Mitochondrial genome diversity in parasites

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Abstract

Mitochondrial genomes have been sequenced from a wide variety of organisms, including an increasing number of parasites. They maintain some characteristics in common across the spectrum of life—a common core of genes related to mitochondrial respiration being most prominent—but have also developed a great diversity of gene content, organisation, and expression machineries. The characteristics of mitochondrial genomes vary widely among the different groups of protozoan parasites, from the minute genomes of the apicomplexans to amoebae with 20 times as many genes. Kinetoplastid protozoa have a similar number of genes to metazoans, but the details of gene organisation and expression in kinetoplastids require extraordinary mechanisms. Mitochondrial genes in nematodes and trematodes appear quite sedate in comparison, but a closer look shows a strong tendency to unusual tRNA structure and alternative initiation codons among these groups. Mitochondrial genes are increasingly coming into play as aids to phylogenetic and epidemiologic analyses, and mitochondrial functions are being recognised as potential drug targets. In addition, examination of mitochondrial genomes is producing further insights into the diversity of the wide-ranging group of organisms comprising the general category of parasites.

Introduction

The history of mitochondrial genome sequencing “projects” is a long one, with the sequence of the human[1]and bovine[2]mitochondrial genomes being completed in 1981 and 1982, not long after the first complete viral genome sequences were obtained. Today, there are at least 130 complete or near- complete mitochondrial genome sequences in the databases. A substantial proportion of these are from vertebrates, but a growing number come from other groups, including invertebrates and protozoans. Indeed, the Organelle Genome Megasequencing Project (OGMP; http://megasun.bch.utoronto.ca/ogmp/) is engaged in systematic sequencing of complete mitochondrial genomes from a variety of single-celled eukaryotes to evaluate the origin and evolution of the mitochondrial genome. This is proving to be a rich source for new insights into the great diversity of mitochondrial genomes. Reviews of animal[3]and protistan[4]mitochondrial genomes have appeared recently.

Mitochondrial genomes vary greatly in size, from the 6-kb length of a single unit of the tandemly repeated Plasmodium DNA to the estimated 2400-kb size of mitochondrial DNA in muskmelons[5]. Despite the disparity in size, all mitochondrial genomes encode ls and ssrRNAs and two components of the mitochondrial electron transport chain: apocytochrome b (CYb)1 and cytochrome c oxidase I (COI)[6](but see the discussion on Phytomonas serpens below). Most also encode other components of the electron transport chain, subunits of the mitochondrial ATPase, and tRNAs. Size of the mitochondrial genome does not necessarily correlate with coding content, however, as much of the large plant mitochondrial genomes consist of non-coding sequence[7]. Instead, the current honours for the most varied content for a mitochondrial genome goes to the jakobid protozoan Reclinomonas americana[8]. In addition to the “expected” content noted above, the 69-kb Reclinomonas genome encodes an additional ATPase subunit, a cytochrome oxidase assembly protein, a translation elongation factor, a secretory path protein, numerous ribosomal proteins, RNase P, and a multi-subunit, eubacterial-like RNA polymerase. The full list of Reclinomonas mitochondrial genes includes all genes which have been described from all other mitochondrial genomes known to date, as well as some that have not been reported previously, and thus arguably represents the closest approach yet to the gene content of the ancestral mitochondrion. Recently, the 1.1-Mb genome of Rickettsia prowazekii, an α-proteobacterium, has also been completely sequenced[9]. The α-proteobacteria are believed to be most closely related to the mitochondrial progenitor and the Rickettsia, as obligate intracellular pathogens, have a reduced genome size compared with most bacteria. Comparisons between the Rickettsia genome and the Reclinomonas mitochondrial genome show that the Reclinomonas genes are a subset of the Rickettsia genes[10]. These examples suggest the gene content and organisation of the earliest mitochondrial DNAs. A wide diversity has since developed, influenced by a proliferation of unusual expression mechanisms.

Interest in obtaining mitochondrial sequences falls into several categories. Initial sequencing efforts defined the gene content of mitochondrial DNA and permitted the pairing of mutant phenotypes with specific DNA alterations. One offshoot of the sequencing of the human mitochondrial genome is that a variety of human genetic diseases have now been found to arise from mitochondrial mutations11, 12. Analysis of mitochondrial DNA sequences and the transcripts made from them have provided seminal and sometimes startling insights into gene expression mechanisms. The identification of introns in mitochondrial genes from Saccharomyces cerevisiae[13]occurred shortly after introns were first recognised[14]. Many mitochondrial introns are similar in structure to the Tetrahymena rRNA intron and, like it, are capable of self-splicing[15]. Identification of proteins encoded within introns also dates from this time; such proteins are generally implicated in splicing of the intron in which they are located[13]. Arguably most startling of all is the phenomenon of RNA editing, first described in trypanosomatid mitochondria16, 17and now known to occur, in varying forms, in other mitochondria, in chloroplasts and in some nuclearly encoded genes (reviewed in Ref.[18]).

Mitochondrial genes have also been commonly used for phylogenetic analyses, considered both as single genes (e.g. Refs19, 20) and, in some cases, assessing changes in gene order to evaluate relationships[21]. The rapid reorganisations which appear to occur in some mitochondrial genomes mean that conclusions based on gene order must be carefully considered[7]. Additionally, mitochondrial sequences have been employed for speciation and epidemiological studies (e.g. Refs22, 23, 24, 25, 26). These are particularly important when related species appear phenotypically very similar but vary in their host range or the severity of the epidemiological consequences (e.g. Refs27, 28). Sequences from individual mitochondrial genes from helminths are commonly used in such comparisons, often paired with a nuclear ribosomal internal transcribed spacer sequence as a second marker. While there are a few exceptions, the vast majority of mitochondrial genomes are inherited uniparentally from the female parent and thus represent a set of genes not subject to sexual recombination, assisting such studies.

Finally, mitochondrial genes present possible targets for disease intervention (e.g. Refs29, 30). The drug atovaquone provides a good example. It targets complex III of the electron transport chain, which contains CYb, and is effective against both toxoplasmosis and malaria. However, resistance rises easily[31]and is accompanied in Plasmodium by changes in the sequence of the CYb gene[32], confirming the mitochondrial target of the drug. Despite this, atovaquone has been shown to be synergistic with proguanil and the combination has been reported to be effective in clinical trials[31]. Atovaquone has also been shown to be effective against exo-erythrocytic Plasmodium falciparum[33]and, as such, may be a good choice for prophylaxis. The potential for other anti-mitochondrial therapeutics remains relatively underexplored.

The extent of mitochondrial genome sequences available is a shifting target, as new submissions to the databases are made regularly. In addition to projects designed to specifically sequence mitochondrial genomes, the proliferation of both genome projects and expressed sequence tag projects is providing a rapid increase in the amount of parasite mitochondrial sequence. In the following comments, I will concentrate primarily on complete or near-complete mitochondrial genomes, with information from less complete genomes as appropriate. By far the most data on parasite mitochondrial genomes are available for the kinetoplastid protozoa, a fact that is unsurprising in light of the longevity of kinetoplastid studies and the intense interest generated by RNA editing. More recently, apicomplexan mitochondrial genomes have also received substantial attention, those from amoebae have come under scrutiny, and new data are available from nematodes, trematodes and cestodes. (Note that mitochondrial sequences from ectoparasites are not included in this review.) In combination, the available parasite mitochondrial genomes span a wide variety of gene content and organisation.

Section snippets

Genome organisation

Mitochondrial genomes in the trypanosomatid branch of kinetoplastid protozoa have a highly unusual organisation, consisting of two separate sizes of circular DNAs catenated together with each other; the complex is called kinetoplast DNA or kDNA. The larger molecule, called the maxicircle, ranges from ∼20 to 40 kb in size and is present in ∼50 copies per cell[34]. The smaller but more abundant molecule, the minicircle, is ∼0.65–2.5 kb and there are 5000–10 000 copies per cell[34]. In the less

Mitochondrial genome organisation

A circular DNA approximately 30–35 kb in size was observed in electron micrographs of Plasmodium lophurae[122]in 1975, and in Plasmodium berghei[123]and Toxoplasma gondii[124]almost a decade later. Because its size was in the range known for other protozoan mitochondrial genomes, and because circularity was the expected form for a mitochondrial DNA, a number of early studies refer to it as the mitochondrial DNA122, 123, 124, 125, 126. In the early 1990s, sequencing data from this DNA predicted

Genome organisation and gene content

The mitochondrial genomes of Acanthamoeba castellani and Naegleria gruberi, a non-pathogenic relative of Naegleria fowleri, have been completely sequenced by the Organelle Genome Megasequencing Project, but only the A. castellani sequence has been released to date (Table 1). The size of the A. castellani mitochondrial genome has been reported to vary from 38.7 to 44 kb in different strains[175]. The 41.6-kb genome of the Neff strain encodes 33 protein coding genes and an additional eight ORFs,

Genome organisation and gene content

Complete mitochondrial sequences have been determined for five nematodes: the classic nematode model Caenorhabditis elegans[180], the mammalian parasites Ascaris suum[180]and Onchocerca volvulus[181], the mosquito parasite Romanomermis culicivorax182, 183, and the plant root knot nematode Meloidogyne javanica25, 184. The first three are available in the databases, but only portions of the latter sequences have been released to date (Table 1). A partial sequence (1900 nt) from Meloidogyne hapla

Genome organisation and gene content

There is an active Schistosoma mansoni mitochondrial genome project, with plans to also include analyses of other Asian and African species for phylogenetic and population genetic studies (DP McManus, personal communication). Indeed, phylogenetic analyses of schistosome species, based in part on COI sequences, have already been reported196, 197, as well as detailed analyses of subpopulations of Schistosoma japonicum using ND1 sequences198, 199, 200, 201. A review on schistosome evolution,

Cestodes

There are very few mitochondrial DNA sequence data from cestodes, but some information comes from studies of Echinococcus and Taenia. A 1992 study used COI sequences to examine Echinococcus mitochondrial DNA as a potential marker for speciation[23]. Based on sequencing data from 56 isolates and comparison of the Echinococcus COI sequence to that from other organisms, the study found that AGA and AGG probably encode serine, similar to findings in nematodes and trematodes. A molecular phylogeny

Perspectives

Mitochondrial genomes among parasites are as diverse as the parasites themselves. Indeed, given the evolutionary diversity presented by the organisms discussed here, it is the similarities among these genomes, rather than the differences, which should be surprising. All have a small common core set of genes, most are tightly packed with coding sequence and segregate the majority of intergenic sequences to a single region, and a number have short overlaps of coding sequence which are probably

Acknowledgements

I thank Drs DP McManus and TR Unnasch for providing preprints. Work from my laboratory was supported by NIH grant AI40638.

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