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Diversityinmitochondrion-derivedorganellesoftheparasiticdiplomonadsSpironucleusandGiardia

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Diversity in mitochondrion-derived organelles of theparasitic diplomonads Spironucleus and Giardia

Catrin F. Williams, Coralie O.M. Millet, Anthony J. Hayes,Joanne Cable, and David Lloyd

School of Biosciences, Cardiff University, Cardiff CF10 3AT, Wales, UK

Diplomonads are binucleate, anaerobic, or microaerophilicflagellated protists that inhabit low O2 environments.These habitats include anoxic freshwater sediments(e.g., Hexamita inflata), and the intestinal tracts of animals(e.g., Spironucleus vortens) including humans (e.g.,Giardia intestinalis) [1]. Parasitic diplomonads cause se-vere pathologies in companion and farmed animals, andare of considerable concern in both medical and veterinarypractice. Diplomonads, together with other free-living andparasitic protists found in O2-deficient niches, have mis-takenly [2] been described as primitive eukaryotes due totheir alleged ‘ancient amitochondriate ancestry’, unusualcytological features and, often, by lateral gene transfer ofbacteria-like metabolic characteristics, for example thearginine dihydrolase pathway [3]. However, mitochondri-on-derived genes and/or mitochondrion-derived organelles(MDOs) have been found in all examined amitochondriateeukaryotes [4]. These MDOs have arisen by processes ofsecondary adaptation to an anaerobic lifestyle, wherebyfully functional respiration-competent mitochondriahave undergone reductive evolution resulting in double-membraned hydrogenosomes or mitosomes, as studiedextensively in the parabasalid trichomonads and G. intes-tinalis, respectively. Whereas mitochondria often possessas many as 1500 proteins, only 569 were characterised inTrichomonas vaginalis hydrogenosomes (for amino acidmetabolism, substrate-level ATP generation, H2 produc-tion, and Fe–S cluster assembly, etc.) [4]. Unlike mitochon-dria, common features of hydrogenosomes include lack of agenome, a cytochrome-mediated respiratory chain, and aproton-translocating ATP synthase. Mitosomes in G. intes-tinalis, having only 20 functionally defined proteins (nineinvolved in Fe–S cluster assembly, diflavoprotein NADHreductase, vesicle-associated membrane protein, and outermembrane protein translocase) [4] represent, as far as isknown, the ultimate evolutionary reduction of mitochon-dria. For the diplomonads, only Giardia mitosomes havebeen studied: here we summarise evidence for MDOs in S.vortens.

S. vortens is a diplomonad parasite of particular impor-tance in aquaculture because it can lead to devastatingoutbreaks of systemic infections in both ornamental andfood fish; parasite control requires understanding of itsredox biochemistry [1]. Its rapid doubling time of 1.79 hand high rate of H2 production (77 nmol/min/107 cells) [5],far greater than that of its closest examined diplomonad

relative, G. intestinalis (2 nmol/min/107 cells) [6], is evenhigher than T. vaginalis (20 nmol/min/107 cells) [6], whichcontains well-defined hydrogenosomes. Hence, a diiron(Fe–Fe) hydrogenase and the probable presence of MDOshas been suggested [5] and prompted further investigationinto the source of H2 production by S. vortens. No detect-able cytochromes and no evident cristate mitochondriawere detected, but membrane-potential generating(Figure 1A), large (200–1000 nm) hydrogenosome-like orga-nelles (Figure 1B) and, possibly, small (100–150 nm) puta-tive mitosomes (Figure 1C) were observed in S. vortens usingtransmission electron microscopy (C.O.M.M., PhD Thesis,Cardiff University, 2009). Two mitochondrial genes in thegenome of Spironucleus salmonicida encode proteins(Hsp60 and Nifsp) [7], which are both present in the Giardiamitosome. Thus, there is compelling evidence to support thepresence of MDOs in the genus Spironucleus.

The activity of O2-sensitive S. vortens Fe hydrogenase[5] requires anaerobiosis, and the organism has an O2

consumption rate of 62 nmol/min/107 cells, thus detoxifica-tion by the parasite is highly effective and mediated by aflavodiiron protein [7]. Glutathione is the major antioxi-dant, intracellular non-protein thiol [8]. This is in contrastwith G. intestinalis, which utilizes cysteine, and bearssimilarities to aerobic protists such as Plasmodium spp.[4]. The presence of such effective O2 scavenging mecha-nisms reflects the fluctuating O2 tensions S. vortens islikely to encounter during systemic fish infections andthe ability of this parasite to survive for prolonged periodsoutside the host in the faeces of the infected individualwithout encystation [8].

The 5-nitroimidazole, metronidazole, is the main treat-ment for anaerobic/microaerophilic pathogens, includingG. intestinalis, and was also for S. vortens before its banfrom use in farming of food-fish in the EU and USA [9].Drug activation is by pyruvate:ferredoxin oxidoreductase-mediated nitro group reduction. This inhibits H2 produc-tion and disrupts redox balances in S. vortens [5]. A secondmode of metronidazole activation via thioredoxin reduc-tase has been documented in Entamoeba histolytica,T. vaginalis, and most recently in S. vortens [10]. Garlic-derived compounds have also been documented as analternative therapy for spironucleosis, a manifestationwhich can result in systemic infection and occurs in bothornamental (e.g., angelfish) and food-fish (e.g., salmon).Proteome analysis indicates that garlic derivatives act by adiversity of mechanisms independently of those attribut-able to metronidazole, and also disturb redox balance [10].

Letter

Corresponding author: Williams, C.F. (williamscf@cardiff.ac.uk).

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These findings highlight the differences between twopathogenic diplomonads, the potential variation whichexists in mechanisms for generating ATP in the absenceof mitochondrial oxidative phosphorylation, and theversatility of Spironucleus spp. [11]. Understandingthe diverse biochemistry and physiology of these patho-genic diplomonads, and especially their MDOs, is crucialin the development of new chemotherapeutics and tack-ling drug resistance for effective disease control. Theimminent completion of the S. salmonicida genome[12,13] will not only ease proteomic interpretation butalso make possible further investigation of general char-acteristics and differences between individual diplomo-nads, thereby providing new insights into bothSpironucleus and Giardia.

References1 Williams, C.F. et al. (2011) Spironucleus species: economically-

important fish pathogens and enigmatic single-celled eukaryotes. J.Aquac. Res. Dev. S2, http://dx.doi.org/10.4172/2155-9546.S2-002

2 Lloyd, D. and Harris, J.C. (2002) Giardia: highly evolved parasite orearly branching eukaryote? Trends Microbiol. 10, 122–127

3 Andersson, J.O. et al. (2007) A genomic survey of the fish parasiteSpironucleus salmonicida indicates genomic plasticity amongdiplomonads and significant lateral gene transfer in eukaryotegenome evolution. BMC Genomics 8, 51–76

4 Mu ller, M. et al. (2012) Biochemistry and evolution of anaerobicenergy metabolism in eukaryotes. Microbiol. Mol. Biol. Rev. 76,444–495

5 Millet, C.O.M. et al. (2010) The diplomonad fish parasite Spironucleusvortens produces hydrogen. J. Eukaryot. Microbiol. 57, 400–404

6 Lloyd, D. et al. (2002) Giardia intestinalis, a eukaryote withouthydrogenosomes, produces hydrogen. Microbiology 148, 727–733

7 Andersson, J.O. et al. (2008) Gene transfers from nanoarchaeota toan ancestor of diplomonads and parabasalids. Mol. Biol. Evol. 22,85–90

8 Williams, C.F. et al. Diseases of Aquatic Organisms, in press9 Buchmann, K. (2013) Impact and control of protozoan parasites in

maricultured fishes. Parasitology 1, 1–1010 Williams, C.F. et al. (2012) Disrupted intracellular redox balance of the

diplomonad fish parasite Spironucleus vortens by 5-nitroimidazolesand garlic-derived compounds. Vet. Parasitol. 190, 62–73

11 Kolisko, M. et al. (2008) Molecular phylogeny of diplomonads andenteromonads based on SSU rRNA, alpha-tubulin and HSP90genes: implications for the evolutionary history of the doublekaryomastigont of diplomonads. BMC Evol. Biol. 8, 205–219

12 Jeristsom-Hultquvist, J. et al. (2012) Stable transfection of thediplomonad parasite Spironucleus salmonicida. Eukaryot. Cell 11,1353–1361

13 Roxstrom-Lindquist, K. et al. (2010) Large genomic differencesbetween the morphologically indistinguishable diplomonadsSpironucleus barkhanus and Spironucleus salmonicida. BMCGenomics 11, 258–272

1471-4922/$ – see front matter � 2013 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.pt.2013.04.004 Trends in Parasitology, July 2013, Vol. 29, No. 7

(A)

13.06 µm

(B)

(C)

TRENDS in Parasitology

Figure 1. (A) Confocal micrograph of Spironucleus vortens trophozoites. The

trophozoites are fluorescently stained with DAPI (40,6-diamidino-2-phenylindole;

blue) and TMRE (tetramethylrhodamine, ethyl ester; red) for paired nuclei and

membrane potential of mitochondrion-derived organelles (MDOs), respectively.

Transmission electron micrographs of S. vortens showing: (B) large (200–1000 nm;

scale bar 200 nm); and (C) small (90–170 nm; scale bar 100 nm) electron-dense,

double-membraned organelles (putative hydrogenosome- and mitosome-like

organelles, respectively).

Letter Trends in Parasitology July 2013, Vol. 29, No. 7

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