INTRODUCTION

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The acceptance of Entamoeba histolytica and Entamoeba dispar as distinct species (2, 11) has had a major impact on our views of amebiasis, in particular its clinical management and epidemiology. It is likely that at least 90% of the infections previously ascribed to E. histolytica are actually E. dispar, while only the remaining 10% are infected with E. histolytica in its new sense. However, it also appears that many E. histolytica infections never progress to become symptomatic and are spontaneously lost. This observation raises some important questions. Are the organisms that produce invasive, symptomatic disease genetically distinct from those that give rise to asymptomatic infections? Or do all E. histolytica isolates have the potential to become invasive? Do certain invasive isolates show tropism for specific organs, with some preferentially ending up in the intestinal wall while others reach extraintestinal sites? To address the possibility of a relationship between parasite variation and infection outcome the ability to differentiate isolates of E. histolytica is necessary.

Our present knowledge of intraspecies variation in E. histolytica is limited. Isoenzyme analysis provided the first markers (25), but it now appears that isoenzyme patterns are not fixed (5) and therefore that many `zymodeme' assignments are unreliable (16). A limited number of DNA markers have been shown to exhibit intraspecies diversity. Variation has been observed in the number of rRNA transcription units present on the extrachromosomal ribosomal DNA circles; only one rRNA gene copy has been seen in some strains, while the majority have two (27). Variation has also been detected in the noncoding families of short tandem repeats found both upstream and downstream of the rRNA genes (20, 21, 26). However, variability in the occurrence and instability in the length of some of these sequences limits their use for isolate identification (4). PCR amplification of the Strain-Specific Gene (6) or Tr (27), which is present upstream of one rRNA transcription unit and contains tandemly repeated internal elements, has revealed considerable variation in the number of repeats among strains of E. histolytica (8). However, the complete absence of this locus in certain strains (27) makes it a poor candidate for intraspecies typing.

At present, the most polymorphic gene of E. histolytica is that encoding the serine-rich E. histolytica protein (SREHP or K2) a surface antigen with tandem 8- and 12-amino-acid repeats (17, 28). Repeat number, sequence, and restriction enzyme site polymorphisms have been reported among different E. histolytica isolates (8, 14). However, more than one-third of the isolates tested gave the same restriction fragment pattern (8). The chitinase gene also encodes tandem repeats of a degenerate 7-amino-acid sequence (10), and a report on the use of chitinase repeat polymorphisms to distinguish isolates of E. histolytica has been published recently (14). However, there still exists a need for additional reliable polymorphic E. histolytica DNA markers.

The use of microsatellite locus analysis has gained considerable popularity as a tool for detecting intra- and interspecies variations in a number of organisms, including protozoan parasites such as Trypanosoma (22), Leishmania (24), and Plasmodium (1) spp. Using a method designed to isolate microsatellite loci, we have obtained two new polymorphic DNAs containing tandemly repeated sequences from E. histolytica. We present here the preliminary characterization of the two loci and the interstrain variations they display. In addition, three other loci showing the presence of tandemly repeated sequences have been studied for their potential as polymorphic markers for use in investigating the molecular epidemiology of E. histolytica.

	MATERIALS AND METHODS

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E. histolytica isolates. Except for HM-1:IMSS clone 9, the axenic isolates were provided by John Ackers (London School of Hygiene and Tropical Medicine) (Table 1). All axenic isolates were cultured in the casein-free medium YI-S (12) with 15% heat-inactivated adult bovine serum (Sigma-Aldrich).

Isolation of nucleic acids. DNA was isolated as previously described (7, 9), dissolved in 10 mM Tris-Cl (pH 8.5) and passed over a Microspin S-200 HR column (Amersham Pharmacia Biotech, Inc). RNA was removed by the addition of RNase A (Promega) to 0.05 µg ml¹.

Isolation of repeated DNA containing sequences. A nonradioactive method designed for rapid isolation of microsatellite sequences (13, 22) was adapted. Genomic DNA of isolate HM-1:IMSS (ca. 500 ng) was digested for 2 h with a restriction enzyme, either AluI or RsaI (10 U/20-µl reaction) (Gibco-BRL), followed by incubation at 65°C for 15 min to inactivate the enzyme and passage through a S-200 column to remove the salts.

PCR product size polymorphisms and nucleotide sequence comparison. Primers were designed from repeat flanking region sequences of all the loci. The genomic DNA of E. histolytica was amplified using the primers listed in Table 2 and 30 cycles of 1 min at 94°C, 1 min at the primer-dependent annealing temperature, and 2 min at 72°C, with a final extension of 5 min at 72°C. Amplified products were analyzed using 2.4% NuSieve 3:1 agarose gels (FMC) in 1× Tris-boric acid-EDTA buffer (TBE).

Southern blot analysis. Genomic DNA of isolate HM-1:IMSS clone 9 was digested overnight with 10 U each of restriction enzymes AluI, DdeI, DraI, EcoRI, and RsaI (Gibco-BRL or MBI Fermentas), and fragments were separated by electrophoresis using 0.8% agarose gels in 1× TBE buffer and transferred to BiodyneA membranes (Gibco-BRL) using standard methods. [-³²P]dCTP-labeled double-stranded DNA probes were prepared by using the Rediprime II random prime labeling system (Amersham Pharmacia Biotech). Filters were hybridized overnight at 65°C in a solution of 1 M NaCl-1% sodium dodecyl sulfate-10% dextran sulfate and then washed to a final stringency of 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate at 65°C before autoradiography at 70°C.

	RESULTS

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Isolation of repeated DNA containing sequences from the E. histolytica genome. To try and obtain DNA fragments containing microsatellites, we employed a nonradioactive method based on affinity capture of single-stranded restriction fragments annealed to biotinylated microsatellite oligonucleotides, with attachment to streptavidin-coated magnetic beads (Dynal), followed by adapter-mediated PCR (13, 22). A total of twenty two PCR fragments ranging in size from 250 to 700 bp were gel purified from the total amplification products of AluI or RsaI restriction fragments annealed to one of seven biotinylated oligonucleotides. These fragments were chosen on the basis of their apparent enrichment compared to control amplification products and were cloned, sequenced, and examined for the presence of microsatellites.

Characterization of loci 1-2 and 5-6. The complete sequence of the locus 1-2 clone (Fig. 1A), not including the adapter sequence, is 402 bp long and contains a single repeat block with two related direct repeats arranged in tandem (Fig. 1B). In addition to the major repeat block, tandem duplications of 8 to 12 bp are present in the flanking regions. The complete sequence of the locus 5-6 clone (Fig. 2A), not including the adapter sequence, is 424 bp long and contains a single repeat block (Fig. 2B). As in locus 1-2, other tandem duplications in the regions flanking the repeat block are also evident. BLAST search results revealed no identity of either locus 1-2 or locus 5-6 to any previously reported E. histolytica sequences.

View larger version (41K): [in this window] [in a new window]   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.

View larger version (35K): [in this window] [in a new window]   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).

PCR product size polymorphisms at loci 1-2 and 5-6. Primers were designed in the regions flanking the repetitive blocks for both locus 1-2 and locus 5-6, and the PCR amplification products were analyzed on 2.4% NuSieve agarose gels to look for fragment size polymorphism among the 13 E. histolytica isolates (Fig. 3).

View larger version (36K): [in this window] [in a new window]   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).

Nucleotide sequence analysis and characterization of the observed size polymorphism. In order to study the underlying nature of the observed size polymorphisms, the amplification products of all five axenic isolates at locus 1-2 and locus 5-6 were cloned and sequenced. This analysis revealed differences in the number and sequence of the repeat units, as well as sequence variation in the regions flanking the repeat blocks (Fig. 4).

View larger version (35K): [in this window] [in a new window]   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.

Southern blot analysis. Southern hybridization analysis was performed using HM-1:IMSS clone 9 genomic DNA digested with five enzymes (Fig. 5) and either locus 1-2 or locus 5-6 as specific probes. The specificity of the probes was ensured by using PCR products produced from plasmid DNAs (clone 4 and clone 1, respectively).

View larger version (65K): [in this window] [in a new window]   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).

Characterization of other loci containing internal repeats. A number of other DNA elements containing internal tandem repeats have been reported in E. histolytica. No attempts have been made to study their potential for the detection of intraspecies polymorphisms. We selected three of these internally repetitive loci for study: a 978-bp element described by Michel et al. (19; GenBank accession number M77091; our designation, locus 3-4), a 931-bp DNA element isolated by J. Rosales-Encina and D. Eichinger (personal communication; GenBank accession number AF265348; our designation, locus 9-4), and a 964-bp element reported by Huang et al. (15; our designation, locus 16-17). Schematic representations of the repeat arrangements seen at these loci are given in Fig. 6A, B, and C, respectively.

View larger version (25K): [in this window] [in a new window]   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.

PCR product size polymorphisms at loci 3-4, 9-4 and 16-17. Primers were designed to amplify all three loci, and the products were analyzed on 2.4% NuSieve agarose gels to look for size polymorphisms among the 13 E. histolytica isolates. In each case primers were also designed in the regions between the two main repeat blocks to look additionally for size variations in each half of the locus (Fig. 6).

View larger version (34K): [in this window] [in a new window]   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).

View larger version (36K): [in this window] [in a new window]   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).

View larger version (35K): [in this window] [in a new window]   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).

	DISCUSSION

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We were unsuccessful in our attempt to clone microsatellite loci using a modification of a method that was successful in other organisms. We did, however, isolate two novel loci containing internal tandem repeats (1-2 and 5-6). Whether this reflects an absence or a reduced population of the classical di- and trinucleotide microsatellites in the E. histolytica genome or simply their relative abundance is at present too early to say. A (GA)₂₇ stretch in an expressed sequence tag has been reported by Azam et al. (3), suggesting that microsatellites do exist in this organism. Three other, previously reported internally repetitive loci were also studied.

The genomic organization of the loci was investigated by Southern blotting. The large fragments generated by DraI and/or EcoRI detected with the locus 1-2 and 5-6 specific probes and the presence of major fragments of the same size with two or more restriction enzymes suggests that both loci are arranged in long tandem arrays. The fact that some enzymes generate very large fragments also was reported by Michel et al. (19) for locus 3-4, and these authors also suggested that it was tandemly arrayed. Similarly, Southern hybridization with locus 16-17 indicated that this element was tandemly repeated (15). The successful amplification of locus 9-4 using the primer orientation indicated in Fig. 6B suggests that this element is in tandem arrays also.

Significant levels of identity exist between the sequences of loci 3-4 and 9-4, in the repeat domains as well as the flanking regions. Similarity has also been noted between some of the repeat units of locus 1-2 and those of loci 3-4 and 9-4. Two other internally repetitive DNA elements were reported independently by Lohia et al. (18) and Willhoeft and Tannich (30) which also bear remarkable similarity in their repeat domains to loci 3-4 and 9-4. However, complete sequence alignment of all five DNA sequences shows enough variation to suggest that they are distinct members of the same family of repeat containing DNA elements (data not shown). That loci 3-4 and 9-4 are indeed distinct is clearly seen from comparing Fig. 7B and Fig. 8B.

Size variations within the repeated domains were studied, and all loci studied showed PCR product length polymorphism. At most loci, amplification results in two or three bands in at least some isolates. Present evidence suggests that the Entamoeba genome is tetraploid (29). It is possible that the multiple bands we observe reflect polymorphism among homologous loci on allelic chromosomes. Multiple amplification products have also been reported for the SREHP gene in a number of E. histolytica isolates (8), a finding consistent with the isolation of cDNAs containing variable numbers of internal tandem repeats (17). SREHP gives the pattern expected of a single copy gene when analyzed by Southern blotting indicating that the length differences are allelic variations. Alternatively, the presence of multiple bands in this study could be explained by the existence of these repeat loci at multiple locations in the Entamoeba genome, each with a characteristic PCR product size.

The observed size variation was further characterized by nucleotide sequence comparison of five isolates at loci 1-2 and 5-6. Isolates 200:NIH and H-303:NIH are easily differentiated by PCR from most of the other isolates but cannot reliably be separated from each other by gel electrophoresis. However, DNA sequence comparison at locus 5-6 revealed that the two isolates are distinguished by the absence of one copy of an 8-bp tandem duplication from strain 200:NIH; this difference is too small to be detected on gel electrophoresis. Thus, PCR product sizes alone may not discriminate among all distinct isolates, at least under these conditions.

From our results it appears that a number of loci showing size polymorphism are present in E. histolytica. The degree of diversity seen varies, with some loci showing more polymorphism and thus having a greater potential for detecting interstrain polymorphism among E. histolytica isolates than others. Despite being from geographically restricted regions, the Bangladeshi and South African samples could be differentiated with ease. For the most part variations in PCR product size appear to be a result of differences in the numbers of tandem repeat units. While no single locus discriminates between all the samples, the collective use of multiple loci allows differentiation of a majority of the E. histolytica isolates.

We believe that the polymorphisms we describe here have potential as tools to answer many of the outstanding questions surrounding the epidemiology of E. histolytica. We are currently studying samples from a broader geographic range to validate the general utility of these loci and are examining samples from infected family groups and amebiasis outbreaks for shared polymorphisms. In the present work, all of the isolates came from individuals who had invasive disease or were likely infected by someone who had invasive disease. Whether invasive and noninvasive strains of E. histolytica exist remains to be established, but hopefully the polymorphic loci described here will be useful in answering this important question.