The genes encoding cAMP-dependent protein kinase catalytic subunit homologues of the microsporidia Encephalitozoon intestinalis and E. cuniculi: molecular characterisation and phylogenetic analysis

Parasitology International 53 (2004) 277 – 285 www.elsevier.com/locate/parint

The genes encoding cAMP-dependent protein kinase catalytic subunit homologues of the microsporidia Encephalitozoon intestinalis and E. cuniculi: molecular characterisation and phylogenetic analysis Leila Equinet a,1, Eric Bapteste b,1, Marc Thellier c, Meryem Ouarzane-Amara c, Christian P. Vivare`s d, Isabelle Desportes-Livage c, Christian Doerig a,* a

INSERM U609, Wellcome Centre for Molecular Parasitology, University of Glasgow, 56 Dumbarton Road, Glasgow G11 6NU, Scotland, UK b Phyloge´nie, Bio-informatique et Ge´nome, UMR CNRS 7622, Universite´ Pierre et Marie Curie, Paris 6, Paris, France c INSERM U511, Centre Hospitalier et Universitaire Pitie´-Salpeˆtrie`re, Universite´ Pierre et Marie Curie, Paris 6, Paris, France d Laboratoire de parasitologie mole´culaire et cellulaire, UMR CNRS 6023, Universite´ Blaise Pascal, Aubie`re, France Received 21 December 2003; received in revised form 11 February 2004; accepted 3 March 2004 Available online 11 May 2004

Abstract A gene encoding a protein kinase was identified by homology-based PCR amplification in Encephalitozoon intestinalis, a microsporidian parasite pathogenic to humans, and its orthologue has been identified by database mining in the genome of the related species E. cuniculi, whose sequence has been recently published. Phylogenetic analysis revealed that the proteins encoded by these genes are homologues of the cAMP-dependent protein kinase catalytic subunits (PKAc). Southern blot analysis indicated that the EiPKAc gene is present in two copies in the E. intestinalis genome, whereas the E. cuniculi orthologue (EcPKAc) is a single copy gene. RT-PCR data showed that the EiPKAc gene is expressed in at least one of the intracellular stages during infection of the mammalian host cell by E. intestinalis. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Microsporidia; Protein kinase; Protein kinase A; Phosphorylation; Phylogeny

1. Introduction Microsporidia are obligate intracellular eukaryotic parasites found in a wide diversity of animal hosts [1]. These organisms have emerged as opportunistic pathogens during the AIDS pandemics, and several species including Encephalitozoon intestinalis and E. cuniculi were found to cause infections in humans [2]. These parasites are disseminated by spores, which possess a unique invasive apparatus consisting of the so-called polar tube, the extrusion of which ensures the

Abbreviations: bp, base pair; cAMP, cyclic adenosine monophosphate; EST, expressed sequence tag; kb, kilobase; kDa, kilodalton; ORF, open reading frame; PCR, polymerase chain reaction; PKA, protein kinase A (or cAMP-dependent protein kinase); PKAc, PKA catalytic subunit; PKAr, PKA regulatory subunit; RT, reverse transcriptase. * Corresponding author. Tel.: +44-141-339-8855x6201; fax: +44-141330-5422. E-mail address: [email protected] (C. Doerig). 1 Leila Equinet and Eric Bapteste have contributed equally to this work. 1383-5769/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2004.03.001

discharge of the infectious sporoplasm into the host cell. The lack of mitochondria and peroxysomes and the atypical organisation of Golgi membranes led to the conclusion that microsporidia were primitive amitochondriate eukaryotes [3]. However, the subsequent finding of genes encoding mitochondrial proteins in microsporidian species, including E. cuniculi, whose genome has recently been entirely sequenced, together with recent phylogenetic analyses, suggest that these parasites are related to fungi [4– 6]. The genome of these organisms is dramatically reduced in size [7], and it is now widely accepted that their primitive features result from secondary losses reflecting high level of specialisation to intracellular parasitism [8]. The intracellular development of microsporidia begins with a proliferative phase, merogony, which generates cells that subsequently develop into sporonts. Sporonts also divide, but their daughter cells arrest their cell cycle and differentiate into mature spores that are released after host cell death [1]. The molecular mechanisms controlling proliferation and development in microsporidia are still

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largely unknown. We are interested in determining whether regulators of cell division and differentiation can be considered as targets for novel anti-parasitic compounds. In this context, the effect on microsporidia of compounds known to be anti-mitotic in other systems has been characterised [9]. A complementary approach consists of identifying potential microsporidian signal transduction pathway components and cell cycle regulators, and using such elements in chemical library screening procedures. In several eukaryotes, including numerous protists, the cAMP pathway plays a central role in many developmental processes. For example, cAMP triggers the mating responses of Chlamydomonas reinhardtii gametes [10], and is thought to play a crucial role in the differentiation of bloodstream forms in Trypanosoma brucei [11]. cAMP or activators of adenylyl cyclase enhance metacyclogenesis in T. cruzi [12] and cAMP appears to be implicated in Plasmodium falciparum sexual differentiation [13]. During aggregation of Dictyostelium discoideum, extracellular cAMP acts as chemotractant and inducer of cellular differentiation, whereas intracellular cAMP presumably acts by activation of the catalytic subunit of the protein kinase A, to facilitate early development [14]. cAMP-dependent protein kinase (protein kinase A or PKA) activity has been implicated in numerous cellular processes in unicellular fungi [15], including virulence in a pathogenic Cryptococcus species [16]. The eukaryotic cAMP pathway is generally activated by the binding of a ligand to a membrane receptor, which causes dissociation of heterotrimeric GTP-binding proteins (G-proteins). This leads to activation of a membrane-bound adenylate cyclase, which results in an increase in intracellular cAMP concentration. In eukaryotic cells, PKA exists as an inactive complex of catalytic and regulatory subunits. In unstimulated mammalian cells, the catalytic subunit of the PKA (PKAc) is bound to inhibitory regulatory subunits (PKAr) in a tetrameric complex composed of two PKAr and two PKAc molecules. Binding of cAMP dissociates the complex and thereby activates the kinase function of the catalytic subunit, which then phosphorylates a number of substrates including transcription factors, resulting in changes in the gene expression pattern (reviewed in Ref. [17]). Protein kinase genes have undergone many duplication events through evolution. This large multigenic family displays a wide variety of functions and is classified into several groups on the basis of amino acid sequences (http:// www.sdsc.edu/kinases/). Furthermore, supplementary duplications occurred inside clearly identified functional groups, complicating the history of this gene family. For example, in the PKA group, two isoform sequences are present in Leishmania major [18,19] and Drosophila [20], while Saccharomyces cerevisiae and mammals contain three copies (Tpk1, Tpk2, Tpk3 and a, b, d, respectively) [21,17]. Clues about the function of a novel kinase may be gained from

determination of its phylogenetic position relative to wellcharacterised groups. Here, we report the identification, characterisation and phylogenetic analysis of putative PKAc genes in E. intestinalis and E. cuniculi.

2. Methods 2.1. Culture of parasites and DNA isolation E. intestinalis and E. cuniculi spores were collected from monolayers of rabbit kidney cells (RK13) [22]. Spores were harvested every 3 days and centrifuged at 1500  g for 5 min to eliminate cellular fragments. Sieved spores were then sedimented by centrifugation at 4000  g for 20 min and washed twice in sterile phosphate-buffered saline (PBS). Spores were purified on Percoll-gradient as described in Ref. [23] and stored at 80 jC until use. Genomic DNA was obtained from spores using the Invitrogen DNA extraction kit following recommendations from the supplier. 2.2. Molecular cloning of EiPKAc and EcPKAc Degenerate primers (forward: ggNgCNTAYggNgTNgT; reverse: CKNgCNARNCCRAARTC) designed to hybridise to conserved regions of serine/threonine kinases were used to amplify part of the kinase catalytic domain from E. intestinalis genomic DNA (see Fig. 1). Conditions for PCR amplification of gDNA with Taq polymerase (Gibco) were 94 jC for 1 min, 50 jC for 45 s, and 72 jC for 1 min for 35 cycles. PCR products were inserted into pGEMTEasy vector (Promega), transformed into BL21 Escherichia coli, and sequenced using an ABI Prism Sequencer. Additional sequences flanking the first PCR product were obtained using an inverse PCR approach [24]. The E. cuniculi ORF encoding the putative PKAc homologue was identified by BLAST analysis and amplified from genomic DNA (no intron predicted from the NCBI database) using the following primers: forward, CgCggATCCATgTTgAAgATCCAggACTT); reverse, CCggAATTCTTAATTTACAgTCCCTTCgAg (restriction sites: italic, Start and Stop codons: bold italic). The PCR product was digested with BamH1 and EcoR1 and inserted into the pGEX-4T-3 expression vector (Amersham Pharmacia Biotech). The entire insert of the resulting pGEX-4T-3EcPKAc construct was verified by DNA sequencing prior to expression in E. coli. Expression was induced by 0.5 mM isopropyl-h-thiogalactopyranoside (IPTG) at 37 jC during 3 h, and purification of recombinant GST-EcPKAc was performed as previously described [25]. Protein concentration was estimated by using the Bio-Rad dye reagent according to the manufacturer’s recommendations. Aliquotes of purified protein were analysed by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining.

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Fig. 1. CLUSTALW alignment of the catalytic domain of E. intestinalis (AJ507058) and E. cuniculi (NP_585980) PKA with that of T. gondii PKA (AF288604), P. falciparum PKA (U78291), S. pombe PKA (P40376), H. sapiens PKAc-like (X85545) and D. melanogaster DC2 (X16961). The 15 invariant residues of serine/threonine kinase [36] are indicated by an (o) at the top, and the two protein kinase signatures [35] are underlined. Black and grey shadings label residues that are identical or similar, respectively, in at least five of the aligned sequences. Dashes (-) indicate gaps introduced in the sequences to optimise alignment, and dots (.) in the E. intestinalis sequence indicate the region of the protein whose sequence was not determined. The FxxF motif at the C-terminus discussed in the text is boxed. The conserved regions from which the degenerate primers used to identify the E. intestinalis sequence were designed are indicated by arrows.

2.3. Kinase assays

2.4. Southern blot analysis

PKA activity was assayed by measuring 32P incorporation into a synthetic peptide (Kemptide, Sigma), as previously described [26]. Recombinant mouse PKAc (a kind gift from F. Traincard, Institut Pasteur, Paris) was used as a positive control.

Genomic DNA from E. intestinalis spores and from rabbit kidney (RK13) cells DNA was digested overnight with EcoRI or BamHI or RsaI (Biolabs), and separated by agarose electrophoresis (1 – 5 Ag/lane). Southern blot procedure was performed as described in Ref. [27]. The probe

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was a 433 bp PCR product prepared using forward (172gACTTTggggAAATCCTTAAg-192) and reverse (605gCACgTCCATCATCTTCgCTC-585) oligonucleotides, and radiolabelled using the megaprime DNA labelling system according to the manufacturer’s recommendations (Amersham). 2.5. RT-PCR analysis RNA was isolated from a T-25 culture flask at 50 h postinfection. Cells were treated with TRIzolR Reagent according to the manufacturer’s protocol (GibcoBRL), followed by phenol – chloroform extraction and ethanol precipitation. Reverse transcription reactions were carried out with 200 U of reverse transcriptase (Clontech laboratories) for 1 h at 37 jC. cDNA samples were subjected to PCR amplification using the EiPKAc-specific primers and conditions described above. 2.6. Phylogenetic analyses of the microsporidian PKA[c] ORFs 2.6.1. Construction of a global dataset Serine/threonine protein kinases of the AGC group form a large multigenic family (see ttp://pkr.sdsc.edu). To place the E. cuniculi sequence, we decided to retain first only some representatives of plants (Arabidopsis thaliana), fungi (S. cerevisiae and Schizosaccharomyces pombe), and metazoa (Drosophila melanogaster, Caenorhabditis elegans and Homo sapiens). Related sequences were identified through BLAST searches [28] and retrieved from NCBI (http://www.ncbi.nlm.nih.gov/blast/). This led to the construction of a first dataset of 474 sequences that were aligned, together with the new sequence determined in this study, using CLUSTALW [29]. The resulting multiple alignments were manually edited using the program ED from the MUST package [30], and 183 partial and redundant sequences were excluded from our alignment. 2.6.2. Definition of paralogous groups Preliminary phylogenetic analyses of this dataset, excluding ambiguously aligned positions, were pursued to identify ancient groups of paralogs resulting from duplication events in the common ancestor of plants, fungi and metazoa. Phylogenetic trees were constructed using distance (neighbour-joining [NJ] [31] and maximum-parsimony [MP]) with the MUST version 1.0 package (program NJ) [30] and PAUP 4b8, respectively. Phylogenetic distances were computed with the method of Kimura [31]. MP trees were obtained from 100 random addition heuristic search replicates and the tree bisection –reconnection branch swapping option. This allowed the identification of a minimum of 26 paralogous groups.

2.6.3. Addition of protist sequences in the paralogous groups Complete genome and expressed sequence tags (EST) projects were also examined, with the ‘est’ program. This program (H. Philippe, personal communication) seeks homologous EST sequences for various protists and Opisthokonta, starting from a seed in each predefined group of paralogy, and incorporates the new sequences if their BLAST score is inferior to a threshold determined by the user. Each new sequence was then automatically aligned, using a blast alignment on the most similar aligned sequence of the file as reference (baba program, H. Philippe, personal communication). This process allows to easily add a large number of closely related sequences to a complex alignment. EST sequences were then manually edited, and when overlapping, concatenated to reconstruct a larger sequence. In this way, the sequences of D. discoideum (http://genome.imb-jena.de/dictyostelium), Entamoeba histolytica (http://www.ncbi.nlm. nih.gov/dbGSS/index. html), and Toxoplasma gondii (http://www.ncbi.nlm.nih.gov/dbEST/index.html) were retrieved and are included in the final phylogenetic analysis. 2.6.4. Preliminary phylogenetic analyses to identify which paralogous groups are close to the microsporidian sequence These preliminary phylogenetic analyses also indicated that the E. cuniculi sequence was closer to four paralogous groups among the 26 identified. This phylogenetic proximity was confirmed by a tBLASTn from E. cuniculi, launched on the whole genome of S. cerevisiae. A careful examination of the BLAST score of the homologous sequences retrieved revealed that two gaps (of 1e 4 and of 1e 5) separated the sequences of S. cerevisiae of the same four groups from those of the rest of the alignment (data not shown). 2.6.5. Construction of the studied dataset Among these four groups, 48 sequences were retained for further phylogenetic analyses. Our choice of species was aimed at representing the diversity of eukaryotes in the paralogy group and the numerous duplication events intervened in the fungi (up to three) and in the metazoa (up to seven) for the different paralogy groups. These secondary duplications were represented each time by two sequences from fungi (S. cerevisiae and S. pombe) and two sequences from metazoa (H. sapiens and D. melanogaster or C. elegans, if Drosophila was lacking or too divergent). See Fig. 4 for a list of the sequences retained for analysis. 2.6.6. Phylogenetic analysis Maximum likelihood (ML) trees were constructed with the program PROTML from the MOLPHY 2.3 package, using the quick search option and the JTT substitution model (options -jf -q -n 1000) [32], and with the program

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ProML from the PHYLIP package, version 3.6 [33], using the JTT substitution model with a G-law for eight rate categories. Branch lengths were re-calculated on the best topology with the program PUZZLE [34] using a G-law to correct for among-site rate variation. Bootstrap proportions were estimated using the RELL method upon the 1000 topranking trees obtained by ProtML and using the NJBOOT program of MUST package, allowing to take alpha parameter into account. We also tested further the positioning of E. cuniculi on seven topologies manually reconstructed from the best one, moving E. cuniculi at the base of every group of fungi in the best MLtree. These different tree topologies were statistically compared to the best one using the AU tests with the programs PROTML [32], PUZZLE [34], and CONSEL (http://www.ism.ac.jp/ f shimo/).

3. Results and discussion 3.1. Identification of E. intestinalis and E. cuniculi PKAc homologues

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3.2. Gene copy number For Southern blot analysis, genomic DNA isolated from E. intestinalis spores and from RK13 host cells was digested with EcoRI and BamHI (for which no site exists in the known portion of the EiPKAc ORF), and with RsaI, which cuts once within the ORF. With the EcoRI and the BamHI digests (Fig. 2, lanes 1 and 2), the probe we used gave two bands of identical intensity. The RsaI restriction site is located within 45 bp of the 3V end of the 433 bp probe, so we expect that only one RsaI band per gene copy in the genomic DNA should give a strong signal. Two strong bands are indeed visible in lane 3, which is consistent with the data obtained with EcoRI and BamHI patterns. The lanes containing DNA obtained from host RK13 cells as a negative control yielded no signal, confirming that the EiPKAc ORF used as a probe is of parasitic origin. Our interpretation of these data is that the EiPKAc gene is present in two copies in the E. intestinalis genome. In contrast, the E. cuniculi genomic database indicates that EcPKAc is represented only once in the genome of this species. 3.3. Expression of EiPKAc during infection of the host cell

In the context of a PCR-based search for protein kinase genes from E. intestinalis, a 315 bp PCR product was obtained from genomic DNA using degenerate primers designed to hybridise to conserved regions of the protein kinase catalytic domain (see Fig. 1). This PCR product contained a continuous ORF, which appeared, upon BLASTP analysis, to have been amplified from a gene encoding a protein related to cAMP-dependent protein kinases. An inverse PCR approach allowed us to sequence a total of 663 bp at the N-terminal part of the ORF (including the putative start codon), i.e. approximately 70% of the inferred full length EiPKAc ORF (based on a comparison with other members of this family, including the 996 bp orthologue from the closely-related species E. cuniculi, see below and Fig. 1). At this stage, the genome sequence of E. cuniculi was published [7], and we identified in the resulting database the orthologue of this enzyme, EcPKAc (accession number: AL590447), which displays 86% identity with the E. intestinalis sequence at the aminoacid level (Fig. 1). Both the EiPKAc and EcPKAc ORFs carry the two signatures, which, when present in the same polypeptide, unambiguously assign it to the protein kinase family [35]. In addition, all 15 invariant residues that are conserved in protein kinases [36] are present in both sequences (Fig. 1). Moreover, all highest scores obtained after BLASTP analysis correspond to PKAc subunits from a variety of organisms. Although this suggests that these genes encode true orthologues of PKAs, we performed a detailed phylogenetic analysis of these sequences (see below) to further ascertain this point. It is of interest to mention here that examination of the E. cuniculi database reveals the presence of a putative PKA regulatory subunit homologue (NP_597223).

We used RT-PCR analysis of total RNA from infected or uninfected RK13 host cells to determine whether the EiPKAc gene is expressed during the intracellular phase of the life cycle of the parasite. The data clearly show that EiPKAc mRNA is present in infected cells (Fig. 3, lane 4). The samples in which reverse transcriptase had been omitted from the reaction (lane 1 and 2) yielded no amplified product, allowing us to exclude that the signal in lane 4 was due to contamination with genomic DNA. RT-PCR from uninfected RK13 DNA was negative (lane 3), excluding the possibility that the signal in lane 4 was due to amplification from host cell mRNA.

Fig. 2. Southern analysis. DNA from uninfected RK13 host cells (left panel) or from E. intestinalis spores obtained from RK13 cell cultures (right panel) was digested with EcoR1 (lane1), BamH1 (lane 2) or Rsa1 (lane 3). Following gel electrophoresis and transfer, the membrane was hybridised to a radiolabelled 433 pb probe spanning positions 172 – 605 of the EiPKAc sequence (AJ507058). Sizes are indicated to the right.

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Fig. 3. RT-PCR analysis. RT-PCR products obtained from total RNA from uninfected (lanes 1 and 3) or E. intestinalis-infected (lanes 2 and 4) RK13 cells using EiPKAc-specific primers. Reverse transcriptase was omitted in the reactions corresponding to lanes 1 and 2. Lane 5 presents the PCR product amplified from E. intestinalis genomic DNA using the same primers.

The parasite develops asynchronously in the host cells [1], and therefore several developmental stages coexist in any given preparation of parasite material. This renders a study of the time-course of gene expression impossible using RT-PCR or Northern blot approaches. Nevertheless, our data demonstrate that E. intestinalis expresses PKAc at least during some stage of infection of its host cell. 3.4. Recombinant protein and kinase activity assays By analogy with the role of the cAMP pathway in other unicellular eukaryotes, it seems reasonable to propose that microsporidian PKAc may represent interesting potential drug targets. We attempted to produce an active recombinant EiPKAc protein as a tool to screen chemical libraries, as has been done with other parasitic protein kinases [37]. A recombinant protein containing the entire EcPKAc fused to glutathione S-transferase (GST) was expressed in E. coli and purified on glutathione beads. A protein with the expected apparent 60 kDa molecular weight was readily obtained from induced bacteria, and as expected, reacted with an anti-GST antibody (data not shown). This recombinant protein was used in in vitro kinase assays, using the Kemptide peptide (a classical substrate for PKAs) as an exogenous substrate. No activity was detected, although a recombinant murine PKAc, used as a positive control, was strongly active in these conditions. This may be due to a requirement for essential co-factors or post-translational modifications, and may reflect possible differences in the modalities of regulation of activity between the mammalian and microsporidian (and plasmodial) enzymes. The recombinant PKAc subunit from P. falciparum also appears to lack kinase activity (Merckx, Parzy, Langsley, Doerig, unpublished). Both E. cuniculi and P. falciparum PKAc lack the FXXF motif at the C-terminus (Fig. 1), which has been shown to be important for activity and stability of the Dictyostelium homologue [38] and is conserved in PKAc subunits from most organisms. 3.5. Phylogenetic analysis The history of the protein kinase (PK) gene family is complex. Our phylogenetic analysis of kinases sequences related to EcPKAc revealed a large number of duplication

events of very different ages as well as several independent gene losses. For example, an ancient duplication led to the PKA group: this event took place in an early ancestor of eukaryotes, as suggested by the presence of PKA in Opisthokonta (a monophyletic group of fungi and metazoa) and of various protists such as Kinetoplastids [e.g. L. major [18,39] and T. cruzi [39]], Apicomplexa (e.g. T. gondii, P. falciparum [26]) and Conosa (D. discoideum), whereas it appears to be absent from plants (Fig. 4). Moreover within this group, metazoa underwent supplementary duplication events (one of them prior to the arthropods and mammals divergence), leading to the presence of two PKAc copies in Drosophila and three copies in humans; likewise, duplication events also occurred in other lineages, leading for example to the three isoforms present in yeast. One of the biological consequences of duplication is the production of gene copies evolving at various speeds, since a duplicated gene would be more free of selection pressure and one copy could thus accumulate more mutations than the other. Such various evolutionary rates are well known to bias phylogenetic reconstructions, since the presence of fast evolving sequences can result in a long branch attraction artefact (LBA). LBA tends to misplace the fast evolving sequences, which emerge with a very good statistical support at basal nodes in the phylogenetic trees [40]. Multiple gene duplications in the kinase family have (probably) affected the evolutionary rates, making phylogenetic inferences difficult. Hence the phylogenetic analysis of the microsporidian PKA sequence must be considered with caution, because of its probable high rate of evolution. Since our phylogenetic analysis aimed at finding protein kinases homologous to the E. cuniculi sequence, we retained only the most closely-related sequences obtained from preliminary phylogenetic analyses, to constitute a subsample of the multiple paralogous groups of protein kinases. In our tree (Fig. 4), E. cuniculi emerges robustly at the base of the PKAc group (bootstrap value of 82% with the RELL method, a likely overestimation). Interestingly, the same phylogenetic position was obtained with two ML approaches (PROTML [Fig. 4] and ProML [data not shown]), both of which lead to very close and equally likely tree topologies, as evaluated by an AU test. This, together with the presence of conserved features of protein kinase catalytic domains, suggests that the sequence encodes a PKAc gene, even though cAMP-dependent kinase activity has not yet been demonstrated. We decided not to include the E. intestinalis sequence in the analysis, because of concerns that, being the only partial sequence in the set, its inclusion might bias the results. However, preliminary analysis (not shown) indicated that the phylogenetic position of this sequence follows exactly that of EcPKAc, and that its presence does affect neither branch length nor LBA. Unexpectedly, the E. cuniculi sequence does not appear to emerge any closer to PKA homologues of fungal origin than to those of other eukaryotes. To test if the early emergence

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Fig. 4. ML tree of protein kinases (see Section 2) based on 200 positions, using a JTT model correcting branch length to take into account sites rate variation (JTT + G model). Bootstrap values indicated in bold characters were obtained by RELL method, and boostrap values indicated in italics were calculated with the NJBOOT program of the MUST package. Only bootstrap values > 70% are reported. Functionally identified groups are indicated to the right. Triangles indicate alternative phylogenic positions for the E. cuniculi sequence, close to fungi groups. The AU test showed that these positions are significantly worse than the inferred one, at the base of PKA group. Accession numbers and the corresponding species are listed in the order they appear in the tree, starting at the top: S. pombe [AL109834], S. cerevisiae [Z71662], A. thaliana [AF232236], A. thaliana [AB019228], H. sapiens [AJ010119], D. melanogaster [AE003546], S. cerevisiae [Z35897], S. pombe [Z97052], H. sapiens [U08316], D. melanogaster [L28945], D. melanogaster [U67304], H. sapiens [M60724], A. thaliana [AC012562], H. sapiens [M80776], D. melanogaster [A41615], H. sapiens [BC017272], D. melanogaster [P32866], D. discoideum [est], H. sapiens [X85545], D. melanogaster [X16961], L. major [U91743], L. major [U43906], T. gondii [est/27488656], S. pombe [P40376], S. cerevisiae [Z28166], P. falciparum [U78291], H. sapiens [M34181], D. melanogaster [AE003625], E. histolytica [est], S. cerevisiae [X57629], S. pombe [T38040], S. pombe [AL157991], S. cerevisiae [Z28126], D. discoideum [est], D. discoideum [est], C. elegans [Z81140], H. sapiens [AJ000512], H. sapiens [AF085234], D. melanogaster [Z26242], H. sapiens [L33881], D. melanogaster [AF288482], H. sapiens [X65293], D. melanogaster [AE003768], H. sapiens [D26181], D. melanogaster [AE003834], S. cerevisiae [Z35866] and S. pombe [D14337].

of our microsporidian sequence in the PKAc group was incongruent with a fungal relationship, we compared alternative E. cuniculi positions, close to fungal groups of the tree. In all cases, the basal position of E. cuniculi in the PKAc group was significantly preferred (data not shown). However, our observation that the E. cuniculi gene is located at the basal position of the PKA branch (rather than

within the cluster of fungal PKA homologues) is not sufficient to challenge the previously reported relatedness of microsporidia to fungi. Indeed, the LBA phenomenon is expected to result in anchoring this long branch at the basal position. Therefore, the present analysis does not allow us to propose new insights in the difficult question of the general phylogenetic position of microsporidia.

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Acknowledgments We thank Herve´ Philippe for access to his phylogenetic analysis programmes, Dominique Dorin for help with recombinant protein expression, and Simonetta Gribaldo and David Moreira for critical reading of the manuscript. Work in C.D. laboratory is supported by INSERM, the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR), the French Ministe`re de la Recherche (PRFMMIP and PAL+ programs), the French Ministe`re de la De´fense (De´le´gation Ge´ne´rale pour l’Armement [DGA]), the French – South African joint program on Science and Technology (financed by the French Ministe`re de l’Education Nationale, the Ministe`re des Affaires Etrange`res and the South African Foundation for Research and Development). L.E. is the recipient of a studentship awarded by the French De´le´gation Ge´ne´rale pour l’Armement (DGA), French Ministe`re de la De´fense.

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