Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: a potential anti-microbial agent target

International Journal for Parasitology 34 (2004) 297–308 www.parasitology-online.com

Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: a potential anti-microbial agent target Craig W. Robertsa,b, Fiona Robertsb, Fiona L. Henriqueza, Donna Akiyoshic, Benjamin U. Samuelb, Thomas A. Richardsd, Wilbur Milhouse, Dennis Kylee, Lee McIntoshf, George C. Hillg, Minu Chaudhurig, Saul Tziporic, Rima McLeodb,* a

Department of Immunology, Strathclyde Institute for Biomedical Life Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, Scotland G4 0NR, UK b Department of Ophthalmology and Visual Sciences, University of Chicago, Chicago, IL 60637, USA c Tufts University School of Veterinary Medicine, North Grafton, MA 01536, USA d Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK e Walter Reed Army Institute of Research, Bethesda, MD 20170, USA f Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA g Department of Microbiology, School of Medicine, Meharry Medical College, Nashville, TN 37208, USA Received 5 August 2003; received in revised form 6 November 2003; accepted 6 November 2003

Abstract The observation that Plasmodium falciparum possesses cyanide insensitive respiration that can be inhibited by salicylhydroxamic acid (SHAM) and propyl gallate is consistent with the presence of an alternative oxidase (AOX). However, the completion and annotation of the P. falciparum genome project did not identify any protein with convincing similarity to the previously described AOXs from plants, fungi or protozoa. We undertook a survey of the available apicomplexan genome projects in an attempt to address this anomaly. Putative AOX sequences were identified and sequenced from both type 1 and 2 strains of Cryptosporidium parvum. The gene encodes a polypeptide of 336 amino acids and has a predicted N-terminal transit sequence similar to that found in proteins targeted to the mitochondria of other species. The potential of AOX as a target for new anti-microbial agents for C. parvum is evident by the ability of SHAM and 8-hydroxyquinoline to inhibit in vitro growth of C. parvum. In spite of the lack of a good candidate for AOX in either the P. falciparum or Toxoplasma gondii genome projects, SHAM and 8-hydroxyquinoline were found to inhibit the growth of these parasites. Phylogenetic analysis suggests that AOX and the related protein immutans are derived from gene transfers from the mitochondrial endosymbiont and the chloroplast endosymbiont, respectively. These data are consistent with the functional localisation studies conducted thus far, which demonstrate mitochondrial localisation for some AOX and chloroplastidic localization for immutans. The presence of a mitochondrial compartment is further supported by the prediction of a mitochondrial targeting sequence at the N-terminus of the protein and MitoTracker staining of a subcellular compartment in trophozoite and meront stages. These results give insight into the evolution of AOX and demonstrate the potential of targeting the alternative pathway of respiration in apicomplexans. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Alternative oxidase; Immutans; Phylogeny; Apicomplexan; Mitochondria; Cryptosporidium parvum; Plasmodium; Toxoplasma gondii; Antimicrobial

1. Introduction Apicomplexan parasites which include Plasmodium (species), Toxoplasma gondii, Neospora caninum and * Corresponding author. Tel.: þ 1-773-834-4152; fax: þ1-773-834-3577. E-mail address: [email protected] (R. McLeod).

Cryptosporidium (species), Theileria (species) and Babesia (species) are a diverse phylum of considerable medical and veterinary importance (Current and Bick, 1989). There are a number of inadequacies in current treatments for apicomplexans. Notably, multiple drug resistance is a major obstacle for the control and treatment of infection by the four species of Plasmodium (Plasmodium

0020-7519/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2003.11.002

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falciparum, Plasmodium vivax, Plasmodium malaria and Plasmodium ovalae) that cause malaria in humans. Collectively these species account for over 1 million deaths per year, mostly in tropical Africa (Ridley, 2002). Many of the drugs used to treat apicomplexan infections, such as the sulphonamides, are poorly tolerated due to allergy and others such as pyrimethamine are bone marrow toxic, causing neutropenia. Moreover, the inability of present therapeutic regimens to eliminate the bradyzoite stage of T. gondii or the hypnozoites of Plasmodium, each responsible for disease persistence and reactivation, is a major deficiency (Roberts et al., 2002). Finally, in the case of Cryptosporidium parvum there is no effective treatment available, making it a potentially fatal disease, especially in the young, elderly and immune compromised (Hunter and Nichols, 2002; Coombs and Muller, 2002). The benefits of new anti-microbial agents capable of eliminating these parasites and therapeutic problems are clearly evident. A number of potential targets for new anti-microbial agents have recently emerged, including a plethora of prokaryotic and plant like biochemical pathways, some of which are associated with the plastid organelle (apicoplast) (Clough et al., 1997; Fichera and Roos, 1997; Murphy et al., 1997; Lang-Unnasch et al., 1998; McFadden and Roos, 1999; Murphy and Lang-Unnasch, 1999; McLeod et al., 2001; Surolia and Surolia, 2001; Hunter and Nichols, 2002) that has been described in most apicomplexans examined to date (Lang-Unnasch et al., 1998). Analyses so far have not revealed an apicoplast in C. parvum. Consequently, the identification of targets unrelated to this organelle is vital. One such target is the alternative pathway of respiration, which in addition to being present in higher plants, has been described in fungi, green algae and in a limited number of protozoa including Trypanosoma brucei (McIntosh, 1994; Murphy et al., 1997; Murphy and Lang-Unnasch, 1999; Nihei et al., 2002). The alternative pathway of respiration has been most extensively studied in higher plants, which have two pathways for mitochondrial electron flow: the cytochrome respiratory pathway, which is found in all mitochondria and the alternative respiratory pathway, which is absent from mammals (McIntosh, 1994). During conventional mitochondrial respiration electrons are passed from complexes I or II to ubiquinone and then on to the cytochrome oxidases, complex III and IV. At each point protons are transferred across the inner membrane creating a transmembrane potential that is coupled to ATP production. The alternative pathway diverges from the cytochrome pathway after the ubiquinone complex. Electrons flowing through the AOX are donated directly to oxygen to form water (McIntosh, 1994). In addition to the mitochondrial located AOX, many plants have also been demonstrated to have an additional similar oxidase, termed immutans, which is located in their chloroplasts (Wetzel et al., 1994; Carol et al., 1999; Josse et al., 2000).

Recently, cyanide insensitive oxidase activity has been described in P. falciparum and a number of compounds known to inhibit this process in plants, fungi and protozoa have been demonstrated to curtail the growth of P. falciparumin vitro (Murphy et al., 1997; Murphy and Lang-Unnasch, 1999). Although these observations provided evidence for the existence of an alternative pathway of respiration, they do not provide any information regarding the molecular nature of the oxidase involved. Moreover, AOX is notably absent from published annotation of the P. falciparum genome project (Gardner et al., 2002). We therefore surveyed the available apicomplexan genome projects for candidate AOXs and identified the first molecular evidence for an AOX in the apicomplexa. Curiously this evidence comes from C. parvum where the presence of a mitochondrion has been the subject of much recent debate and the enzymes necessary for oxidative phosporylation are apparently absent (Coombs, 1999). These studies also provide information regarding the likely evolution of AOX and the related immutans proteins.

2. Materials and methods 2.1. C. parvum genome projects Preliminary data was obtained from Virginia Commonwealth University/Tufts University School of Veterinary Medicine C. parvum Genome Sequencing Project for the human isolate (genotype 1, NEMC1 strain), web site: http:// www.parvum.mic.vcu.edu/. For the genotype II, IOWA strain, preliminary sequence data was obtained from the University of Minnesota C. parvum Genome (MCPG) sequencing project web site: http://www.cbc.umn.edu/ ResearchProjects/AGAC/Cp/. Each database was searched for DNA sequences that may code for proteins with homology with known AOXs using the tBLASTn algorithm. In the case of the genotype I sequences, where chromatograms are available these were edited and assembled using Sequencher 4.1 (Genecodes, USA). 2.2. Amplification, cloning and sequencing of C. parvum AOX and expression in cultured C. parvum Genomic DNA from oocysts was extracted as previously described (Widmer et al., 1998). Briefly, TU502 (Akiyoshi et al., 2002) and GCH1 oocysts were purified from feces collected from infected calves as previously described (Tzipori, 1998). The putative AOX open reading frame was amplified from genomic DNA using PCR. Primers used to amplify AOX were: sense [50 -ccgctcgtgctgacatgaa-30 ] and antisense [50 -gttcattacctgattatgaaataataacaatctcaag-30 ]. The expression of AOX mRNA in C. parvum-infected Madin-Darby bovine kidney (MBDK) cells in culture was

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examined by reverse transcriptase (RT)-PCR using specific primers for C. parvum AOX, (CpAOXRT sense; 50 -tattacccgctcgtgctcac-30 and CpAOXRT antisense; 50 -cctctggtaagccatagtagacg-30 ). Confluent monolayers of MBDK cells were infected with C. parvum genotype 2 (GCH1 isolate; 5 £ 106) oocysts (Tzipori, 1998). A control flask containing mock-infected MDBK cells was maintained in parallel and was treated in the same manner as the infected cells. The cultures were incubated at 37 8C and 5% CO2 and 95% N2 for 48 h. The uninfected- and infectedMDBK cells were collected by centrifugation (1000 £ g for 5 min) following trypsination and washed once in PBS. The cell pellets were resuspended in PBS (200 ml) and 2.2 ml of RNAlater (Qiagen, UK) added and the cells immediately frozen at 2 80 8C. RNA was extracted using a protocol based on the single-step acid guanidinium thiocyanate – phenol – chloroform RNA isolation method (Chomczynski and Sacchi, 1987). cDNA was produced from 7 mg of RNA using Moloney Murine leukemia virus (MMLV) reverse transcriptase (Invitrogen, Paisley, Scotland, UK) as previously described (Lyons et al., 2001). In a 90 ml reaction volume, 7 mg of RNA was combined with 18 ml of 5 £ first strand buffer (250 mM Tris– HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), 18 ml of deoxynucleoside triphosphate mix (10 mM), 9 ml of 0.1 M dithiothreitol, 80 units of RNAsin ribonuclease inhibitor (Promega), 500 ng of random hexamer primers (Promega) and 1200 units of M-MLV reverse transcriptase. Following a 10 min pre-incubation at 27 8C, the mixture was incubated at 42 8C for 60 min, followed by reaction termination by heating at 95 8C for 5 min. Control reactions that omitted MMLV were carried out simultaneously. All cDNA was stored at 2 20 8C until used in PCR. All PCR reactions were performed in a 25 ml reaction volume and contained a final concentration of 1 £ PCR buffer (50 mM KCl, 10 mM Tris –HCl pH 9.0, 0.1% Triton X-100), 1.5 mM MgCl2, 200 mM of each dNTP, 0.5 mM of each primer and 0.25 units Pfu polymerase (Stratagene). Cycling conditions were an initial denaturation at 94 8C for 3 min followed by 35 cycles of 94 8C for 30 s, 58 8C for 30 s and 60 8C for 1 min. Reactions finished with a final extension period for 10 min at 72 8C. PCR products were separated on a 1.5% agarose gel and visualised by ethidium bromide staining under UV illumination. Following excision from the gel, products were purified using the QIAquick Gel Purification kit (Qiagen) according to the manufacturer’s instructions and ligated into pDRIVE using the Qiagen PCR Cloning Kit (Qiagen). Two microlitres of the ligation reaction was used to transform DH5a using the heat-shock transformation method of Cohen et al. (1972). Automated sequencing was performed on cloned products (MWG biotech, Milton Keynes, UK or MWG Biotech, High Point, NC, USA) and sequences assembled using Sequencher (Genecodes).

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2.3. The effect of SHAM and 8-hydroxyquinoline on growth of C. parvum To assess the effect of SHAM and 8-hydroxyquinoline (8-HQ) on C. parvum, oocysts were added at 50,000/well to confluent MDBK F5D2 cell monolayers in 96-well tissue culture plates, as previously described (Tzipori, 1998). These were incubated at 37 8C under air with 8% CO2 in medium containing serial dilutions of inhibitor. After 48 h the level of infection was determined following immunostaining using rabbit anti-C. parvum serum, followed by fluorescein-conjugated goat anti-rabbit antibody. Sixteen fields were counted at £ 100 magnification and mean parasite count per field determined. Paromomycin (2000 mg/ml) was used in parallel wells as a positive control. 2.4. The effect of SHAM and 8-hydroxyquinoline on growth of T. gondii The effect of SHAM and 8-HQ on in vitro growth of T. gondii was tested by a modification of the method of Mack and McLeod (1984) (Roberts et al., 1998). Briefly, T. gondii (RH strain) were harvested from human foreskin fibroblasts (HFF) infected 5 – 7 days previously and following filtration through a 3 mm filter, counted in a haemocytometer and 2 £ 104 tachyzoites added to the wells of a 96-well tissue culture plate containing confluent HFF monolayers or to four chamber labtek slides. Potential inhibitor compounds were added 1 h later and the plates incubated for 72 h before adding [3H]uracil. Pyrimethamine (0.4 mM) and sulphadiazine (46 mM) were used in combination as a positive control. After incubation for a further 24 h cultures were harvested onto filter paper using an automatic cell harvester and [3H]uracil incorporation assessed by liquid scintillation. Microscopic preparations were also fixed in aminoacridine, stained with Giemsa and analysed as previously described. 2.5. The effect of SHAM and 8-hydroxyquinoline on growth of P. falciparum The effect of SHAM and 8-HQ on the growth of P. falciparum was measured using a modification of the semi-automated micro-dilution technique for assessing antifolates (Milhous et al., 1985). Two clones were used for these tests; the W2 clone is susceptible to mefloquine, but resistant to chloroquine, pyrimethamine and sulphadoxine and quinine; the D6 clone is susceptible to chloroquine, pyrimethamine and sulphadoxine, but is resistant to mefloquine (Oduola et al., 1988). SHAM or 8-HQ was dissolved in DMSO and diluted 400-fold into RPMI 1640 media supplemented with 10% Albumax1 (Gibco). Serial dilutions of SHAM or 8-HQ were added to parasites in culture. The cultures were incubated at 37 8C in 5% CO2 and 90% N2 for 48 h. [3H]hypoxanthine incorporation was

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measured as described previously (Milhous et al., 1985). Then IC50 and IC90 for each clone were determined. 2.6. MitoTracker staining of C. parvum and fluorescence microscopy Sterile glass coverslips, cleaned by sonication and airdried were seeded with 3.75 £ 104 MDBK host cells in complete media and incubated at 37 8C with 8% CO2 for 48 h, following which the host cells were infected with GCH1 strain oocysts and incubated for an additional 48 h, as before. After incubation, the media was replaced with fresh, pre-warmed (37 8C) media containing either 100 or 500 nM of MitoTracker Green FM (Molecular Probes Cat No: M-7514) and incubated, protected from light, for 30 min at 37 8C, as above. The monolayer was then washed thrice with phosphate buffered saline (PBS), fixed in 3% paraformaldehyde in PBS for 15 min at room temperature. To detect host cell and parasite nuclei the coverslips were incubated with the DNA staining dye Hoechst 33342 (Molecular Probes Cat Ni: H 3570), as per manufacturer’s instructions. These were washed three times with PBS, mounted on glass slides and processed for fluorescence microscopy. Coverslips were examined using Zeiss Axiovert inverted fluorescence microscope equipped with cooled CCD camera (MicroMAX). Image capture and deconvolution were performed with Slidebook or Openlab (Improviosn Ltd) on Macintosh Dual Processor G4. Optical sections were taken through the depth of the cell and the software used to deconvolve these images and construct 3D volume views. 2.7. Phylogenetic analyses of alternative oxidase and immutans genes All available AOX and immutans protein sequences were downloaded from GenBank, BLAST searches were used to detect further homologous sequences amongst the submitted and annotated genome data (searched 04/03). These sequences were aligned using the automated alignment program ClustalX (Thompson et al., 1997). The amino acid alignment was refined manually using the program Genetic Data Environment (GDE) (Maidak et al., 1996). The alignment was masked to select sequence regions that were conserved; hyper-variable sequence regions that could not be aligned with confidence were removed. A final alignment of 47 eukaryotic taxa and three prokaryote taxa with 172 amino acid characters was used for phylogenetic analysis. Phylogenetic trees were calculated using the program Mr Bayes 2.01 (Huelsenbeck and Ronquist, 2001) with a gamma correction for variable site rates, with the shape parameter estimated from the data using the JTT matrix. Tree and parameter space was sampled using the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) method implemented in the Mr Bayes program. The MCMCMC analysis was initiated

on a random starting tree and was run for 500,000 generations with four chains run in parallel. The preliminary generations were discarded (the ‘burn in’) and only the generations that constituted a plateau in the MCMCMC were retained and used to calculate the consensus trees shown in Fig. 4. Bootstrap values from a distance analysis were calculated with the program PUZZLEBOOT (Holder, M., Roger, A.J., PUZZLEBOOT version 1.03. http// hades.biochem.dal.ca/rogerlab/software/software.html) using the gamma correction and proportion of invariant sites parameters derived and averaged from the plateau in the MCMCMC search.

3. Results 3.1. Identification and expression of alternative oxidase in C. parvum The genotype 1 C. parvum (TU502 isolate) genome project was searched for DNA sequences with potential to code an AOX using the T. brucei AOX amino acid sequences (Genbank accession: Q26710) and the tBLASTn alogrithim. Four short genomic DNA sequences C. parvum genome project were identified as potential partial genes for C. parvum AOX (cp011120_a331_c11_082.r, cp011130_a354-f03_027.r, cp010319_a015_f01_016.r, and cp011120_a331_c11_082.f). The chromatograms of these sequences were edited and assembled using Sequencher 4.1 (Genecodes). The nucleotide sequence was used to search the C. parvum, genotype II (IOWA strain) genome project and yielded two contigs, gn\CVMUN_5807lcparvum-Contig1622 and gn\CVMUN_5807lcparvum-Contig649, that shared almost complete identity with the 30 and 50 flanking regions, respectively of the sequence assembled for the genotype 1 strain, confirming the presence of this gene in both type 1 and type 2 strains of C. parvum. A region including the ORF was amplified from both strains using PCR and both strands sequenced. The ORFs from the GCH1 (Genbank Accession AY312954) and TU502 (Genbank Accession AY312955) strains are 97.71% identical at the nucleotide level and 97.02% identical at the amino acid level. Each of the resulting sequences yield an open reading frame of 1008 bp that encodes a polypeptide of 336 amino acids in length with a predicted molecular weight of 39.2 kDa. The proteins are predicted to have an N-terminal mitochondrial targeting sequence of 14 amino acids using MitoProt II (Claros and Vincens, 1996), TargetP version 1.0 (Emanuelsson et al., 2000) and Predator (www.inra.fr/servlets/WebPredotar). The predicted mature polypeptide has a theoretical molecular weight of 37.46 kDa. The C. parvum AOX protein has significant identity with previously described AOXs (Fig. 1) including the four highly conserved regions characteristic of alternative oxidases, the LET, NERMHL,

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Fig. 1. Multiple sequence alignment of alternative oxidases from diverse species including the Cryptosporidium parvum alternative oxidase (TU502). Sequences were aligned using CLUSTAL W, within MacVector 7.0. The predicted C. parvum mitochondrial targeting sequence is in bold.

LEEEA and R_DE_H regions, which contain the postulated ligands to the diiron center (Berthold et al., 2000). It has all of the amino acids, previously described as invariant among AOXs (Berthold et al., 2000) in the areas surrounding these four regions with the exception of two, the first of these is an isoleucine instead of a threonine in position 228 of the sequence which it shares with the closely related immutans proteins and the second is an arginine instead of an alanine at position 315. In contrast immutans has an asparagine at this position (Berthold et al., 2000; Fig. 1). Using RT-PCR, and the primers CpAOXRT sense and CpAOXRT antisense, a product of 1070 bp was amplified from mRNA isolated from cultured GCH1 isolate indicating that this gene is transcribed. Control reactions that used RNA isolated from non-infected MBDK cells or where MMLV reverse transcriptase was omitted from the RT reaction did not yield any product, confirming that cDNA was amplified.

100 mg/ml inhibiting approximately 90% of growth (Fig. 2A-B). Paromomycin (2000 mg/ml) used as a positive control inhibited just over 20% of parasite growth. 3.3. The effect of SHAM and 8-hydroxyquinoline on growth of T. gondii SHAM was found to significantly inhibit the in vitro growth of T. gondii over control cultures in a dose dependent manner with 0.78 mg/ml inhibiting over 90% of growth ðP , 0:0001Þ (Fig. 2B). Similarly 8-HQ also inhibited the in vitro growth of T. gondii with concentrations of 2.5 mg/ml inhibiting approximately 80% of parasite growth ðP , 0:005Þ (Fig. 2C). Pyrimethamine and sulphadiazine used in combination as a positive control inhibited greater than 95% of parasite growth. Microscopy (Fig. 3) confirmed these results.

3.2. The effect of SHAM and 8-HQ on growth of C. parvum

3.4. The effect of SHAM and 8-hydroxyquinoline on growth of P. falciparum

SHAM was found to significantly inhibit the in vitro growth of C. parvum over untreated control cultures in a dose dependent manner with 10 mg/ml concentration inhibiting approximately 50% of growth and 100 mg/ml inhibiting approximately 90% of growth ðP , 0:05Þ: 8-HQ also inhibited the in vitro growth of C. parvum with 1 mg/ml concentration inhibiting approximately 50% of growth and

SHAM was found to inhibit the in vitro growth of the W2 and D6 strains of P. falciparum (IC50s of 5.7 mg/ml and 6.2 mg/ml, respectively and IC90s of 43 and 25 mg/ml, respectively). 8-HQ also inhibited the in vitro growth of the W2 and D6 strains of P. falciparum (IC50s of 1.6 mg/ml and 1.2 mg/ml, respectively and IC90s of 4.5 mg/ml and 1.9 mg/ml, respectively) (Table 1).

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Fig. 2. The effect of SHAM and 8-HQ on the in vitro growth of Toxoplasma gondii and Cryptosporidium parvum. SHAM and 8-HQ inhibited the growth of C. parvum (A and B) and of T. gondii (C and D) ðP , 0:05Þ compared with untreated cultures (Media). (PRM, paromomycin, P/S, pyrimthamine/sulphadiazine and RH, T. gondii strain).

3.5. Phylogenetic analyses of alternative oxidase and immutans genes Phlyogenetic analysis was used to investigate the evolutionary origins of eukaryotic alternative oxidase and the plant immutans protein. BLAST searches of all annotated and submitted genome sequence data from GenBank (searched 04/03), revealed three putative prokaryote homologs, two from the Cyanobacterium Nostoc sp. and Synechcoccus sp. and a sequence from the alpha proteobacterium Novosphingobium aromaticivorans. These were aligned with all known eukaryotic AOX and immutans genes and the C. parvum AOX gene reported here. Immutans genes have so far only been detected in genomes of plants, in the case of Capsicum annum and Lycopersicum esculantum the immutan proteins have been shown to functionally locate to the chloroplast organelle (Wetzel et al., 1994; Carol et al., 1999; Josse et al., 2000).

Phylogenetic analysis shows that the immutans genes cluster with the cyanobacterial sequences (see Fig. 4) suggesting that the plant immutans gene have been derived from the endosymbiotic incorporation of the cyanobacterium that led to the establishment of the plant chloroplast (Milani et al., 2001). This evolutionary scenario is consistent with the taxonomic distribution of the immutans gene and the functional localisation of this protein. The alpha proteobacterial putative AOX gene groups within the eukaryotic alternative oxidase phylogenetic cluster. A phylogenetic relationship that suggests the eukaryotic AOX genes have been derived from the endosymbiotic genome of the alpha proteobacterium that led to the mitochondrial organelle (Viali and Arakaki, 1994). Eukaryotic AOX proteins have been demonstrated to function within or closely associated to the mitochondrion organelle (McIntosh, 1994; Nihei et al., 2002). This observation accompanied by the phylogenetic evidence

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Fig. 3. The effect of SHAM on the growth of Toxoplasma gondii in HFF. (i) Control cultures showing large groups of parasites, destruction of the fibroblast monolayer and numerous extracellular parasites (arrow). (ii) Cultures treated with SHAM (0.8 mg/ml). No parasites are identified and the fibroblast monolayer is intact. (Giemsa, £ 120).

makes the eukaryotic AOX a good candidate for an endosymbiotic gene transfer from the mitochondrial or mitochondrial progenitor genome, although other evolutionary scenarios could explain this phylogenetic tree topology, such as horizontal gene transfer (HGT). 3.6. MitoTracker staining of C. parvum trophozoites and merozoites Having shown the presence of alternative pathway of respiration in C. parvum, it was of interest to see if a respiring mitochondrial-like structure(s) could be identified. MitoTracker Green FM is a mitochondrion-selective stain that is concentrated by active mitochondria and well retained during cell fixation. The different morphological forms of C. parvum in culture were identified using differential interference contrast (DIC) microscopy. The adequacy of mitochondrial staining was assessed by looking to see if the host mitochondria are stained, under the experimental conditions used (data not shown). As shown in Fig. 5A, in trophozoites-stage parasites the dye is concentrated and shows a discrete, but beaded staining (pseudocolored-red) pattern. This raises the possibility that the mitochondrion during this stage of intracellular growth is likely to be branched and/or segmented. During the intracellular life-cycle, the fully developed trophozoitesstage parasite undergoes first generation schizogony resulting in the formation of eight merozoites. Fig. 5B shows merozoite-stage parasites with a more diffuse Mitotracker staining pattern. Remarkably, this staining pattern resembles host cell mitochondrial staining, indicating that during this stage of the life cycle the parasite mitochondrial

organelle has undergone a morphological change. Fixing cells with 3% formaldehyde, thus abolishing mitochondrial membrane potential prior to MitoTracker loading, showed only non-specific background staining of cells (data not shown) strongly suggesting that the pattern of fluorescence signal shown in Fig. 5 is mitochondrial specific. The association of host cell mitochondria with a parasitophorous vacuole as described in T. gondii infection has not been shown to occur in C. parvum infection. The images obtained were carefully analysed by deconvolution microscopy with multiple optical Z- sections and 3-D volume views. These views showed that the MitoTracker staining associated with the parasites is within the parasite-body and specifically in relationship with the nucleus of the parasite. This is evident in Fig. 5B (the merozoite-stage).

4. Discussion The presence of an AOX in apicomplexans has been suggested by a number of previous studies (Murphy et al., Table 1 The IC50s and IC90s of SHAM and 8-HQ for the pyrimethamine-resistant (W2) and pyrimethamine-sensitive (D6), Plasmodium falciparum clones P. falciparum clone

Inhibitor

IC50 (mg/ml)

IC90 (mg/ml)

W2

SHAM 8-HQ

5.7 1.6

43 4.5

D6

SHAM 8-HQ

6.2 1.2

25 1.9

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Fig. 4. Phylogenetic tree of the alternative oxidase and immutans genes reveal two potential endosymbiotic gene transfers. The eukaryotic alternative oxidase genes cluster with the alpha proteobacteria suggesting a mitochondrial origin for the eukaryotic alternative oxidase. The plant immutans genes cluster with the cyanobacteria suggesting a chloroplast origin for the plant immutans genes. The phylogenies were calculated from a masked alignment of 50 taxa and a sampling of 172 amino acid characters. Posterior probabilities and Bootstrap values are shown in the respective order (Posterior probability/Bootstrap value) on the branch labels. For full phylogenetic methods see Section 2. AOX sequences that have been reported to have a putative mitochondrial targeting peptide are marked on the phylogeny with (M-tp). Immutans proteins that have been shown to localise to the chloroplast are marked (P-L). The three prokaryote sequences were recovered from annotated genome projects, the Synechoccus sp. and Novosphingobium aromaticivorans are listed as hypothetical proteins in GenBank (see gi 23133458 and gi 23109030).

1997; Murphy and Lang-Unnasch, 1999). Notably, cyanide, an inhibitor of complex IV and thus conventional respiration, was only able to inhibit 70% of oxygen consumption in P. falciparum. Furthermore, SHAM, an inhibitor of AOX, was able to inhibit some of the remaining oxygen consumption (Murphy et al., 1997). These observations are consistent with the ability of SHAM or propyl gallate to potentiate the inhibitory effect of atovaquone, an inhibitor of complex III of the conventional respiratory system, on the in vitro growth of P. falciparum (Murphy and Lang-Unnasch, 1999). However, the recent completion of the P. falciparum genome project has not assisted in identification of any strong candidate AOX, based on sequence comparison with any of the published sequences from plants, fungi or a small

selection of protozoans. We therefore undertook a survey of the available, completed and ongoing, apicomplexan genome projects and identified an AOX in C. parvum. The alternative oxidases thus far described from plants, fungi, and protozoan, have a high level of amino acid conservation and a number of conserved features (Vanlerberghe and McIntosh, 1997; Nihei et al., 2002). These include a cleavable, N-terminal signal sequence and four predicted helices containing regions of high amino acid identity that are postulated to contribute to the diiron binding site proposed in structural models (Vanlerberghe and McIntosh, 1997; Berthold et al., 2000). The C. parvum AOX would appear to share all of these features including an N-terminal mitochondrial targeting sequence. Our future

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Fig. 5. Morphological evidence for the presence of mitochondrion in different life-cycle stages of Cryptosporidium parvum. MDBK host cells infected with GCH1 strain of C. parvum were stained with the mitochondrial dye, MitoTracker Green FM and processed for deconvolution fluorescence microscopy, as detailed in Experimental Procedures. Composite A: Two trophozoites inside parasitophorous vacuoles showing discrete but beaded staining with the mitochondrial dye MitoTracker Green FM (pseudocolored red). Note that the staining is localised to one region of the parasite. Composite B: Collection of merozoites inside a parasitophorous vacuole (arrow). The parasites take up the mitochondrial dye MitoTracker GreenFM (pseudocolored red). Host mitochondria are also evident. HC-host cytoplasm containing host mitochondria. Scale Bar: 5 mm.

work will determine the ability of this N-terminal region to function as a mitochondrial targeting sequence and determine localisation of the C. parvum AOX by empirical means. The presence of mitochondria in C. parvum has been a contentious issue. Early studies suggested the absence of this organelle (Current and Bick, 1989) and a more recent 3D ultrastructural analysis of the sporozoites of C. parvum failed to reveal any structure similar morphologically to mitochondria found in other apicomplexan parasites (Tetley et al., 1998). A small ribosome-studded organelle has been reported to have some structural similarities with mitochondria (Coombs, 1999; Riordan et al., 1999). However, most functional biochemical studies are consistent with C. parvum having an anaerobic metabolism and being amitochondriate (reviewed Coombs, 1999). Ultrastructural studies have shown the presence of mitochondrion-like structure in the merozoites of a closely related species, Cryptosporidium muris (Uni et al., 1987). Cryptosporidium has a complex life cycle. It is conceivable that during differentiation the putative mitochondrion undergoes changes in form and metabolic function in different life cycle stages making it difficult to visualise by morphological analysis. For example, in a related apicomplexan parasite P. falciparum, the number and morphological

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features of mitochondrial structures differ markedly (Divo et al., 1985; Krungkrai et al., 2000) with possible close association with the plastid organelle (Hopkins et al., 1999) making visualisation difficult. Nonetheless, classical inhibitors of the respiratory chain such as cyanide and azide are not active against the sporozoite stages of C. parvum in vitro (Brown et al., 1996) and atovaquone is not active in a murine model (Coombs, 1999). Furthermore, enzyme activities associated with the TCA cycle would appear to be absent at least from the sporozoite stage (Denton et al., 1996; Entrala and Mascaro, 1997). More recent studies have reported a number of normally mitochondrial located nuclear encoded proteins including valyl-tRNA synthase, adenylate kinase and chaperonin (Cpn) 60 (Coombs, 1999; Riordan 1999). However, C. parvum has been shown to possess a pyruvate:ferredoxin oxidoreductase/NADPHcytochrome P450 reductase (PFOR) similar to the protein found in Euglena gracilis, with the major difference being the absence of a N-terminal mitochondrial targeting sequence (Rotte et al., 2001). The apparent loss of the Nterminal sequence has been suggested as evidence of evolutionary adaptation accompanying the loss of the mitochondrion. However HSP70 in the microsporidian, Trachipleistophora hominis has been shown to localise to the remnant mitochondrion in the absence of an identifiable mitochondrial targeting peptide (Williams et al., 2002). The present study supports the presence of a mitochondrial compartment both through the identification of a C. parvum AOX and through MitoTracker staining of structures in both the trophozoite and merozoite stages. Available antibodies to AOX from one heterologous species (a monoclonal antibody to an alternative oxidase from the aroid lily Sauromatium guttatum) is known to recognise the specific AOX peptide RADEAHHRDVNH (Finnegan et al., 1999). The A2 and seven C terminal amino acids are essential for the interaction. This antibody did not recognise C. parvum alternative oxidase as would be predicted since in C. parvum AOX this peptide is RRDESHHRDVNH (i.e., the C. parvum peptide lacks the A2). Ablation of mitochondrial activity (using formalin) did make the MitoTracker signal disappear (data not shown). Although the existence of a mitochondrial compartment in C. parvum has been controversial, our data described herein and additional very recent data further supports existence of a mitochondrial compartment. Riordan et al. (2003) have sequenced Cpn60 and demonstrated by immunolocalisation that this mitochondrial associated molecule is present in an organelle that they refer to as a mitochondrial relic. With the absence of any good candidate AOXs identified from the P. falciparum genome project, we further investigated the role of known inhibitors of this molecule on the growth of P. falciparum and T. gondii. This confirmed the previously reported ability of SHAM to inhibit the in vitro growth of P. falciparum and demonstrated that 8-HQ, another known inhibitor of this molecule,

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restricts the growth of this parasite at similar concentrations. This was independent of the two strains of P. falciparum investigated. In addition, we find that these two compounds are active in vitro against T. gondii. Previous studies failed to detect cyanide insensitive respiration in extracellular T. gondii tachyzoites (Murphy et al., 1997), however, the studies reported herein examined the effect of AOX inhibitors on the replication of intracellular tachyzoite stages. The apparent lack of AOX activity in extracellular T. gondii may reflect differences in oxygen consumption between intracellular and extracellular tachyzoites. In support of this, a reduction in mitochondrial membrane potential is evident after tachyzoites invade a host cell (Tanabe and Murakami, 1984). This is a hypothesis that could be tested. In future studies it would be of interest to determine whether intracellular T. gondii tachyzoites, tachyzoites converting to bradyzoites, bradyzoites or C. parvum have such activity. These studies would be challenging, if possible at all, because of the large numbers of parasites required. Cyanide insensitive respiration that can be inhibited by SHAM has been demonstrated in P. falciparum (Murphy et al., 1997), although no P. falciparum AOX has been identified in the genome project that currently detects , 70– 80% of ORFs. Both SHAM and 8OH quinoline inhibit AOXs. Interestingly, we found that both SHAM and 8OH quinoline inhibit all three apicomplexan parasites with similar IC50 s, although, as for any inhibitor study, selectivity is not proven. The presence of AOX in T. gondii was further investigated by western blotting of tachyzoites with antibodies raised to AOXs from a number of heterologous species. In preliminary studies (data not shown), we found that five distinct antibodies (two distinct monoclonals, called IA2 and 7D3, to T. brucei AOX; a monoclonal to Voo Doo Lilly AOX; a murine polyclonal to Voo Doo Lilly AOX; and a murine polyclonal to T. brucei AOX) reacted with a 66 kD T. gondii protein in parasite lysates under nonreducing conditions and a 33 kD protein under reducing conditions. AOXs are 66 kDa dimers which reduce to 33 kD monomers in exactly these conditions and react with these antibodies. Examination of the T. gondii genome, currently at 10 x coverage, and the vast number of ESTs available from different strains and life cycle stages did not definitively identify an AOX although a number of candidates are currently being pursued and this task may become easier when gene prediction has been optimised for this organism. However, the apparent absence of any strong candidate AOX in the P. falciparum genome project (Gardner et al., 2002), raises the possibility that the molecule responsible for cyanide resistant respiration and susceptible to inhibitors of AOXs may bear little sequence similarity with previously described AOXs. The description of the first apicomplexan AOX raised the question of the origin of AOX in these parasites and

prompted phylogenetic analyses of the newly discovered sequence and available AOX sequences and immutans sequences. These analyses tested the hypothesis that both AOX and immutans are derived from separate endosymbiotic gene transfers (EGT). Support for this hypothesis was first obtained through identification of putative AOXs in alpha proteobacteria and two putative cyanobacterial immutans proteins. These prokaryote groups include the probable progenitors of the mitochondrial and chloroplasts organelles (Josse et al., 2000; Milani et al., 2001; Richards et al., 2003). We find no evidence to reject this hypothesis, indeed phylogeny suggest that these genes are derived from endosymbiotic gene transfers, although other evolutionary scenarios such as independent horizontal gene transfer events could explain the phylogenetic relationships observed (Richards et al., 2003). However, the subcellular localisation of these proteins, where studied, combined with our phylogenetic investigation suggests that these genes are derived from endosymbiotic origins from the mitochondria in the case of AOX and the chloroplast in the case of immutans. This is the most parsimonious evolutionary scenario given that the bacterial sampling available does not enable a comprehensive phylogenetic evaluation of these putative EGTs. Further sequencing of bacterial homologues may necessitate the re-evaluation of the phylogeny and the hypothesised evolutionary origin of these eukaryote genes. The role of the AOX C. parvum remains to be determined. Electron flow via the AOX does not contribute to transmembrane potential and two of the three potential coupling sites for proton transport and thus ATP production are lost (Vanlerburghe and McIntosh, 1997). Since the energy of electron flow through the pathway is not conserved as chemical energy, it is lost through the generation of heat (Vanlerburghe and McIntosh, 1997). The ability of this heat to assist in the dispersion of insect attractants and thus pollination during the flowering of Sauromatum guttatum (Voodoo Lily) is the only proven function of AOX in higher plants (Vanlerburghe and McIntosh, 1997). Nonetheless, a number of other possible advantages of using AOX have been suggested. As many plants release cyanide following damage or infection, the AOX would provide a cyanide resistant means to maintain at least partial mitochondrial function (Vanlerburghe and McIntosh, 1997). In addition, AOX has been suggested to function in an energy overflow system, maintaining a partial electron transport chain to allow TCA cycle to proceed in the absence of adenylate regulation (Vanlerburghe and McIntosh, 1997). In T. brucei, the AOX and the conventional cytochrome oxidases are expressed in a stage specific manner. The procyclic stages, found in the tsetse fly, have well developed cristae in their mitochondria and synthesise ATP by oxidative phosporylation. In contrast, long slender forms of the parasite, found in the blood stream of the mammalian host are reliant on glycolysis, which takes place in

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the glycosome. The NADH produced as a result of glycolysis is re-oxidised in the mitochondria by a system comprising, glycerol-3-phosphate dehydrogenase, ubiquinone and AOX. The mitochondria of the procyclic forms lack cytochrome oxidases and are incapable of oxidative phosphorylation (Nihei et al., 2002). The apparent absence of a conventional respiratory chain in C. parvum raises the possibility that it may also posses a modified respiratory chain similar to that observed in T. brucei. The advantage conferred in these circumstances would be the ability to reoxidise NADH in the anaerobic environment in which it survives for most of its life cycle. In the case of T. gondii and P. falciparum that are capable of oxidative phosporylation, the advantage of employing an AOX for energy demands would not be clear. However, use of the AOX by plants has been demonstrated to reduce the levels of mitochondrial reactive oxygen species (ROS) which may confer advantages to protozoan parasites which in addition to their endogenously produced reactive oxygen species have to contend with host immune response derived ROS (Vanlerburghe and McIntosh, 1997). It has also been reported that while the conventional terminal cytochrome oxidases are susceptible to inhibition by nitric oxide, the AOX is resistant (Millar and Day, 1996). This would confer a considerable advantage to many intracellular protozoan parasites as this molecule is produced during the immune response and has been shown to restrict their growth (Alexander and Hunter, 1998). Acknowledgements Preliminary data was obtained from Virginia Commonwealth University/Tufts University School of Veterinary Medicine Cryptosporidium parvum Genome Sequencing Project for the human isolate (genotype 1, TU502 strain), web-site: http://www.parvum.mic.vcu.edu/. For the genotype II, IOWA strain, preliminary sequence data was obtained from the University of Minnesota Cryptosporidium parvum Genome (MCPG) sequencing project web-site: http://www.cbc.umn.edu/ResearchProjects/ AGAC/Cp/. We thank Susan Chapman for technical support for the Madin Darby cell work and Robert P. Hirt for guidance on phylogenetic analyses. This work was funded by NIH, USA RO1 AI-43228 (R.McL, C.W.R), NO1 AI-25466, RO1 AI-50471 (S.T), Koshland, Breenan, Blackmon, Langel and Kiewit families. T.A.R. is supported by a BBSRC studentship. R. McLeod is recipient of the Jules and Doris Stein Research to Prevent Blindness Professor at the University of Chicago. References Akiyoshi, D.E., Feng, X., Buckholt, M.A., Widmer, G., Tzipori, S., 2002. Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species. Infect. Immun. 70, 5670–5675.

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