Cardiff School of Biosciences, Cardiff University, Main Building, PO Box 915, Cardiff CF1 3TL, UK
Correspondence
David Lloyd
(Lloydd{at}cf.ac.uk)
An interesting and important cohort of protists, now widely referred to as anaerobic protists, has become a focus of interdisciplinary research. Although there are many free-living species (e.g. Metopus contortus in marine sediments), those best studied are parasites that cause some of the most widespread diseases of humans and animals. Giardia intestinalis is the most prevalent water-borne protist, parasitic in humans and cattle worldwide; its cysts survive in water supplies for months. Trichomonas vaginalis, a sexually transmitted flagellate, is the most common of all human parasitic protists; Tritrichomonas foetus, causing abortion in cattle, leads to enormous economic losses in large herds in Australia and the USA. The fact that these organisms have poorly developed mitochondria (Fenchel & Finlay, 1995; Lloyd et al., 2002
) has encouraged the view that they may be unusual, as being representative of present-day counterparts of organisms that evolved soon after an endosymbiotic
-proteobacterium became the energy-yielding organelle of the earliest eukaryotic lineage. Accumulating evidence, obtained by biochemical investigations, improved ultrastructural studies and work on molecular phylogeny (Lloyd & Harris, 2002
), invalidates this hypothesis. We still know little of the events that occurred to make possible the development and emergence of lower eukaryotes, plants and animals. As yet, we know nothing about the long line of ancestral forms of present day lower eukaryotes, so that we cannot identify a missing link (Fenchel, 2002
).
Although it has been clear for more than 20 years that lack of understanding of some basic biological principles, as well as inadequate citation of available literature in some influential reviews, has biased textbook accounts of this field, current advances are also often still prefaced by incorrect statements. Here I emphasize basic details concerning the wide range of functions of mitochondria, and the distinction between anaerobic and microaerophilic lifestyles that are misunderstood, as well as terms that are misused. Further details have been published previously (Lloyd, 1989).
Mitochondria, even if highly modified, do occur in all well-studied protists
These double-membraned organelles occur ubiquitously in aerobic eukaryotic single-celled organisms and in the metazoa. Mitochondria sometimes have diminished (reduced) structural organization and accompanying functions where and when environmental or in situ energy demands do not require the full expression of their repertoire. Major functions include electron transport chain-mediated substrate oxidations from reduced donors to terminal electron acceptors. In aerobic organisms, when O2 is present at concentrations sufficient to sustain a rate of respiration adequate to provide energetic needs, this system will be highly organized and will contribute a complement of at least 80 polypeptides to the inner membrane together with ubiquinone, the mobile hydrophobic electron transfer component and antioxidant, at concentrations 20-fold higher than that of the terminal oxidase (cytochrome c oxidase). Together with ATP synthase, the respiratory chain forms an integrated system for energy conservation. All the components of this system, together with the proximal ones of aerobic oxidation, the tricarboxylic acid cycle and the -oxidation spiral of fatty acid oxidation, can be down-regulated in response to diminished bioenergetic need in circumstances where alternative systems become more appropriate. Examples of where this is so include high sugar concentrations, during hypoxia, or when inhibitors of cytochrome c oxidase (e.g. S2, CN or NO) are present.
Ultrastructural modifications accompany decreased function, even to the point where the characteristic ultrastructural features cannot be discerned in electron micrographs. These promitochondria originally characterized in glucose-repressed or anaerobically grown yeast do, however, still possess a range of functions necessary for the continuing cellular survival, even when energy generation is entirely due to glycolytic ATP generation. These are (i) ATP-driven generation of inner mitochondrial membrane potential necessary for transport functions for the exchange of metabolites and coenzymes between the mitochondrial matrix compartment and the cytosol, import/export flux of nascent proteins; and (ii) ion transport processes of H+, K+, Ca2+ and substrate anions between these compartments.
Another situation in which mitochondrial structure and function becomes de-differentiated is in some parasitic organisms where structure, energy-generating and ion-transport functions may become difficult to detect. Terms recently introduced (e.g. mitosome, mitochondrial remnant or relict mitochondria) are hardly necessary and confuse neophytes to this area of research. The term hydrogenosome is useful as it emphasizes a major characteristic of a highly specialized mitochondrion and, as it has been used for 30 years, will probably continue to be used.
The anaerobic condition
Anaerobiosis is a widely misunderstood term. Avogadro's number is very large and it is difficult to exclude O2 from experimental systems. Similarly, many natural environments have just a little O2 and, at those very low concentrations, detectability becomes difficult. It becomes increasingly apparent that, in eukaryotes, the anaerobic condition is a rare attribute or may even be non-existent. Even where an anaerobic style of metabolism is evident, O2 may be detectable or even plentiful (e.g. in the Crabtree Positive yeasts, those that carry out aerobic glycolysis). In some parasitic worms or protists, where O2 is inadequate to maintain energy metabolism, sulphide may provide reducing power and an alternative electron acceptor, fumarate, may be used for electron transport-driven energy transduction. In many of the so-called anaerobic protists, the most anaerobic that we know are the methanogen-containing ciliates that inhabit the often highly sulphide-rich marine sediments. Ciliates and chytrid fungi that inhabit the rumen live in the most important of all fermentative habitats, but this is not the anaerobic compartment of textbook descriptions. Rather it is one where high input of reducing equivalents, when the host animal is feeding, alternates with an ingress of considerable levels of O2 (1 µM). In the caecum or large intestine, parasites adherent on the mucosal lining epithelium inhabit a region where O2 downloads from the oxyhaemoglobin of the erythrocytes flowing through adjacent peripheral capillaries. In the jejunum, the O2 levels experienced, for instance by Giardia species, may reach 60 µM. In the vagina, O2 levels fluctuate, but sometimes reach 12 µM. Careful experiments with laboratory continuous cultures indicate that growth of Trichomonas vaginalis is more rapid at 1 µM O2 than under strictly anaerobic conditions (Paget & Lloyd, 1990
). Perhaps, like Trichomonas vaginalis, all these organisms are microaerophilic (Lloyd & Williams, 1993
). Fluctuating environmental conditions are the rule rather than the exception. Complex life cycles provide variation in environmental O2 supplies.
Early eukaryotic evolution
The concept of a Tree of Life provides a useful model for tracing the ancestry of extant organisms. However, the common occurrence of lateral gene transfer events, especially during the earlier stages of evolution, and changes of evolutionary rates are factors that place a growing burden of justification on claims for connectivities and phylogenetic reconstruction. For the earliest stages of the 2-billion-year period that followed the endosymbiotic origin of eukaryotes, we still have no information on the succession of changes. Even our knowledge of the atmospheric environment on Earth is incomplete (Fenchel, 2002; Lane, 2002
). Extrapolation from extant organisms to early eukaryotic evolutionary progression is hardly possible. Issues are compounded by extensive lateral gene transfer, which may account for as much as 10 % of the protist genome, as observed, for example, in Entamoeba histolytica. Convergent evolution is particularly evident in hydrogenosomes where cognate functions have arisen separately in different lineages (i.e. are polyphyletic in origin) (Biagini et al., 1997
).
Giardia as a specific example
The ultrastructural features of this common, water-borne parasite appeared unusually simple in electron micrographs. Similarly, isolation of particulate fractions on subcellular fractionation after harsh disruption by (motor-driven) homogenization was not possible. More-refined methods of investigation of cellular structure and function (Lloyd et al., 2002), using non-invasive approaches (confocal laser scanning microscopy with a range of specific fluorophores and less-disruptive cell-breakage techniques), give more reliable information on intra-cellular compartmentation. Its bacteria-like metabolism and enzymes have further encouraged its description as a primitive, early branching eukaryote. Recent reviews refute this view (Lloyd & Harris, 2002
; Biagini & Bernard, 2000
) and provide evidence for de-differentiated mitochondria that contain electron transport components and can generate a membrane potential. Giardia is in fact a highly successful parasite, moulded by its very special environmental situations as either a trophozoite or an encysted protist and by the changing ecosystems it experiences during its survival in water and passage through the gut. Its exquisite structurefunction adaptations have been developed to match its lifestyle (Lloyd & Harris, 2002
).
REFERENCES
Biagini, G. A. & Bernard, C. (2000). Primitive anaerobic protozoa: a false concept? Microbiology 146, 10191020.
Biagini, G. A., Finlay, B. J. & Lloyd, D. (1997). Evolution of the hydrogenosome. FEMS Microbiol Lett 155, 133140.[CrossRef][Medline]
Fenchel, T. (2002). Origin and Early Evolution of Life, p. 123. Oxford University Press.
Fenchel, T. & Finlay, B. (1995). Ecology and Evolution in Anoxic Worlds, p. 127. Oxford University Press.
Lane, N. (2002). Oxygen, the Molecule that Made the World, p. 83. Oxford University Press.
Lloyd, D. (1989). Aerotolerantly anaerobic protozoa. In Biochemistry and Molecular Biology of Anaerobic Protozoa, pp. 121. Edited by D. Lloyd, G. H. Coombs & T. Paget. Chur, Switzerland: Harwood Academic.
Lloyd, D. & Harris, J. C. (2002). Giardia: highly evolved parasite or early branching eukaryote? Trends Microbiol 10, 122127.[CrossRef][Medline]
Lloyd, D. & Williams, A. G. (1993). Biological activities of symbiotic and parasitic protists in low O2 environments. Adv Microb Ecol 13, 211262.
Lloyd, D., Harris, J. C., Maroulis, S., Wadley, R., Ralphs, J. R., Hann, A. C., Turner, M. P. & Edwards, M. R. (2002). The primitive microaerophile Giardia intestinalis (syn. lamblia, duodenalis) has specialized membranes with electron transport and membrane-potential-generating functions. Microbiology 148, 13491354.
Paget, T. A. & Lloyd, D. (1990). Trichomonas vaginalis requires traces of oxygen and high concentrations of carbon dioxide for optimal growth. Mol Biochem Parasitol 41, 6572.[CrossRef][Medline]