INTRODUCTION

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Microsporidia (phylum Microspora Sprague, 1977) are small spore-forming unicellular eukaryotes with an obligate intracellular parasitic lifestyle. These parasites, characterized by 70S ribosomes and the absence of mitochondria, were thought to be very ancient (10). However, data accumulated from recent molecular phylogenies lend credit to a close relationship of these organisms with fungi (36). Several species are of medical and veterinary significance, infecting animals and humans (7). Three species from the Encephalitozoon genus (E. cuniculi, E. hellem, and E. intestinalis) are known to be involved in AIDS-associated pathologies (13). Disorders in immunocompetent individuals also have been reported. For example, E. intestinalis was found in travelers, not infected with human immunodeficiency virus, presenting with chronic diarrhea (33). Serological studies with blood donors and pregnant women revealed a prevalence of about 8% (37).

Microsporidia exhibit a remarkable invasion mechanism depending on the extrusion of a specific organelle called the polar tube, originally coiled within the spore. The polar tube discharges from the anterior pole of the spore like an everting glove finger (25) and then is used to transfer the sporoplasm inside a potential host cell. The whole process of in vitro spore germination is completed in less than 2 s (17). Very little information is available about the primary structure of polar tube proteins (PTPs) and the extent of interspecies sequence variability. Molecular characterization of the polar tube is therefore of importance for improving diagnostic and defining therapeutic strategies.

The polar tube resists dissociation in detergents, urea, and acids but dissociates in the presence of thiol-reducing agents, e.g. 2-mercaptoethanol or dithiothreitol (DTT) (19, 40). A Glugea americanus 43-kDa PTP, differentially solubilized with 2% DTT and purified by high-pressure liquid chromatography, was shown to contain a large amount of proline residues (19). Proline-rich PTPs were similarly isolated from Encephalitozoon species, with the apparent molecular sizes varying from 45 to 55 kDa (20). We previously described the first complete sequence of a proline-rich 55-kDa PTP in E. cuniculi (11). The predicted protein has 395 amino acids (aa), with a central region consisting of four 26-aa repeats, and shows no homology with known proteins. A similar ptp gene encoding a 453-aa protein in E. hellem has been also sequenced (21). The repeated region (six 20-aa repeats) is very divergent relative to that in E. cuniculi (22). Since the molecular sizes calculated from sequences (43 kDa in E. hellem and 37 kDa in E. cuniculi) are not consistent with those deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) migrations (50 to 55 kDa), we propose the designation PTP1 for members of this new protein family.

We identified an E. cuniculi PTP (PTP2) assumed to be more conserved than PTP1 among microsporidian species, as judged by immunological cross-reactivity with a 34- to 35-kDa protein from a species of the genus Glugea (12). As reported in the present paper, the genes encoding PTP2 in the three human-infecting Encephalitozoon species were fully sequenced. To complete the comparison of the two different PTPs, the ptp1 gene was also cloned and then sequenced in E. intestinalis. The conservation of the PTP2 sequence within a microsporidian genus suggests that this protein plays a basic role in the construction of the polar tube and may be of interest for medical applications.

	MATERIALS AND METHODS

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Growth of parasites and isolation of DNA. E. cuniculi, E. hellem, and E. intestinalis were grown in vitro in either Madin-Darby canine kidney (MDCK), human lung fibroblast (MRC-5), or rabbit kidney (RK13) cells as described elsewhere (2). Spores collected from supernatants were harvested (5,000 × g for 10 min), washed, purified as described previously (11), and stored in phosphate-buffered saline (PBS) at 4°C. Genomic DNA was released by boiling purified spores at 100°C for 10 min.

Antibody production. Polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) to microsporidian proteins were described previously (11). BALB/c mice were immunized with the recombinant E. cuniculi (EcPTP2) expressed in E. coli. After expression, the recombinant protein was purified by chromatography on Ni-nitrilotriacetic acid (Ni-NTA) resin (Qiagen). Recombinant protein was then excised from Coomassie blue-stained gels and crushed in PBS with a Potter homogenizer. Mice were injected intraperitoneally with samples homogenized with Freund complete adjuvant, and identical injections were given on days 14 and 21 with Freund incomplete adjuvant. Sera were collected 1 week after the last injection and stored at 20°C.

SDS-PAGE and Western blotting analysis. SDS-PAGE was performed using standard methods (24). Crude extracts from microsporidia or recombinant bacteria were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blot analysis was carried out using 10 to 12% polyacrylamide gels run under reducing conditions with 5 to 10% 2-mercaptoethanol in the loading samples. After electrophoresis, proteins were transferred to polyvinylidene difluoride (Immobilon-P; Polylabo). For detection, the membranes were incubated with MAbs or PAbs and then with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG), IgA, and IgM (Sigma), and bound antibodies were visualised using the ECL system (Amersham).

Indirect immunofluorescence (IFA). Intracellular parasites grown for 24 to 48 h in MRC-5 cells on glass slides were fixed with 4% paraformaldehyde and 0.05% glutaraldehyde for 20 min at room temperature or with methanol for 10 min at 70°C. Cells were then permeabilized with 70% ethanol-0.5% Triton X-100 and blocked with 5% skim milk in PBS. Slides were incubated for 1 h with primary antibodies (PAbs or MAbs) diluted in PBS-0.1% Tween 20, washed, and incubated further for 1 h in fluorescein isothiocyanate-labeled goat anti-mouse IgG, IgA, and IgM (Sigma). Slides were mounted and preparations were examined with a BH2 Olympus epifluorescence microscope.

Peptide sequencing. The 35-kDa protein isolated from two-dimensional gels was digested with 0.8 µg of endoprotease-LysC in 0.1 M Tris-HCl (pH 8.6)-0.5 M EDTA-0.03% SDS at 35°C for 18 h, and peptides were then separated using high-pressure liquid chromatography on DEAE-C₁₈ columns with a gradient of acetonitrile-0.1% trifluoroacetic acid. One peptide of 15 aa (AVQGTDRCILAGIID) was sequenced using Edman degradation (Applied Biosystems 473A sequencer).

Cloning and sequencing of ptp genes. The different PCR and single-specific primer PCR (SSP-PCR) amplification steps are described in Fig. 2. The primers used were A (5'-CAGGGIACIGAYMGITGYATHYTIGC-3'), B (5'-GTACTTGCGCTTGTTCACC-3'), C (5'-GAGGAGACAAGCTAATTGC-3'), D (5'-GACATACAGAAGACGGGG-3'), E (5'-CTTATCAGAGCAGATGTTC-3'), F (5'-CCATGCGAACCTAAGAAG-3'), G (5'-GGCTGAAGTCCATAGTCAAC-3'), H (5'-GAAGGAGATCAAGGAGAGCCC-3'), I (5'-ATGAAAGGTATTTCTAAG-3'), J (5'-GATTGTTTTTAGAGGGATCTG-3'), K (5'-CATTGTCATTGTCGACATCG-3'), L (5'-GGCGAGAAGTAACAACAT-3'), M (5'-GAGATTTCTAACGGCGAGG-3'), N (5'-ATRCAICKRTCIGTICCYTG-3'), and O (5'-GCAATGGTTCAAAGAGCC-3'). Amplified products were cloned into pGEM-T Easy Vector System I (Promega). Recombinant plasmids were sequenced using the ABI Prism Dye Terminator Cycle Sequencing kit according to the recommendations of the manufacturer (Perkin-Elmer). Thermocycling of the sequencing reactions and electrophoresis were carried out on a GeneAmp PCR system 2400 and a ABI Prism 377 sequencer (Perkin-Elmer), respectively. Gel readings were processed using the Staden package (35), and the resulting contigs were compared with databases using BLAST (1). Staden package and BLAST programs are available on the French molecular biology server Infobiogen.

EcPTP2 heterologous expression in E. coli. The coding sequence of EcPTP2 was amplified by PCR to introduce a BamHI site at the start codon. The oligonucleotide 5'-GGATCCGCAGCACCTCTCCATG-3' was combined with the antisense oligonucleotide primer 5'-CACTTGAAGATTCAATCC-3' for PCR amplification. The PCR product was first cloned into pGEM-T Easy vector (Promega) and subcloned after BamHI-SacI digestion into the bacterial expression vector pQE-30 (pQE expression system from Qiagen). Expression of the recombinant protein was analyzed in E. coli strain M15 after induction with 1 mM IPTG (isopropyl--D-thiogalactopyranoside).

RNA extraction and RT-PCR. Total RNA was extracted from E. cuniculi-infected MRC-5 cells. Cells were lysed in 5 ml of a lysis buffer containing 180 mM Tris, 90 mM LiCl, 4.5 mM EDTA, and 1% SDS (pH 8.2). After incubation at 55°C for 30 min in 1 volume of phenol-chloroform and three phenol-chloroform-isoamyl alcohol extractions, RNA was precipitated, resuspended in 200 µl of diethyl pyrocarbonate-distilled water, and stored at 80°C. DNA was eliminated by treatment with 10 U of DNase (Promega) in the presence of 40 U of RNAsine (Promega) for 1 h at 37°C. Reverse transcription (RT) was done using 5 µg of total RNA. After denaturation for 2 min at 65°C, RNA was incubated with 40 U of avian myeloblastosis virus reverse transcriptase (Promega) in 50 mM Tris (pH 8.3)-8 mM MgCl₂-30 mM KCl-10 mM DTT-0.5 mM deoxynucleoside triphosphates-RNAsine-100 pmol of oligo(dT) (5'-GACTCACTATAGGGCATGCTTTTTTTTTTTTTTTTTT-3'). The reaction mix was incubated for 90 min at 42°C. PCR amplification of the cDNA 3' end was done with a 1:20 dilution of the RT reaction mixture with a primer specific for either Ecptp1 (D, 5'-GACATACAGAAGACGGGG-3') or Ecptp2 (C, 5'-GAGGAGACAAGCTAATTGC-3') and the primer corresponding to the adapter sequence (5'-GACTCACTATAGGGCATGC-3').

Sequence analysis. Protein sequence alignments were done using the GeneStream alignment program, which is accessible via an electronic mail server (http://vega.igh.cnrs.fr/bin/nph-align_query.pl). The prediction of signal peptide cleavage sites was done using the algorithm of von Heijne (39) and the PSORT program (30).

Nucleotide sequence accession numbers. The nucleotide sequences of EcPTP1, EcPTP2, EiPTP1, EiPTP2, and EhPTP2 have been deposited in GenBank under accession numbers AX007049 through AX007053, respectively (C. Vivarès, A. Danchin, and F. Delbac. July 1998. Patent WO0001724; FR no. 98/08692, 07.07.1998. Protéines de tube polaire de microsporidie, acides nucléiques codant pour ces protéines et leurs applications).

	RESULTS

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An E. cuniculi gene encodes a lysine-rich 30-kDa PTP (EcPTP2). We previously showed that the MAb Ec102, directed against the polar tube of E. cuniculi and reacting with the proline-rich EcPTP1 (a protein with an apparent size of 55 kDa in SDS-PAGE), cross-reacted in Western blotting with two other protein bands of 35 and 28 kDa in size (12). In addition, a PAb (PAb anti-35) raised against an E. cuniculi 35-kDa protein band specifically labeled the polar tube mainly after extrusion.

View larger version (111K): [in this window] [in a new window]   FIG. 1.   (A) SDS-PAGE analysis of E. coli-expressed recombinant EcPTP2 (Coomassie blue staining). Lanes 1 and 2, total E. coli lysate before (lane 1) and after (lane 2) IPTG induction; lane 3, recombinant EcPTP2 after purification on an Ni-NTA column; lane M, molecular mass standards in kilodaltons. (B) Immunoblotting reactivity of MAb Ec102 with E. coli proteins 4 h after IPTG induction. The blot was probed with a 1:10,000 dilution of MAb Ec102 and developed using ECL (Amersham). (C) Lane 2, immunoblot of E. cuniculi whole-cell homogenates probed with anti-recombinant EcPTP2 antiserum. Lane 1, total E. cuniculi proteins stained with Coomassie blue. (D) Indirect immunofluorescence of E. cuniculi spores with extruded polar tubes (arrows). (1) Labeling with an antiserum against total proteins; (2) specific labeling of polar tubes with anti-recombinant PTP2. Bars, 5 µm.

A ptp1-ptp2 gene cluster exists in the three Encephalitozoon species. The chromosomal colocalization of the E. cuniculi ptp1 and ptp2 genes led us to test possible clustering of these genes, through PCR experiments with pairs of primers designed to correspond to the 5' end of one gene and the 3' end of the other gene. Sequencing of a 1.4-kbp amplified fragment showed that the Ecptp2 gene is located downstream of the Ecptp1 gene, with the respective ORFs being on the same DNA strand and separated by 860 nucleotides (Fig. 2). There is no sequence homology of this interval with known genes, while the 3' flanking region of the ptp2 gene shared homology with an RNA-binding protein-encoding gene (on the complementary strand). RT of RNAs from E. cuniculi-infected MRC-5 cells was performed using a poly(T) oligonucleotide coupled with an adapter sequence in the 5' region. Specific PCR amplification of 3' regions of the cDNAs corresponding to Ecptp1 and Ecptp2 mRNAs was then done with primers D and C, respectively, and the primer corresponding to the adapter sequence. Sequencing of the corresponding PCR products (260 and 480 bp) provided evidence for short 3' untranslated regions (UTRs) (25 nucleotides in Ecptp1 and 27 to 29 nucleotides in Ecptp2) with polyadenylation signals and poly(A) tails (Fig. 3). This confirms that the genomic sequence is intronless (at least in the 3' end) and supports independent transcription of the two genes.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Schematic representation of ptp1 and ptp2 gene clusters with positions of PCR primers (A to O) in the three Encephalitozoon species, E. cuniculi (Ec), E. intestinalis (Ei), and E. hellem (Eh). The peptide obtained by microsequencing of EcPTP2 (AVQGTDRCILAGIID) is indicated by a black square. Sense primer D, designed 186 bp upstream from the stop codon of Ecptp1, combined with antisense primer B, designed 380 bp downstream from the initiation codon of Ecptp2, were used to amplify a 1.4-kbp DNA fragment. Two primers deduced from the Ecptp2 sequence (C and E, positions 419 to 699 in the Ecptp2 ORF) were used to amplify a 280-bp DNA fragment in E. intestinalis. The corresponding sequence shared about 90% identity with that of Ecptp2 but showed some differences that were useful to determine specific oligonucleotides. Downstream and upstream regions of the 280-bp known sequence were amplified with PstI and HindIII ligation products, respectively, using two specific primers (F and G) and the reverse vector primer. Thus, SSP-PCR experiments led to 120-bp (3' with PstI) and 1,150-bp (5' with HindIII) amplicons, respectively. The 3' end of Eiptp2 was completed through another SSP-PCR step with primer H, resulting in a 570-bp amplification from the ApaI ligation product. The final sequence is 1,968 bp in length with a predicted 825-bp ORF coding for EiPTP2. The Eiptp1 gene was amplified using a combination of an antisense primer in the 5' flanking region of Eiptp2 (J) and a sense primer (I) determined from the alignment of conserved regions encoding signal peptides of EcPTP1 and EhPTP1. For E. hellem, primer K, determined in the 3' known flanking region of Ehptp1, was combined with the reverse primer L, determined by the alignment of the highly conserved N-terminal sequence encoding the PTP2 signal peptide in E. cuniculi and E. intestinalis. The whole coding sequence of Ehptp2 and its 3' UTR were completed by SSP-PCR amplification with primer O. The 1,696-bp sequence is from reference 21.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Alignments of ptp1 and ptp2 gene 5' and 3' flanking regions showing conserved signals. Start and stop codons are underlined. Identical nucleotides are indicated by asterisks. The AT-rich consensus sequence in the 5' region and potential site of polyadenylation are boxed. Partial E. cuniculi cDNAs with short 3' UTRs (less than 30 nucleotides) are in boldface lowercase letters.

Comparison of PTP2-coding regions. PTP2s are basic proteins of similar size (close to 30 kDa), with the maximal difference between EcPTP2 and EhPTP2 being only five residues (Table 1). The degree of conservation at the amino acid level is higher than for PTP1 and extends throughout the entire coding region (Fig. 4). There is more than 80% identity between EcPTP2 and EiPTP2, 58% identity between EcPTP2 and EhPTP2, and 60% identity between EiPTP2 and EhPTP2. Thus, the PTP2 of E. intestinalis is more closely related to that of E. cuniculi than to that of E. hellem.

View larger version (45K): [in this window] [in a new window]   FIG. 4.   Alignment of the three complete PTP2 amino acid sequences. Amino acids are numbered on the right. Identical residues are indicated by asterisks. The common putative cleavage site for the signal peptide is shown by an arrow. The eight cysteine residues are shaded. The common putative N-glycosylation site and the RGD sequence are indicated in boldface. The lysine-rich central region and the acidic tail are underlined.

A shorter PTP1 with degenerate repeats in E. intestinalis. The PTP1-coding region in E. intestinalis, representing a 371-residue polypeptide (35 kDa), is shorter than those in other species (43 kDa in E. hellem and 37 kDa in E. cuniculi). Sequence alignment confirmed the highest homology in the N- and C-terminal domains (Fig. 5A). The N-terminal signal peptide is remarkably conserved (17 identical residues over 22). The whole sequence of EiPTP1 showed only 48 to 49% identity with those of EhPTP1 and EcPTP1, mainly because of the divergent central domain (delimited by common boundaries PGYY/GQ in Fig. 5A) The repetitive character of this core is less evident in E. intestinalis. Only two major, highly degenerated repeats of 27 or 28 aa were indeed distinguishable (Fig. 5B), contrasting with the nearly perfect repeats seen in EhPTP1 (six 20-aa repeats) and EcPTP1 (four 26-aa repeats) (11, 21). This suggests a rather minor role of such repeats in PTP1 organization and function.

View larger version (69K): [in this window] [in a new window]   FIG. 5.   (A) Alignment of the PTP1 amino acid sequences from E. cuniculi (Ec), E. hellem (Eh), and E. intestinalis (Ei). Amino acids are numbered on the right. Identical amino acids are indicated by asterisks. The highly conserved 22-aa signal peptide is boxed. The three PTP1s share common N- and C-terminal parts but diverge in their tandemly repeated sequences, forming a central core delimited by common boundaries (in boldface). Conserved cysteine residues are shaded. (B) Alignment of the tandemly repeated sequences of EiPTP1 (two repeats) and EcPTP1 (four repeats). Identical amino acids are shaded.

	DISCUSSION

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The polar tube is a typical microsporidian spore structure whose extrusion is absolutely required for the invasion of a host cell. Its protein heterogeneity has been supported by biochemical and immunological data (3, 12, 20). However, only one PTP (here referred to as PTP1) was defined at the primary structure level, after isolation of the corresponding single-copy gene in two species of the family Encephalitozoonidae, E. cuniculi (11) and E. hellem (21). We describe here a gene encoding another antigenic protein (PTP2) located in the polar tubes of three Encephalitozoon species. For a better comparison, we have also cloned and sequenced the ptp1 gene of E. intestinalis, a major human-infecting microsporidian mainly responsible for intestinal disorders. Alignment of the deduced amino acid sequences confirmed that the three species exhibit two different PTPs reflecting two novel structural protein families. While PTP1 is a proline-rich acidic protein with a highly variable repeat-containing core, PTP2 is a more conserved lysine-rich basic protein with an acidic tail. The conserved charged residues in PTP sequences can be thought to be involved in some protein-protein ionic interactions required for the assembly of the polar tube. The role of disulfide bonds in protein-protein interactions has been postulated through in vitro polymerization of a purified PTP (23 kDa) in Ameson michaelis (40) and dissociation of the polar tube in the presence of thiol-reducing agents (19, 40). It seems very likely, therefore, that conserved cysteine residues in PTP2 as well as PTP1 can form intermolecular disulfide bridges of primary importance for the high tensile strength and functioning of the polar tube. This somewhat resembles the case of proline-rich minicollagens found in extrusive organelles (nematocysts) involved in prey capture and locomotion of cnidarians (23). However, it should be stressed that no evident homology exists between these unusual collagens and known PTPs, despite the fact that PTP1 is rich in glycine and proline residues.

The two PTPs are characterized by hydrophobic leader sequences suggestive of secreted proteins. The N terminus of PTP1 consists of a signal sequence of 22 aa cleaved during maturation, probably for targeting to the endoplasmic reticulum, in E. cuniculi and E. hellem (11, 21). This peptide also exists in E. intestinalis PTP1, and the high sequence conservation strongly argues for a similar processing. The signal peptide for PTP2 is predicted to be cleaved between the 13th and 14th aa. The reduced length relative to that in PTP1 cannot be due to a misplaced start codon. No in-frame ATG is found in the upstream region, and an A occupies the 3 position as observed in 5' UTR of protozoa (41). Interestingly, the start codon is preceded by a trinucleotide, AAG, which is common to both ptp genes (Fig. 3) and the recently characterized 5' UTR of an E. cuniculi gene encoding the spore wall protein SWP1 (6).

Several potential glycosylation sites can be deduced from PTP1 and PTP2 sequence analysis, suggesting their glycoprotein nature, which is in agreement with cytochemical data indicating the presence of glycoconjugates in the polar tube (38). Recent electron microscope studies indicate that the microsporidian spore can attach to the host cell membrane prior to the invasion and that entry of sporoplasm occurs by phagocytosis like for other intracellular parasites (8, 28). In addition, the basolateral domain of enterocytes would be the preferential site of penetration for E. intestinalis, as suggested by confocal microscopy observations of the colocalization of the parasite and F-actin at the periphery of host cells (16). Some N-linked oligosaccharides of PTPs might be essential for early interactions between the polar tube and the host cell surface.

Encephalitozoon species possess the smallest nuclear genomes so far identified: 2.9, 2.6, and 2.3 Mb in E. cuniculi, E. hellem, and E. intestinalis, respectively (4, 5). A report on a 4.3-kb chromosomal region of E. cuniculi has revealed that some intergenic regions can be less than 50 bp (15). Our data provide additional information about gene organization and transcription of microsporidian genes. All Encephalitozoon ptp1 and ptp2 genes are present as a single copy with a common clustering on one chromosome, with conserved orientation and spacing. This is the first example of conservation of synteny between microsporidian genomes. The products of the two contiguous genes are involved in the construction of the same cellular structure, the polar tube. This supports the conception that a strong selection pressure maintains the evolutionary conservation of the order of genes whose products are physically associated, as frequently invoked for prokaryotic gene clusters. Conserved synteny has been also reported among the chromosomes of all four species of human malaria parasites (9). We demonstrated that E. cuniculi ptp1 and ptp2 mRNAs are polyadenylated and display reduced 3' UTRs. Short 3' UTRs have been described for Giardia, an amitochondriate parasitic flagellate with a small genome (26, 29). It seems likely that the 5' UTRs of ptp mRNAs are very short, as is the case for the SWP1 gene (6). In Trypanosoma, genes are transcribed polycistronically, with the pre-mRNA being processed by 3' -end formation and trans splicing to create conventional eukaryotic monocistronic mRNAs (18). Caenorhabditis elegans also contains numerous polycistronic clusters with intergenic regions less than 200 bp in length (31). Considering the rather large interval between the ptp1 and ptp2 ORFs, short untranslated regions, and mRNA polyadenylation, we conclude that there are two separate transcription units. Studying the differential expression of PTP1 and PTP2 during development of the parasite within parasitophorous vacuoles is a future challenge for a better understanding of sporogenesis events.

Microsporidia are ubiquitous parasites, suggested to be waterborne pathogens (14). There is need for new detection techniques. Serological diagnosis using recombinant PTPs as antigens, in Western blotting or enzyme-linked immunosorbent assay, might be a potential tool to evaluate the prevalence of microsporidia. Through PCR amplifications of the PTP1 repeated regions in different E. cuniculi isolates, we provided the first data about the variability in both the sequence and repeat number of this protein (32). Further search for PTP1 and PTP2 homologues in species from other microsporidian genera should be undertaken to identify the most characteristic signatures. Immunological cross-reactions with proteins of the fish microsporidian species Glugea atherinae have been observed (12). This species, characterized by a larger genome (19.5 Mb), could be used to determine whether the synteny for the two ptp genes is maintained. Cloning and molecular characterization of new ptp genes, in conjunction with development of techniques for genetically manipulating microsporidia, will be required for elucidation of the structure of the microsporidian polar tube and its extrusion mechanism. Are PTPs truly unique to the phylum Microspora? The identification of sequences having homologies with ptp genes in lower metazoa, especially the myxozoa, which exhibit polar tube-like extrusomes, might help us to understand the evolutionary history of PTPs.