Review ArticleMolecular EpidemiologyMethods for the Investigation of Diversity in Entamoeba histolytica
Introduction
Intraspecific diversity is studied for a number of reasons, ranging from identifying genetic linkage to following patterns of transmission in a population. In the case of Entamoeba histolytica, however, it had an unusual outcome, namely, the recognition that what was thought to be a single species was in fact two. In 1978, Sargeaunt and Williams (1) began to study the isoenzyme patterns of E. histolytica isolates using starch gel electrophoresis. It became apparent very quickly that the patterns they were observing fell into two categories that correlated with the sample origin. One group included all the isolates from individuals with amebic colitis and liver abscess while isolates from the other group were all from people without invasive disease. Initially called ‘pathogenic’ and ‘nonpathogenic’ E. histolytica (2), with the accumulation of additional evidence from antibody and DNA studies these two groups were eventually recognized as distinct species and are now known as E. histolytica and E. dispar, respectively (3).
Isoenzymes were also used to study variation within the two groups (now species) with about a dozen variants described for each (2). However, it is now realized that those ‘zymodeme’ designations are problematic as most patterns are dependent on the bacterial flora, if not actually bacterial enzymes themselves. In fact, Jackson recognizes only three ‘primary’ zymodemes in E. histolytica and one in E. dispar that can be distinguished in starch gels (4). Other separation systems (such as thin layer agarose gels) allow the detection of a few additional variants but the overall diversity is still very limited (Cunnick and Diamond, unpublished). The investigation of diversity in E. histolytica is now fully dependent on the use of DNA variation detected using the polymerase chain reaction (PCR).
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SREHP and SSG
The first DNA sequences shown to exhibit stable and significant polymorphism were the Strain Specific Gene (SSG) and the Serine Rich Entamoeba histolytica Protein (SREHP) gene (5). SSG (also known as Tr) is a transcribed but untranslated region of the episomal DNA encoding ribosomal RNA (rDNA) in E. histolytica 6, 7. This episome contains several classes of tandemly repeated sequences of which SSG is one (8). SSG contains 26 base pair (bp) repeats, the number of which varies among isolates of
Chitinase
The chitinase gene of Entamoeba species encodes a protein with a repeat structure near the amino terminus (14). This degenerate 7 amino acid repeat region exhibits PCR product size polymorphism between isolates, again dependent on the repeat copy number. In E. histolytica, chitinase behaves like a single copy gene in Southern blotting, but in contrast to SREHP allelic variants appear to be rare or absent. Chitinase is also present and polymorphic in E. dispar (15). Most studies of chitinase
tRNA-Linked Loci
In many eukaryotic pathogens, microsatellite loci have become the target of choice for detecting diversity, and so about 6 years ago an attempt was made to isolate such loci from E. histolytica. Although it failed (and we now know that the E. histolytica genome is essentially devoid of microsatellites), the attempt did yield another type of polymorphic locus. These contained short-tandem repeats (STRs) but of a size and complexity much greater than is associated with ‘traditional’
Comparison among Loci
Although isoenzymes, SSG, SREHP, and chitinase can all be used to detect diversity among E. histolytica isolates, each method had its drawbacks. Isoenzymes require the establishment of cultures and the generation of substantial numbers of cells to obtain the necessary amount of material. The PCR-based methods require smaller amounts of material and are not completely reliant on cultures, but SSG is not always present while SREHP and chitinase require restriction enzymes and/or sequencing to
Stability
One area of significant concern is the stability of the patterns observed at the different loci. In particular, for following transmission patterns in populations, stability is essential. Although rapid changes in repeat number have been observed in other regions of the rDNA episome (19), SSG/Tr usually appears to be stable when present. However, deletion of the whole locus has been observed to occur, making its reliability suspect (20). No such concerns have been raised about the other
Other Entamoeba Species
SSG is absent from E. dispar and all other species as far as it is known (7). SREHP is also present and polymorphic in E. dispar (15) but little has been published on its diversity in this species. In contrast, substantial chitinase sequence variation has been reported in E. dispar 15, 23, at least as much as has been identified in E. histolytica. The level of diversity seen in the tRNA-linked loci of E. dispar also appears to be largely similar to that observed in E. histolytica (24), with one
Applications
The ability to differentiate variants of E. histolytica has several potential applications. One is ‘forensic’—can the origin of an infection be traced to its source? Another is epidemiological—is there a structure to the population of E. histolytica in a given area and does this differ among regions? In the clinical setting the question of whether a post-treatment infection is the result of re-infection or recrudescence can be answered (25). However, perhaps the most interesting question is
Acknowledgments
Work performed in my laboratory has been supported by the Wellcome Trust (Grant #067314).
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