Over the years, biologists have developed a whole battery of techniques for the routine and rapid genetic analysis of traditional model organisms, such as Escherichia coli, yeast, worms, and flies. Researchers working on more exotic organisms, however, are not so fortunate. Often, the methods available for dissecting gene function in these species are unreliable, labor-intensive, or both, and the complex and poorly characterized biology of the organism significantly stymies analysis. In this issue, Stock et al1. report use of antisense peptide nucleic acid (PNA) for the specific inhibition of genes in the protozoan pathogen, Entamoeba histolytica. The availability of a technology for rapidly assessing gene function in E. histolytica promises to facilitate research on an important contributor to acute and chronic diarrheal illness (amebiasis) worldwide.

Protozoan parasites occupy a unique niche in the biosphere because they are adapted to thrive at the expense of a living host. The diseases they cause impose both a heavy toll on human health—amebiasis alone is estimated to kill 100,000 people a year2—and an economic burden on agriculture through the infection of livestock. In the past decade, the development of several molecular genetic methods for studying protozoan parasites (Table 1) has enabled significant advances in our understanding of parasite biology and therefore disease control.

Table 1 Molecular techniques used to determine gene function in parasitic protozoa
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It is important to remember that protozoan parasites are truly a very diverse group of organisms with many different adaptations for succeeding in varied host environments. Many of them have multiple life stages that are dependent on the host, and some require multiple hosts. Moreover, many protozoa have haploid genomes, others are diploid, some are haploid only over part of their life cycle, and yet others have poorly defined ploidies. Each parasite thus requires a set of genetic tools to be specifically developed because of its unique biology.

Entamoeba histolytica, the causative agent of amebiasis, is a case in point. Currently, no techniques for the chromosomal manipulation of this parasite are available, and all existing attempts to manipulate the genetics of this organism have focused on stable episomal transfection of either dominant mutant gene constructs or antisense constructs.

In the present paper, Stock et al1. use antisense PNA oligomers for the specific inhibition of genes in E. histolytica. PNA oligomers have a polyamide backbone in place of the sugar-phosphate DNA backbone and are uncharged and stable to cleavage by enzymes. The authors show that E. histolytica trophozoites readily take up the oligomers, which then specifically block expression either of an episomal neomycin resistance gene or of a chromosomal erd2 homolog.

As with any antisense approach, the inhibitory effects were partial, but in both instances, an effect on cell growth was observed. Although the utility of the technique remains to be proven using other genes, the current data certainly suggest that the method should facilitate rapid functional analysis of genes in this organism. This is particularly important because the E. histolytica genome project is currently well underway. (The combined efforts of The Institute for Genomics Research and the Sanger Centre promise a complete genome sequence shortly.)

In fact, genome projects are in progress for a host of protozoan parasites, including those responsible for malaria (Plasmodium spp.), sleeping sickness (Trypanosoma brucei), leishmaniasis (Leishmania major), and cryptosporidiosis (Cryptosporidium parvum). Additionally, large expressed sequence tag (EST) databases have been established for such parasites as the toxoplasmosis agent, Toxoplasma gondii, and the cause of Chagas disease, Trypanosoma cruzi.

Sequence information from these projects, used in conjunction with molecular techniques (see Table 1), promises to facilitate identification of genes that determine pathogenicity. Genes associated with pathogenicity that encode functions unique to a parasite may serve as potential candidates for diagnosis, therapy, and vaccine development. In addition, sequence comparisons between related organisms may also reveal putative drug targets.

Apart from Entamoeba, molecular genetics approaches are also being used to unravel the biology of kinetoplastid parasites, such as the diploid Leishmania3 and Trypanosoma spp., and of apicomplexan parasites, such as Plasmodium spp. and Toxoplasma gondii. The tremendous potential of DNA chip technology in identifying genes that are expressed during specific developmental stages of the life cycle has already been demonstrated in Plasmodium falciparum4,5 and in T. gondii (J.C. Boothroyd, personal communication). Such arrays promise to provide much information regarding parasite biology and disease pathogenesis in the future.

In light of the rapidly accumulating sequence information from the various parasite genome projects, there is an urgent need for molecular techniques that can replace traditional time-consuming chromosomal gene “knockout” methodologies and enable rapid screening of identified open reading frames (ORFs) for functional significance. One promising approach is that of RNA interference (RNAi), which has been used to systematically analyze predicted ORFs on two different chromosomes of Caenorhabditis.elegans6,7. RNAi has been shown to work for several genes in T. brucei8,9 and will likely prove useful for analysis of other genes in other protozoan parasites. Antisense PNA oligomer technology, as demonstrated by Stock et al., may also be an effective strategy for rapid functional analysis of parasite genes. With protozoan disease and drug resistance an ever-present global problem, the more technologies we have at our disposal for unraveling parasite biology, the better.

Figure 1: Epifluorescence phase contrast micrograph of E. histolytica with capped cell-surface lectin.
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Genetic analysis of the this organism will be facilitated by the work of Stock et al.

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