Molecular Microbiology (1998) 29(3), 825–834

On of the microsporidian invasive apparatus: complete sequence of a polar tube of Encephalitozoon cuniculi

Fre´de´ric Delbac,1 Pierre Peyret,1 Guy Me´te´nier,1 and Lom, 1986), are small spore-forming protozoa that lack Danielle David,1 Antoine Danchin2 and Christian P. mitochondria. They have attracted new attention because Vivare`s1* of the AIDS pandemic, opportunistic infections due to 1Protistologie Mole´culaire et Cellulaire des Parasites some species having been reported in immunocompro- Opportunistes, LBCP, UPESA CNRS 6023, Universite´ mised patients (reviewed by Desportes-Livage, 1996). Blaise Pascal, 63177 Aubie`re Cedex, France. To date, three main genera are known to infect AIDS 2Unite´ de Re´gulation de l’Expression Ge´ne´tique, URA patients: Enterocytozoon, Encephalitozoon and Trachi- CNRS 1129, Institut Pasteur, 75724 Paris Cedex 15, pleistophora. The first of these is the most commonly France. reported and is responsible for chronic diarrhoea and weight loss (e.g. Desportes et al., 1985; Cali and Owen, 1990). More recently, cases have been Summary described in immunocompetent patients (Sandfort et al., The microsporidian Encephalitozoon cuniculi is an 1994; Raynaud et al., 1998), and serological tests have obligate intracellular parasite that can cause oppor- shown that 5–8% of non-immunocompromised patients tunistic infections in AIDS patients. Spore invasion could be infected (Van Gool et al., 1997). of host cells involves extrusion of a polar tube. After These unicellular have developed a specific immunocytochemical identification of several polar process for invading their host, involving the extrusion of a tube proteins (PTPs) in E. cuniculi, a major PTP was very long tubular structure. This polar tube projects from isolated from two-dimensional gels and two peptide the spore and penetrates the host plasma membrane. The fragments were sequenced. The complete nucleotide sporoplasm contents, including the nucleus, are thereby sequence of the corresponding was obtained injected through the tube into the host cell cytoplasm using a combination of PCR amplification and cloning (reviewed by Undeen, 1990). The whole process (germi- techniques. The gene exists as a single copy per haploid nation) can be completed in less than 2 s (Frixione et al., and encodes an acidic proline-rich protein, 1992). One hypothesis for the triggering of the germi- with a deduced molecular mass of 37 kDa, that con- nation is that trehalose hydrolysis occurs in response to tains four tandemly arranged 26-amino-acid repeats. some signal and that the resulting glucose metabolites An N-terminal region of 22 residues represents a strongly elevate spore osmotic pressure (Undeen and cleaved signal peptide, probably involved in the tar- Vander Meer, 1994). Germination may be also associated geting of the PTP. No similarity with known proteins with the swelling of a membranous system (polaroplast) has been found. The protein was expressed in Escheri- appearing as a Ca2þ-sequestering compartment (Weidner chia coli, purified and injected into mice. The antisera and Byrd, 1982). Close interspecies similarities in the struc- reacted specifically with the polar tube in indirect tural organization of the polar tube suggest that some of its immunofluorescence assays and electron microscope molecular components may have been conserved during immunocytochemistry. Further identification of con- evolution of . Initially, a 23 kDa protein was served and variable PTP structural motifs should be identified and proposed as being the single component useful for diagnostic purposes and new therapeutic of the polar tube in Ameson (formerly Nosema) michaelis strategies. (Weidner, 1976). More recently, in the fish microsporidian Glugea americanus, four proteins differentially solubilized with 2% dithiothreitol have been claimed to originate from Introduction the polar tube. This has been demonstrated for a 43 kDa Microsporidia, obligate intracellular parasites infecting a proline-rich protein, the information on the amino acid wide range of invertebrate and vertebrate hosts (Canning sequence being limited to a short N-terminal region (Keo- hane et al., 1996a,b). Several polar tube proteins (PTPs), Received 26 December, 1997; revised 11 May, 1998; accepted 14 May, 1998. *For correspondence. E-mail [email protected] differing in apparent molecular mass (45 kDa, 55 kDa), bpclermont.fr; Tel. (4) 73 40 74 57; Fax (4) 73 40 74 55. were similarly isolated from human microsporidia (Keohane

ᮊ 1998 Blackwell Science Ltd 826 F. Delbac et al. et al., 1996c). In addition, a monoclonal binding two-dimensional gel electrophoresis (Fig. 2A). The com- to the polar tube reacted with two protein bands (60 and mon 55 kDa component recognized by the three above- 120 kDa) in Encephalitozoon intestinalis, which indicates mentioned corresponded to a prominent protein some protein heterogeneity (Beckers et al., 1996). spot with an acidic pI close to 5. Mice were subsequently No complete sequence of any protein component of the immunized using this reactive spot. As expected, the polar tube or other spore organelle has yet been defined. pAbs thus obtained reacted only with the acidic protein Encephalitozoon cuniculi, widespread in rodents, can used for immunization (Fig. 2B) and specifically labelled cause various infections in HIV-infected patients (Terada the polar tube in immunofluorescence (Fig. 2C) and electron et al., 1987; Zender et al., 1989; Hollister et al., 1995) microscope immunocytochemistry investigations. That such and is known to contain a nuclear genome of only 2.9 Mb a protein may be of interest for diagnostic purposes is sup- (Biderre et al., 1995). We first characterized E. cuniculi ported by its strong antigenicity in immunoblotting assays PTPs using an immunoblotting and immunocytochemical with various antisera collected from microsporidia-infected study with monoclonal and polyclonal antibodies (Delbac mice or rabbits (data not shown). et al., 1996). This prompted us to identify the gene encod- ing a major 55 kDa protein. We report here the first cloning The cloned PTP gene encodes a polypeptide with a and sequencing of such a PTP gene. The corresponding 22 residue terminal cleaved signal peptide protein, devoid of significant homology with any known proteins, has been successfully expressed in Escherichia The strategy chosen for identifying the DNA sequence coli. The Encephalitozoon PTP deserves investigation for encoding PTP was based on microsequencing of peptide possible original protein motifs and new diagnostic tools. fragments and PCR amplification of genomic DNA because of the lack of a cDNA library (due to technical difficulties in isolating differentiating cell stages from an intracellular Results parasite). The PTP eluted from two-dimensional gels was digested with Endolysine C and two peptides were sequ- An acidic protein with an apparent size of 55 kDa is enced. The first one was 20-amino-acid residues long located in the polar tube of E. cuniculi (P1, ATALCSNAYGLTPGQQGMAQ) and the second As microsporidian spores have a resistant wall, the proce- one 15-amino-acid residues long (P2, SATQYAMEACA- dure for protein extraction used cell disruption with metal TPTP). Various combinations of degenerate primers for beads in a lysis buffer containing SDS and 2-mercapto- PCR experiments were tried. We succeeded in amplifying ethanol. An example of SDS–PAGE protein pattern is shown in Fig. 1A (lane 1). Spores were treated with various concentrations of 2-mercaptoethanol (5–50%), disulphide bonds having been reported to be required for polar tube integrity (Weidner, 1976; Keohane et al., 1996a–c). Most of the material solubilized at high concentrations of the reducing agent was represented by a band migrating at 55 kDa (Fig. 1A, lane 2). Different protein bands including the 55 kDa band were excised from gels and then used for producing mouse polyclonal antibodies (pAbs). Attempts were also made to raise monoclonal antibodies (mAbs) against the 55 kDa band. Our immunocytochemical local- ization experiments showed a polar tube labelling with pAbs anti-55 kDa and anti-35 kDa, and one mAb anti- 55 kDa (IgM isotype) termed Ec102 (Delbac et al., 1998). However, pAb anti-55 kDa also afforded significant labelling Fig. 1. A. SDS–PAGE separation (12%) of spore proteins. Lane 1, of the spore wall (Fig. 1B). Western blot analysis with this fraction directly solubilized in 2% SDS, 10% 2-mercaptoethanol; lane 2, residual fraction obtained after incubation in 50% pAb revealed no cross-reaction with other protein bands 2-mercaptoethanol up to 48 h. Gels were stained with Coomassie (Fig. 1C, lane 1), indicating protein heterogeneity of the brilliant blue R-250. 55 kDa band. In contrast, mAb Ec102 cross-reacted with B. Indirect immunofluorescence analysis using pAb against the 55 kDa band from SDS–PAGE (1:50 dilution) on E. cuniculi infected the 35 kDa and 28 kDa bands (Fig. 1C, lane 2) and pAb MRC-5 cells. Spores with extruded polar tube are strongly reactive. anti-35 kDa cross-reacted with the 55 kDa band (not The bar represents 5 ␮m. shown). C. Immunoblot reactivity with pAb anti-55 kDa (1:5000 dilution, lane 1) and mAb Ec102 (1:10000 dilution, lane 2). Molecular mass For better characterization of an Encephalitozoon PTP, markers (M) indicated on the left are given in kDa. The blots were spore proteins were subjected to Western blotting after developed using ECL (Amersham).

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 825–834 Microsporidian polar tube protein 827

Fig. 2. Immunoreactivity of an acidic 55 kDa PTP. Two-dimensional gel electrophoresis was performed using isoelectric focusing in the first dimension and 12% gels in the second dimension. The separated proteins were either silver stained (A) or transferred to PVDF membranes and incubated with a 1:5000 dilution of the pAb directed against the 55 kDa acidic spot from 2D-gels (B). Note that the reactive spot is faintly silver stained (arrow). Both molecular sizes (kDa) and isoelectric points (4–8) are indicated. C. Specific labelling with this pAb of extruded polar tubes in immunofluorescence (arrow). The bar represents 5 ␮m. a genomic DNA fragment of about 1 kbp using two primers and corresponded to peptide P1. Consequently, the pre- (EcA and EcB respectively) designed to match regions dicted 22 residue signal peptide must be removed during PGQQGM and TQYAMEACA. The DNA sequence was maturation of the protein. Various methods of secondary then extended in both directions using the single specific structure prediction clearly indicated that the signal pep- primer–PCR technique (Shyamala and Ames, 1993). tide can form an ␣-helix. For this purpose, genomic DNA was completely digested Thus, the encoded polypeptide contains 395 amino by the restriction enzyme BamHI or EcoRI and ligated acids, among which 373 should be present in the mature in pBluescript-II SKþ cloning vector. Downstream and protein. The calculated molecular mass of 37 230 Da is upstream regions of the known sequence were, respec- well below the value given by SDS–PAGE, whereas the tively, amplified with BamHI and EcoRI ligation products deduced pI (4.7) agrees with the pI estimated in isoelectric using two specific primers (EcC or EcD), determined focusing (IEF). To substantiate the assignment of this pro- from the 1 kbp fragment, and the reverse vector primer. tein to the polar tube, a major part of the corresponding The corresponding sequence was validated by PCR gene (1 kbp amplicon corresponding to the sequence amplification with two other oligonucleotides determined between P1 and P2) was subcloned in an expression vec- in the 5Ј and 3Ј non-coding regions of the gene. tor. Here again, the expression in E. coli revealed a protein The complete nucleotide sequence data, with an open band with a size (Ϸ50 kDa) greater than expected: 37 kDa reading frame of 1188 nucleotides (54% GþC), have (Fig. 3A, lanes 2 and 3). After purification by chromato- been submitted to the EMBL database under accession graphy using Ni-NTA resin, the recombinant protein was number AJ005666. An AT-rich region (63%) extending injected into mice and corresponding antisera tested either about 100 bp upstream of the translation start codon is by Western blotting or indirect immunofluorescence assays assumed to contain promoter elements. The flanking 3Ј (IFA). Specific labelling of the 50 kDa recombinant protein region includes a potential polyadenylation sequence and the E. cuniculi 55 kDa protein was observed on blots (AATAAA), five nucleotides downstream from the stop (Fig. 3B, lanes 1 and 2). Extruded polar tubes were strongly codon. The deduced N-terminal region of the protein reactive in IFA (Fig. 3C). Localization in the polar tube shows similarities with a signal peptide presenting a poten- coiled within the spore was confirmed in immunogold elec- tial cleavage site between residues 22 (serine) and 23 tron microscopy (Fig. 3D). We also tested anti-PTP (mAb (alanine), as predicted by the algorithm of von Heijne Ec102 and pAb anti-55 kDa) on blots from bacteria trans- (1986; Nielsen et al., 1997). The peptides P1 and P2 cor- formed with the pQE30-PTP construct before and after respond, respectively, to the regions 23–42 and 351–365. IPTG induction. The two antibodies bound the expressed The amino acid sequence does not account for the com- protein (not shown). plete P1 peptide because of the absence of a N-terminal lysine residue after endolysine C digestion. To test the The PTP is a proline-rich protein that contains four hypothesis of a cleaved signal peptide, the PTP excised tandem repeats of 26 amino acids from two-dimensional gels underwent N-terminal sequen- cing. The obtained sequence was identical to that between The mature PTP amino acid composition is mainly charac- residues 23 and 32 of the deduced amino acid sequence, terized by the prominence of five amino acids, proline

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 825–834 828 F. Delbac et al.

Fig. 3. PTP expression in E. coli. A. SDS–PAGE of proteins extracted from bacteria with pQE30-PTP construct. Uninduced (lane 1), induced (lane 2) and purified recombinant PTP on Ni-NTA resin (lane 3). Coomassie blue staining. B. Immunoblotting with antisera against the recombinant PTP on E. coli proteins 4 h after IPTG induction (lane 1) and on E. cuniculi proteins (lane 2). The blots were probed with a 1:1000 dilution of antisera and developed using ECL. Molecular mass markers are indicated in kDa. IFA (C) and electron microscopy immunogold labelling of sections of E. cuniculi spores (D) with antisera against the recombinant PTP. Extruded polar tubes are indicated by arrows (C). The bars represent 5 ␮m in C and 0.2 ␮minD.

(13.4%), glycine (11.8%), threonine (11.0%), serine (10.7%) them in the extreme C-terminal position, suggest a poss- and glutamine (10.5%). Three aromatic residues (arginine, ible formation of interprotein or intraprotein disulphide tryptophane and phenylalanine) are lacking. Negatively linkages. charged amino acids (glutamate and aspartate) make up 4% of the total residues. The PTP gene is present as a single copy on the The central region of the PTP consists of four tandemly VI repeated sequences of 26 amino acids between residues 179 and 282. Despite a few deviations in seven positions, Chromosomal location of the PTP gene was carried out by the repeat mainly appears rich in hydrophilic residues (Fig. hybridization of the 1 kb cloned genomic fragment with the 4). A shorter repeat (nine amino acids) with a common E. cuniculi separated by pulsed-field gel PGQQ motif can be seen in the two flanking regions. A electrophoresis. As shown in Fig. 5A, only one hybridizing high conservation at the nucleotide level is found through- band corresponding to the chromosome VI was detected out the repetitive region. For example, two codons CAG (lane 2). In addition, Southern blot analysis of E. cuniculi surrounding the alternative codon CAA regularly encode genomic DNA digested with different restriction endonuc- the peptide QQQ. It should be noted that the preferential leases (BamHI, HindIII, EcoRI, PvuII) showed that the use of CAG for glutamine contrasts with the bias towards PTP probe hybridized to a single band (Fig. 5B). This indi- A in the third position for three other abundant residues cates the presence of a single PTP gene copy per haploid (glycine, proline and threonine). Searches in the databases genome. Considering the upstream and downstream sequ- using BLAST (Altschul et al., 1990) did not reveal any signifi- ences (300 nt of 3Ј and 5Ј flanking regions are known), it cant homology with known proteins, including other pro- seems unlikely that several copies are tandemly arranged. line-rich proteins such as collagen and elastin. At the secondary level, the PTP is predicted to consist mainly Discussion of coiled structures (e.g. Geourjon and Deleage, 1995). The C-terminal part may represent a globular domain, as The growing interest in the study of microsporidia is mainly indicated by the hydrophobic cluster analysis method prompted by their role in various human infections and (Callebaut et al., 1997). Three potential sites of N-glycosylation (NETS, NGTS and NISG, respectively, at positions 86–89, 173–176 and 311–314) are deduced from the sequence. The abun- dance of serine and threonine residues (21.6%) and a putative glycosaminoglycan attachment site (SGNG at Fig. 4. Alignment of the tandemly arranged repeats of PTP (amino positions 313–316) let us consider the possibility of O-glyco- acids 174–289). Two repeats of nine amino acids partially sylation. The presence of 17 cysteine residues, two of surrounding the four 26 residue repeats are underlined.

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 825–834 Microsporidian polar tube protein 829

The coding sequence resembles no known protein sequences in any of the major sequence databases. A main feature of its amino acid composition is its richness in five amino acid residues, especially proline and glycine. A high proline content (18%) was also observed for a PTP chromatographically purified from Glugea americanus (Keohane et al., 1996a). Similar results have been obtained for PTPs isolated from human microsporidia (Keohane et al., 1996c). As proline-rich proteins, such as collagen and elastin, are characterized by a high tensile strength, Keo- hane et al. (1996a) have postulated a similar property for PTPs in relation to sporoplasm discharge and flow through the polar tube. We could not predict accurately the nature and location of secondary structure elements. Whether the PTP core domain represented by four large tandem repeats corresponds to an original conformation remains an open question. It should be noted that proline-rich repeats have been reported for some proteins of other Fig. 5. A. chromosomal location of the PTP gene on the chromosome VI (257 kb). Lane 1, electrophoretic karyotype of parasitic or free-living protozoan, e.g. thrombospondin- E. cuniculi with 11 ethidium bromide-stained chromosomal bands; related adhesive proteins in Plasmodium species (Robson lane 2, autoradiograph after transfer and hybridization with a et al., 1997; Templeton and Kaslow, 1997) and membrane specific PTP probe. B. Southern hybridization with the PTP probe after digestion of skeletal proteins termed articulins in Euglena gracilis (Marrs E. cuniculi genomic DNA with BamHI (lane 1), HindIII (lane 2), and Bouck, 1992). The acidic pI and high proline content of PvuII (lane 3) and EcoRI (lane 4) restriction enzymes. Markers such proteins may explain their anomalous migration in are indicated on the right in bp. SDS–PAGE, similar to that observed for Encephalitozoon PTP. The prediction of overall secondary structure of their unique invasion process. The infectious agent (sporo- Euglena articulins was a ␤-sheet with turns that could be plasm) is inoculated into a host cell through the polar tube, related to the margins of subdomains (Marrs and Bouck, initially coiled within the spore and then suddenly extruded 1992). A similar interpretation could not be considered in response to various stimuli (reviewed by Undeen, 1990). for Encephalitozoon PTP. Proteins entering the constitu- Using a combination of biochemical, immunochemical and tion of the sheath of first-stage larvae in viviparous nema- molecular genetic approaches, we have identified in Ence- todes display a few other characteristics common with phalitozoon cuniculi the first whole gene that encodes a PTPs (solubilization with reducing agents, repeats rich in major protein of the polar tube. Difficulties in the isolation proline and glutamine), but the so-called microfilarial of mRNAs from intracellular stages of microsporidian para- sheath proteins are basically involved in the protection of sites hinder the determination of transcription initiation the parasite surface (Zahner et al., 1995). sites. Several other amitochondrial parasitic protozoa Several glycosylation sites can be predicted from the (Giardia, Trichomonas, Entamoeba) have mRNAs with PTP sequence. This is not surprising as cytochemical unusually short leaders (9–18 nt) comprising a common data indicate that the polar tube contains glycoconjugates sequence (T)TCA assumed to bind the 3Ј end of small (Vavra, 1972; Desportes, 1976; Canning et al., 1992). It subunit rRNA (Katiyar et al., 1995). Sequencing data on may be speculated that the hydrophilic character of carbo- microsporidia protein-coding were limited to ␤-tubu- hydrate moities is important for explaining the sudden lin, translation elongation factors and hsp70 genes (Li et al., extrusion of the polar tube in relation to a water influx 1996; Kamaishi et al., 1996; Germot et al., 1997; Peyretail- shown to occur through a specific transmembrane path- lade et al., 1998). The above consensus has not been way (Frixione et al., 1997). The presence of associated found within microsporidian putative short 5Ј untranslated oligo- or polysaccharide chains might also account for regions. The PTP appears to be a major sporal protein, the late mobility of PTP in SDS gels. However, as expres- which suggests a high expression level during polar tube sion in E. coli also reveals a recombinant protein with a formation. Unlike some structural protein-coding genes, size greater than expected, it seems likely that protein fea- the PTP gene occurs as a single copy per haploid genome tures (acidic pI and high proline content) other than post- in E. cuniculi. A single EF-1␣ gene copy is also present in translational carbohydrate modification are responsible the microsporidian Glugea plecoglossi, in contrast to the for this anomalous migration. The solubilization of PTP redundancy observed in most higher eukaryotic species in the presence of a reducing agent leads us to predict a (Kamaishi et al., 1996). role of disulphide bonds in protein–protein interactions,

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 825–834 830 F. Delbac et al. as suggested by data on the ‘in vitro’ assembly of a puri- of microsporidia should be useful to characterize conser- fied PTP in Ameson michaelis (Weidner, 1976). As in col- ved and variable structural motifs of PTPs. The identifica- lagen, this might be a general feature of PTP organization, tion of these motifs may serve to develop new molecular given the presence of several cysteine residues through- tools, expected to be more specific than currently used out the Encephalitozoon PTP sequence. rDNA probes (e.g. Delbac and Vivare`s, 1997a; Ombrouck The comparison between the N-terminal sequence of et al., 1997), for diagnostic purposes. New therapeutic stra- the protein excised from two-dimensional gels and that tegies for the treatment of opportunistic microsporidian predicted from the beginning of the coding region has infections could also be developed, as the efficiency of clearly indicated the presence of a 22 residue cleaved sig- current therapy based on the use of is rather nal peptide. This amino-terminal peptide probably serves limited (e.g. Colbourn et al., 1994). The production of a as the initial signal that targets the PTP to a cellular com- PTP recombinant peptide should form a basis for serologi- partment involved in polar tube formation. The problem cal diagnostic tests and a possible vaccine component. arises of whether this compartment corresponds to the endoplasmic reticulum (ER) or not. For most microsporidian species, the polar tube emerges from a rather enigmatic Experimental procedures reticular structure (Desportes, 1976). Certain vesicles associated with polar tube differentiation have been con- Culture, spore purification and DNA extraction sidered as of Golgi nature, despite the absence of typical A mouse isolate of E. cuniculi (courtesy of Professor Canning, dictyosomes (e.g. Vivares, 1975). Cytochemical localiza- Imperial College of Science, Technology and Medicine, Lon- tion of thiamine pyrophosphatase activity in Glugea ste- don, UK) was grown in vitro in either Madin–Darby Canine phani strongly supports the existence of a reticular Golgi (MDCK) or human lung fibroblast (MRC-5) cells, as apparatus (Takvorian and Cali, 1994). Thus, the intracel- described elsewhere (Beauvais et al., 1994). Supernatants × lular transport of PTP might involve an ER–Golgi classical were collected and spores were sedimented at 5000 g for pathway. However, observations mainly in Enterocyto- 5 min, washed, and stored in phosphate-buffered saline (PBS) at 4ЊC. Spores were treated for 30 min at 65ЊC with 300 mM zoon bieneusi indicate that the polar tube precursors are Tris-HCl pH 9.0, 100 mM EDTA, 1% SDS to lysate residual peculiar vesicles of unknown origin scattered in the cyto- host cells and were incubated with 500 U of DNase I (Gibco plasm of sporogonial plasmodia (Desportes et al., 1985; BRL) in 0.1 M NaCl for 1 h at 37ЊC to eliminate host DNA. E. Cali and Owen, 1990; Hilmarsdottir et al., 1993). The tar- cuniculi DNA was released by boiling purified spores at geting of PTP to an ill-characterized organelle different 100ЊC for 10 min (Ombrouck et al., 1997). from the ER should be also postulated. Electron micro- scopy immunolocalization experiments in Enterocytozoon Gel electrophoresis of proteins and immunoblot should be useful to test this hypothesis. analysis It has been suggested that PTPs are members of the same new protein family (Keohane et al., 1996a–c). Sequ- Standard one- and two-dimensional polyacrylamide gel elec- ence analysis of the E. cuniculi PTP supports this sug- trophoresis was performed in a Bio-Rad minigel apparatus. For SDS–PAGE, proteins were extracted via homogenization gestion. Difficulties in the prediction of known periodic of spores with zirconium beads in a buffer containing 2.5% secondary structure motifs also suggest an unusual con- SDS, 10% 2-mercaptoethanol and 20 mg ml¹1 leupeptine formation for a protein constitutive of a unique organelle followed by heating at 100ЊC for 10 min, and centrifugation specifically observed within the phylum Microspora. That at 18000 × g for 5 min. For two-dimensional electrophoresis, three E. cuniculi polypeptides, different in size, react with spore proteins were extracted in a buffer containing 9 M urea, mAb Ec102 should be of interest for further investigating 40 mM CHAPS and 5% 2-mercaptoethanol (or 1% DTT). IEF common motifs. Possible cross-reactions with PTPs of was carried out at 400 V for 4 h, 600 V for 30 min and 800 V for 30 min using the following ampholyte combination: 40% other microsporidian species were tested using two mono- pH 3–10, 60% pH 4–6.5 (Pharmalyte, Pharmacia Biotech). clonal antibodies directed against the polar tube of Ence- After equilibration of IEF gels in 2% SDS, 5% 2-mercaptoetha- phalitozoon intestinalis or Encephalitozoon hellem, two nol for 10 min, second-dimension SDS–PAGE was carried out species also responsible for human infections. Both showed on 12% polyacrylamide gels (Laemmli, 1970). Proteins were a reactivity with the 55 kDa protein of E. cuniculi and the either silver or Coomassie blue stained. For Western blot recombinant protein expressed in E. coli. Size variations immunodetection, proteins were electrophoretically transferred of these proteins could be related to changes in the num- onto polyvinylidene difluoride (PVDF) membranes (Immobilon P, Polylabo) using a semidry system. Non-specific binding ber and (or) length of some common structural motifs sites were blocked in PBS–5% skimmed milk for 1 h at room such as those found tandemly repeated in E. cuniculi. temperature. After incubation for 3 h with 1:100–1:5000 dilu- Such variations have indeed been found in Plasmodium tions of mice antisera in PBS–0.1% Tween 20 and several thrombospondin-related adhesive proteins (Templeton and washes in PBS–0.5% milk–0.1% Tween 20, the membranes Kaslow, 1997). Studies on PTP genes from other species were treated with a 1:5000 dilution of peroxidase-conjugated

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 825–834 Microsporidian polar tube protein 831 goat anti-mouse IgG (or IgM) (Sigma) for 1 h at room tem- P1 (ATALCSNAYGLTPGQQGMAQ) and P2 (SATQYAMEA- perature. Blots were detected using enhanced chemilumines- CATPTP). N-terminal sequencing of the protein was per- cence (ECL) reagents and exposed to Hyperfilm ECL for 15 s formed after transfer on PVDF membranes. (Amersham). DNA cloning and sequencing Polyclonal (pAb) and monoclonal (mAb) antibody Degenerate primers determined from the amino acid sequ- production ences of the two peptides P1 and P2 were, respectively, EcA (5Ј-CCIGGICARCARGGIATGG-3Ј) and EcB (5Ј-GCRCAIG- Antisera to microsporidian proteins were produced in BALB/c CCTCCATIGCRTACTGIG-3Ј). Amplification was carried out mice. For pAbs preparation, we used antigens from either in 50 ␮l of reaction mixture according to standard conditions a 55 kDa protein band or corresponding major spot (pI 5) (Goldstar, Eurogentec). After denaturating the DNA at 94ЊC in two-dimensional gels. Bands or spots were excised from for 3 min, 35 cycles were run using a Perkin-Elmer DNA ther- Coomassie blue stained gels, and crushed in PBS with a mal cycler 2400 apparatus as follows: denaturing at 94ЊC for Potter homogenizer. Mice were injected intraperitoneally with 20 s, annealing at 50ЊC for 1 min and elongation at 72ЊC for samples homogenized with Freund complete adjuvant, and 1.5 min, with 30 min extension at 72ЊC after the 35 cycles. identical injections were given on days 7 and 21 with Freund Amplified products were analysed electrophoretically on 1% incomplete adjuvant. Sera or ascitic fluids were collected agarose gel, electroeluted for 1 h at 100 V, precipitated with 1 week after the last injection and stored at ¹20ЊC. pAbs 2.5 vols of ethanol, 0.3 M sodium acetate, and cloned into were similarly produced against the protein expressed in pCR2 vector (Invitrogen, TA cloning vector) or pGEM-T Easy Escherichia coli after Ni-NTA resin chromatography and Vector System I (Promega). Recombinant plasmids were SDS–PAGE. The 55 kDa band was also used as a source of sequenced by the Sanger method (Sanger et al., 1977), antigen for producing mAbs. Fusion of myeloma cells with using the ABI Prism Dye Terminator Cycle Sequencing kit spleen cells from immunized mice was performed 6 weeks according to the recommendations of the manufacturer (Per- after the last immunization, and hybridomas were screened kin-Elmer). Thermocycling of the sequencing reactions and by Western blotting and indirect immunofluorescence assays. electrophoresis were, respectively, carried out on a GeneAmp Only mAbs of IgM isotype were obtained. PCR system 2400 and a ABI Prism 377 Sequencer (Perkin- Elmer). Gel readings were processed using the Staden pack- age (Staden, 1996), and the resulting contigs were compared Immunocytochemical procedures with public databases using BLAST (Altschul et al., 1990) to seek possible homologies with known proteins. Staden pack- For IFA, E. cuniculi spores were fixed with methanol for age and BLAST programs are available on the French molecular 10 min, and preparations, saturated with PBS–5% skimmed biology server Infobiogen. milk, were incubated for 1 h at 37ЊC with various dilutions of For application of the single-specific primer PCR (SSP-PCR) pAbs or mAbs in PBS–0.1% Tween 20. After washing in technique (Shyamala and Ames, 1993), digestion of genomic PBS–0.1% Tween 20 and treatment for 1 h at 37ЊC with a DNA (Ϸ200 ng) for 4 h with 100 U of the restriction enzyme 1:200 dilution of FITC-conjugated goat anti-mouse IgG, A, BamHI or EcoRI (Eurogentec) was followed by two phenol– M (Sigma), preparations were examined under a BH2 Olym- chloroform–isoamyl alcohol extractions. A 1 ␮g sample of pus epifluorescence microscope. pBluescript-II SKþ (Stratagene) vector was digested using For electron microscopy immunogold labelling, either puri- the corresponding enzyme and dephosphorylated with 10 U fied E. cuniculi spores or infected MDCK cells were fixed of calf intestine alkaline phosphatase (Eurogentec). After two at 4ЊC with 4% paraformaldehyde, 0.5% glutaraldehyde in phenol–chloroform–isoamyl alcohol extractions, vector DNA 0.1 M phosphate buffer, dehydrated in a gradient series of was co-precipitated with the digested genomic DNA. Ligation ethanol and embedded in Unicryl resin. The ultrathin sections was carried out overnight at 16ЊCin12␮l of a mixture contain- were saturated for 1 h with 1% bovine serum albumin (BSA) ing 6 U of T4 DNA ligase (Pharmacia) and 1 mM ATP in One in PBS, incubated for 3 h with various dilutions of mice anti- Phor All buffer. PCR was carried out in 50 ␮l of reaction mix- sera (or mAbs) and then for 1 h with a 1:100 dilution of goat ture containing 1 ␮l of diluted ligation mixture (1:10), 0.25 ␮M anti-mouse IgG (or IgM) conjugated with 10 nm colloidal gold vector primer (universal or reverse), 0.25 ␮M specific primer particles (Sigma). After uranyl acetate staining, the prepara- (EcC, 5Ј-CACATACGACGGCTGCTG-3Ј or EcD, 5Ј-GACAT- tions were examined in a Jeol 1200 × electron ACAGAAGACGGGG-3Ј), 20 ␮M of each dNTP in Taq DNA microscope. polymerase buffer (2.5 mM MgCl2) and 1 U of Taq DNA poly- merase (Goldstar, Eurogentec). After an initial DNA denatura- tion step at 94ЊC for 3 min, 35 cycles (denaturation at 94ЊC for Peptide sequencing 20 s, annealing at 54ЊC for 30 s, and elongation at 72ЊC for The 55 kDa protein from excised spots of two-dimensional 1 min) were run, with a final 30 min extension at 72ЊC. Ampli- gels was digested with 0.8 ␮g of endoprotease-LysC in 0.1 M fied products were cloned and recombinant plasmids sequ- Tris-HCl pH 8.6, 0.5 M EDTA, 0.03% SDS at 35ЊC for 18 h, enced as described above. and peptides were then separated using HPLC on DEAE- C18 columns with a gradient of acetonitrile/TFA 0.1%. Two Expression in E. coli peptides of 20 and 15 amino acids were sequenced using Edman degradation (Applied Biosystems 473A sequencer): The pCR2 vector containing the 1 kb region amplification

ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 825–834 832 F. Delbac et al. product obtained with primers EcA and EcB was digested Encephalitozoon cuniculi. Antimicrob Agents Chemother by the restriction enzymes SacI and EcoRV and the corre- 38: 240–248. sponding DNA fragment was subcloned into the SacI–SmaI- Beckers, P.J.A., Derks, G.J.M.M., Van Gool, T., Rietveld, digested expression vector pQE30. The recombinant protein F.J.R., and Sauerwein, R.W. (1996) Encephalitozoon was expressed in Escherichia coli strain M15, carrying the intestinalis-specific monoclonal antibodies for laboratory lac repressor producing plasmid pREP4, with QIAexpress diagnosis of microsporidiosis. J Clin Microbiol 34: 282– system (type IV construct with 6× His Tag 5Ј to the polylinker). 285. Bacteria were induced with 2 mM isopropylthio-␤-D-galacto- Biderre, C., Page`s, M., Me´te´nier, G., Canning, E.U., and side (IPTG) for 4 h, and proteins were then solubilized in Vivare`s, C.P. (1995) Evidence for the smallest nuclear 2.5% SDS, 5% 2-mercaptoethanol and analysed by SDS– genome (2, 9 Mb) in the microsporidium Encephalitozoon PAGE. The presence of the histidine tail added to the N-termi- cuniculi. Mol Biochem Parasitol 74: 229–231. nus of the sequences allowed the purification of the recombi- Cali, A., and Owen, R.L. (1990) Intracellular development of nant protein by nickel chelate chromatography on Ni-NTA Enterocytozoon, a unique microsporidian found in the resin (Qiagen). intestine of AIDS patients. J Protozool 37: 145–155. Immunization of BALB/c mice with the expressed protein, Callebaut, I., Labesse, G., Durand, P., Poupon, A., Canard, immunoblotting and immunofluorescence experiments were L., Chomelier, J., et al. (1997) Deciphering protein sequ- carried out as described above. ence information through hydrophobic cluster analysis (HCA): current status and perspectives. Cell Mol Life Sci 53: 621–645. Secondary structure analysis Canning, E.U., and Lom, J. (1986) The Microsporidia of Vertebrates. London: Academic Press. Secondary structure prediction was based on multiple align- Canning, E.U., Curry, A., Lacey, C.J.N., and Fenwick, J.D. ment by the Gibrat, Levin, DPM and SOPMA methods acces- (1992) Ultrastructure of Encephalitozoon sp. infecting the sible via an electronic mail server (http://www.ibcp.fr/predict. conjunctival, corneal and nasal epithelia of a patient with html, Geourjon and Deleage, 1995). The hydrophobic cluster AIDS. Eur J Protistol 28: 226–237. analysis (HCA) method was also applied (Callebaut et al., Colbourn, N.I., Hollister, W.S., Curry, A., and Canning, E.U. 1997). The prediction of signal peptide was achieved using (1994) Activity of albendazole against Encephalitozoon the algorithm of von Heijne (1986; Nielsen et al., 1997). cuniculi in vitro. Eur J Protistol 30: 211–220. Delbac, F., Duffieux, F., Peyret, P., David, D., Me´te´nier, G., and Vivare`s, C. (1996) Identification of sporal proteins in Pulsed-field gel electrophoresis and Southern blotting two microsporidian species: an immunoblotting and immuno- Molecular karyotypes of E. cuniculi, transfer and hybridization cytochemical study. J Euk Microbiol 43: 101S. were performed as previously described (Biderre et al., 1995). Delbac, F., and Vivare`s, C. (1997a) A PCR technique for Genomic DNA samples were digested separately with different detecting and differentiating known microsporidia in AIDS restriction endonucleases, run on 0.8% agarose gel in TBE patients. J Euk Microbiol 44: 75S. buffer and transferred to a Biotrans Nylon membrane (ICN). Delbac, F., David, D., Me´te´nier, G., and Vivare`s, C. (1997b) 32P-labelled DNA probes of the PTP gene were produced First complete amino acid sequence of a polar tube protein by random priming and hybridizations were performed over- in a microsporidian species, Encephalitozoon cuniculi. J night (Sambrook et al., 1989). Euk Microbiol 44: 77S. Delbac, F., Duffieux, F., David, D., Me´te´nier, G., and Vivare`s, C. (1998) Immunocytochemical identification of spore pro- Acknowledgements teins in two microsporidia, with emphasis on extrusion apparatus. J Euk Microbiol 45: 224–231. We thank I. Callebaut for HCA analysis and J. D’Alayer for Desportes, I. (1976) Ultrastructure de Stempellia mutabilis peptide sequencing (Laboratoire de Microse´quenc¸age des Leger et Hesse, microsporidie parasite de l’e´phe´me`re Prote´ines, Institut Pasteur, Paris, France). We are grateful Ephemera vulgata. Protistologica 12: 121–150. to Emmanuel Cornillot for helpful discussions and comments Desportes, I., Le Charpentier, Y., Galian, A., Bernard, F., on the manuscript. This work was supported by a grant from Cochand-Priollet, B., Lavergne, A., et al. (1985) Occur- Sidaction. Fre´de´ric Delbac was supported by a grant from the ence of a new microsporidian: Enterocytozoon bieneusi, French Ministry for Higher Education and Research. A preli- n. g., n. sp., in the enterocytes of a human patient with minary report was presented to the 5th-International Work- AIDS. J Protozool 32: 250–254. shops on Opportunistic (3–7 September 1997 Lille, Desportes-Livage, I. (1996) Human microsporidiosis and Delbac et al., 1997b). AIDS: recent advances. Parasite 3: 107–113. Frixione, E., Ruiz, L., Santillan, M., De Vargas, L.V., Tejero, References J.M., and Undeen, A.H. (1992) Dynamics of polar filament discharge and sporoplasm expulsion by microsporidian Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, spores. Cell Motil Cytoskel 22: 38–50. D.J. (1990) Basic local alignment search tool. J Mol Biol Frixione, E., Ruiz, L., Cerbon, J., and Undeen, A.H. (1997) 215: 403–410. 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