and aligned for intrastrain nucleotide sequence comparisons (Fig. 4B), while the primer pair R5A-R6A was used for studying interstrain PCR product size polymorphisms (Fig. 3B).

FIG. 3

Polymorphic DNA analysis of Entamoeba histolytica isolates. (A) Locus 1-2. Amplification products were generated using primers R1 and R2 at an annealing temperature of 53°C. (B) Locus 5-6. Amplification products were generated using primers R5A and R6A at an annealing temperature of 56°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).

FIG. 4

Schematic representation of locus structure in five axenic isolates of E. histolytica. Variations in number, sequence, and arrangement of repeat units are shown. Gaps have been introduced to optimize alignment. (A) Locus 1-2. (B) Locus 5-6.

FIG. 5

Southern blot analysis. (A) Genomic DNA of E. histolytica isolate HM-1:IMSS digested with restriction enzymes and stained with ethidium bromide. (B) Blot of gel in panel A hybridized with a locus 1-2 specific ³²P-labeled probe. (C) Blot of gel in panel A hybridized with a locus 5-6 specific ³²P-labeled probe. Some of the faint bands seen in Fig. 5B may result from slight cross-hybridization to fragments of the abundant extrachromosomal circular DNA seen in the ethidium bromide-stained gel (Fig. 5A).

FIG. 6

Schematic representation of repeat arrangements at three other loci. (A) Locus 3-4. (B) Locus 9-4. (C) Locus 16-17. Only the major blocks of internal tandem repeats are shown. The positions of the primers for whole- and half-locus amplification are shown for all three loci.

FIG. 7

Polymorphic DNA analysis of E. histolytica isolates. (A) Locus 3-4. Amplification products were generated using primers R3 and R4 at an annealing temperature of 55°C. (B) Half-locus 3-8. Amplification products were generated using primers R3 and R8 at an annealing temperature of 50°C. (C) Half-locus 7-4. Amplification products were generated using primers R7 and R4 at an annealing temperature of 50°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).

FIG. 8

Polymorphic DNA analysis of Entamoeba histolytica isolates. (A) Locus 9-4. Amplification products were generated using primers R9 and R4 at an annealing temperature of 55°C. (B) Half-locus 9-11. Amplification products were generated using primers R9 and R11 at an annealing temperature of 50°C. (C) Half-locus 10-4. Amplification products were generated using primers R10 and R4 at an annealing temperature of 50°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).

FIG. 9

Polymorphic DNA analysis of E. histolytica isolates. (A) Locus 16-17. Amplification products were generated using primers R16 and R17 at an annealing temperature of 55°C. (B) Half-locus 16-19. Amplification products were generated using primers R16 and R19 at an annealing temperature of 54°C. (C) Half-locus 18-17. Amplification products were generated using primers R18 and R17 at an annealing temperature of 54°C. Isolate origins: HM-1:IMSS (Mexico); 200:NIH (uncertain); H-303:NIH (VietNam); IULA:1092:1 and IULA:0593:2 (Venezuela); 8691, 4530, 1320300, and 48286 (Bangladesh); 2596, 26.253, 37.0C, and 39.384C (South Africa).