Identification of two new polar tube proteins related to polar
tube protein 2 in the microsporidian
Antonospora locustae
Valerie Polonais1,2, Abdel Belkorchia1,2, Micha€el Roussel2,3, Eric Peyretaillade4,5, Pierre Peyret4,5,
Marie Diogon2,3& Frederic Delbac2,3
1Clermont Universite, Universite d’Auvergne, Laboratoire “Microorganismes: Genome et Environnement”, Clermont-ferrand, France;2CNRS, UMR 6023, LMGE, Aubiere, France;3Clermont Universite, Universite Blaise Pascal, Laboratoire “Microorganismes: Genome et Environnement”, Clermont-ferrand, France;4Clermont Universite, Universite d’Auvergne, EA 4678, CIDAM, Clermont-Ferrand, France; and5UFR Pharmacie, Clermont Universite, Universite d’Auvergne, Clermont-Ferrand, France
Correspondence: Frederic Delbac, Interactions H^otes-Parasites, LMGE, UMR CNRS 6023, Universite Blaise Pascal, 24 Avenue des Landais 63177 Aubiere Cedex, France. Tel.: +33 4 73 40 78 68; fax: +33 4 73 40 76 70;
e-mail: frederic.delbac@univ-bpclermont.fr Received 28 March 2013; revised 28 May 2013; accepted 7 June 2013. Final version published online 17 July 2013.
DOI: 10.1111/1574-6968.12198 Editor: Albert Descoteaux Keywords
Microsporidia; Antonospora locustae; polar tube; invasion; elastomeric protein.
Abstract
Microsporidia are obligate intracellular eukaryotic parasites with a broad host spectrum characterized by a unique and highly sophisticated invasion appara-tus, the polar tube (PT). In a previous study, two PT proteins, named AlPTP1 (50 kDa) and AlPTP2 (35 kDa), were identified in Antonospora locustae, an orthoptera parasite that is used as a biological control agent against locusts. Antibodies raised against AlPTP2 cross-reacted with a band migrating at 70 kDa, suggesting that this 70-kDa antigen is closely related to AlPTP2. A
BLASTp search against the A. locustae genome database allowed the identification
of two further PTP2-like proteins named AlPTP2b (568 aa) and AlPTP2c (599 aa). Both proteins are characterized by a specific serine- and glycine-rich N-terminal extension with elastomeric structural features and share a common C-terminal end conserved with AlPTP2 ( 88% identity for the last 250 aa). MS analysis of the 70-kDa band revealed the presence of AlPTP2b. Specific anti-AlPTP2b antibodies labelled the extruded PTs of the A. locustae spores, confirming that this antigen is a PT component. Finally, we showed that sev-eral PTP2-like proteins are also present in other phylogenetically related insect microsporidia, including Anncaliia algerae and Paranosema grylli.
Introduction
Microsporidia are obligatory intracellular fungi-related parasites that are ubiquitous in the environment and that can infect a wide range of hosts, from protists to mam-mals (Lee et al., 2008b; Didier et al., 2009). Recognized as emerging opportunistic pathogens (Didier & Weiss, 2011), microsporidia are also known as intriguing and minimalist parasites characterized by extremely reduced metabolic potential and cellular components, making them highly dependent on their host (Corradi & Slamo-vits, 2011; Corradi & Selman, 2013).
Despite having considerably different host cell ranges and cell-type specificities, all microsporidian species pos-sess a functionally conserved, highly specialized organelle called the polar tube (PT) that plays a preponderant role in host cell invasion (Franzen, 2005). Until now, PT proteins (PTPs) belonging to three protein families
(PTP1, PTP2 and PTP3) had been identified in the mam-malian microsporidia Encephalitozoon cuniculi and E. in-testinalis (Delbac et al., 2001; Peuvel et al., 2002; Corradi et al., 2010), in the bee parasite Nosema ceranae (Corn-man et al., 2009) and in the Bombyx mori parasite No-sema bombycis (Wang et al., 2007). Genomic surveys have also allowed the identification of PTP1 and PTP2 orthol-ogous proteins in the locust parasite Antonospora (for-merly Nosema) locustae, as in the case of the cricket microsporidia Paranosema grylli, a parasite phylogeneti-cally related to A. locustae (Polonais et al., 2005).
Minimal data are available concerning the function and assembly of the PT. Encephalitozoon cuniculi PTP1, PTP2 and PTP3 have been shown to interact (Bouzahzah et al., 2010) and, more recently, PTP2 and PTP3 were shown to interact with the spore-wall protein SWP5 in N. bombycis (Li et al., 2012). Each type of PTP shares common characteristics among the identified PTPs, despite the
MICR
OBIOLOGY LETTERS
high degree of sequence divergence. For example, all PTP2 proteins present a basic isoelectric point (pI) and high lysine content and are characterized by the strong conservation of cysteine residues, which are most likely involved in intra- and/or interprotein disulfide bridges (Delbac et al., 2001; Polonais et al., 2005).
To gain new insights into PT organization and function, it is important to complete the molecular characterization of its components. A previous study that led to the identi-fication of PTP1 and PTP2 in A. locustae showed that the anti-AlPTP2 antibodies recognized two bands: a 35-kDa band corresponding to AlPTP2 and a band migrating at 70 kDa (Al70) corresponding to an uncharacterized protein. As Al70 was not detected using the anti-AlPTP1 antibodies, the existence of a PTP1–PTP2 complex could be excluded (Polonais et al., 2005). Since this first study, the A. locustae genome annotation progressed (http:// forest.mbl.edu/cgi-bin/site/antonospora01), leading to the identification of 2600 coding DNA sequences (CDSs). In the present study, we describe the identification of two further PTP2-like proteins in A. locustae named AlPTP2b and AlPTP2c that present elastomeric features.
Materials and methods
Microsporidian spore production and purification
Antonospora locustae spores arising from infected grass-hoppers were commercially available from the M & R Durango, Inc. Insectary (Bayfield, CO).
DNA extraction and expression of recombinant AlPTP2b inEscherichia coli
Antonospora locustae genomic DNA was purified using the ELU-Quick DNA purification Kit (Schleicher and Schuell, Dassel, Germany). PCR primers (5′-CGGGATC CTCGTATTCGAGTAGTTGG-3′ and 5′-CGGAATTCTGC GGATGTATGTTGTTG-3′ containing a BamHI and an EcoRI restriction site, respectively) were designed to amplify a 861-bp DNA fragment encoding the N-terminal serine- and glycine-rich extension (amino acids 20–306) of AlPTP2b. PCRs were performed according to standard conditions (Eurobio, Courtaboeuf, France) with an anneal-ing step at 55°C. The Alptp2b truncated form was cloned in the expression vector pGEX4T-1 modified to provide a C-terminal 8 9 His-tag. The resulting recombinant vector was introduced into the E. coli BL21-codon+. After induc-tion [2 mM isopropylthio-b-galactoside (IPTG), 4 h], the recombinant protein was purified under denaturing condi-tions on a Ni-NTA column according to the manufac-turer’s instructions (Qiagen, Valencia, CA) and analysed by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12%).
Generation of anti-AlPTP2b polyclonal antibodies
Polyclonal antibodies against the purified AlPTP2b recombinant protein and Al70 (70 kDa band) were pro-duced in SWISS mice. The animal house (agreement C63014.19) and the experimental staff (agreement 63– 146) were approved by the French Veterinary Service. The experiments were conducted according to ethical rules. Mice were injected intraperitoneally with samples homogenized in Freund’s complete adjuvant for the first injection and in Freund’s incomplete adjuvant for the next injections (days 14, 21 and 28).
Protein gel electrophoresis and immunoblotting
Total A. locustae sporal proteins were extracted in Lae-mmli buffer (2.5% SDS, 100 mM dithiothreitol) by repeated freeze–thaw cycles in liquid nitrogen and sepa-rated by 10% SDS-PAGE. For MS analysis, gels were fixed in a 7.5% acetic acid/30% ethanol solution, stained with Coomassie Blue (Bio-Rad, Hercules, CA) and destained with 30% ethanol. For immunoblotting, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Blots were probed with the appropriate dilution of polyclonal mouse antibodies and reacted with a phosphatase alkaline-conjugated goat antimouse IgG (H+L, Promega, Madison, WI) at 1 : 10 000. Antibody binding was revealed using the phosphatase alkaline substrates NBT and BCIP (Promega).
Indirect immunofluorescence assays (IFAs)
Antonospora locustae, P. grylli and A. algerae spores were fixed with methanol at 20°C. The slides were incubated with the primary antibody (1 : 100) and then with the anti-mouse-Alexa 488 secondary antibodies (1 : 1000, Molecular Probes, Carlsbad, CA). Preparations were observed with a LEICA DMR epifluorescence microscope.
Trypsin digestion and MALDI-TOF analysis
The 70-kDa band (Al70) was manually excised from a Coomassie Blue-stained gel and washed with destaining solutions (25 mM NH4HCO3-5% acetonitrile (ACN) for
30 min and 25 mM NH4HCO3/50% ACN for 30 min).
After dehydration in 100% ACN, the 70-kDa band was digested at 37°C for 5 h with trypsin (10 ng lL 1, V511;
Promega). After centrifugation, the tryptic peptides were extracted by ACN. For the MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) MS analysis, 1lL of the trypsin peptide mixture was loaded onto the MALDI target and the matrix solution (5 mg mL 1 alpha-cyano-4-hydroxycinnamic acid in 50% ACN/0.1% trifluoroacetic acid, v/v) was added. The MALDI-TOF mass spectrometer (Voyager DE-Pro; Perspective Biosys-tems, Farmingham, MA) was used in the positive ion reflector mode for peptide mass fingerprinting. External calibration was performed with a standard peptide solu-tion (Proteomix), while the internal calibrasolu-tion was per-formed using peptides from porcine trypsin autolysis. The protein database from A. locustae (http://forest.mbl. edu/cgi-bin/site/antonospora01) was explored using MAS-COT software version 2.1 (http://www.matrixscience.com).
Peptide masses were assumed to be monoisotopic.
Sequence analysis
The A. locustae genome sequencing project was completed at the Marine Biological Laboratory (MBL, Woods Hole, MA). The database, which contains 2606 CDSs, is avail-able on the MBL server (http://forest.mbl.edu/cgi-bin/site/ antonospora01). Molecular masses and isoelectric points (pIs) were calculated using the ExPASy Proteomics tools (http://www.expasy.org/tools/). Protein statistical analyses were predicted by SAPS (www.ebi.ac.uk), and peptide leaders were scanned using the SignalP program version 4 (www.cbs.dtu.dk/services/SignalP/). Potential O-glycosyla-tion and phosphorylaO-glycosyla-tion sites were determined using the NetOglyc and NetPhos servers (http://us.expasy.org). Searches for homologous proteins in the databases were conducted using tBLASTx on NCBI (http://www.ncbi.
nlm.nih.gov/BLAST/), on the MicrosporidiaDB (http:// microsporidiadb.org/micro/) and on the Broad Institute website (http://www.broadinstitute.org/annotation/gen-ome/microsporidia_comparative/ToolsIndex.html). Multi-ple protein sequence alignments were performed using
CLUSTALW software (www.ebi.ac.uk), and secondary
struc-ture predictions were performed using thePSIPRED(http://
bioinf.cs.ucl.ac.uk/psipred/) and the BETATPRED2 servers
(http://www.imtech.res.in/raghava/betatpred2/).
Nucleotide and protein sequence accession numbers
The Alptp2, Alptp2b and Alptp2c nucleic acid sequences have been submitted under accession numbers GQ37125, GQ397126 and GQ397127, respectively. The AlPTP2, Al-PTP2b and AlPTP2c protein sequence accession numbers are ACV20865, ACV20866 and ACV20867, respectively. Accession numbers for the three contigs containing A. al-gerae ptp2 sequences are CAIR01005461, CAIR01008152 and CAIR01002286.
Results and discussion
Antibodies against the 35-kDa AlPTP2 cross-reacted with an unknown 70-kDa band
Antibodies raised against AlPTP2 were previously shown to react with two bands from the A. locustae protein extract: a 35-kDa band corresponding to AlPTP2 and an unidentified 70-kDa band named Al70 (Fig. 1a, lane 2), suggesting a strong relationship (most likely conserved epitopes) between AlPTP2 and Al70 (Polonais et al., 2005). However, no further genes showing homology with
(a) (b) 116 96 66 45 31 21 200 kDa 1 2 3 AlPTP2 Al70
Fig. 1. Immunolabelling with antibodies against the 70-kDa band. (a) In Western blots, a similar labelling of two bands, one at 35 kDa (AlPTP2) and one at 70 kDa (Al70), was observed with mouse antibodies raised against AlPTP2 (lane 2) and the 70-kDa band (lane 3). Lane 1: Coomassie Blue-stained profile. (b) Indirect immunofluorescence assay revealed that anti-70-kDa antibodies specifically stained the extruded PTs of Antonospora locustae spores.
Alptp2 could be identified in the first A. locustae database (http://jbpc.mbl.edu/Nosema/index.html). In an attempt to characterize this new protein, mice were immunized with the Al70 antigen. As expected, the corresponding antiserum reacted strongly with the A. locustae extruded PTs (Fig. 1b). On Western blots, as with the anti-AlPTP2 antibodies, anti-Al70 antibodies reacted with both the 70-kDa and the 35-70-kDa bands (Fig. 1a, lane 3). These data prompted us to characterize Al70 as a potential new com-ponent of the PT.
Two further genes coding for PTP2-related proteins are present in theA. locustae genome
Concurrently with our initial study, the A. locustae gen-ome annotation progressed, leading to a final database containing 2600 CDSs (http://forest.mbl.edu/cgi-bin/ site/antonospora01). ABLASTp search against this database
allowed the identification of two CDSs that share high homology with AlPTP2 (CDS 1048 in the A. locustae database). These CDSs, referred to as 1712 and 1329, were named AlPTP2b and AlPTP2c, respectively.
The Alptp2b and Alptp2c genes encode proteins of 568 and 599 aa with deduced molecular masses of 55 399 and 56 664 Da, respectively. These proteins are highly con-served (84.2% identity), larger than AlPTP2 (287 aa), and have basic isoelectric points, a characteristic of all known PTP2 proteins (Table 1; Delbac & Polonais, 2008). As for AlPTP2, a signal peptide could also be identified, with a predicted cleavage site between residues S19 and Y20 for both AlPTP2b and AlPTP2c (Fig. 2).
Comparative analyses of the full-length amino acid sequences revealed three distinct regions: (1) an N-termi-nal serine- and glycine-rich extension that is absent in Al-PTP2 (amino acids 20–310 for AlAl-PTP2b and 20–366 for AlPTP2c), (2) an internal highly conserved region (with a mean 97.3% identity between the three proteins) and (3) a short divergent C-terminal tail (Fig. 2). Analysis of the N-terminal extension revealed the presence of 12 and 17 glycine- and serine-rich degenerated tandem repeats of
12 aa in length in AlPTP2b and AlPTP2c, respectively (consensus sequence: GSGSGTGSGAGT, Fig. 2 and Sup-porting Information, Fig. S1). AlPTP2b and AlPTP2c could be differentiated mainly by the presence of a 60-aa insertion (amino acids 103–162) only found in AlPTP2c, which corresponds to five more repeats of the serine- and glycine-rich repeated motif described above (Figs 2 and S1). A second repeated motif of five amino acids (consen-sus sequence: S/GGGYS/T) can also be observed in both AlPTP2b (amino acids 36–77) and AlPTP2c (amino acids 36–73, Figs 2 and S1).
Because of the glycine and serine richness in the N-terminal region (60.5% and 62.5% in AlPTP2b and Al-PTP2c, respectively), these two residues are the major amino acids found in the AlPTP2-like proteins (Table 1). The serine richness also predicts several potential O-glycosylation sites in this region (43 in Al-PTP2b and 42 in AlPTP2c, amino acids). As in the pre-viously identified PTP2 proteins, eight and nine conserved cysteine residues are present in AlPTP2b and AlPTP2c, respectively, suggesting a potential role in the secondary structure or a cross-linking mechanism. Sec-ondary structure predictions suggest that both helix and coiled-coil domains are the predominant structural fea-tures present in the conserved region. Interestingly, anal-ysis of the N-terminal regions of AlPTP2b/AlPTPT2c revealed that these regions formed mainly b-turn struc-tures (Fig. 2). Structural analysis of already described elastomeric proteins suggested that coiled-coil and par-ticularlyb-turn structures contribute to protein elasticity (Tatham & Shewry, 2002).
In the AlPTP2-like proteins, two common properties, glycine richness and the presence of tandem repeats, sug-gest that both proteins are elastic, in accordance with PT compaction in the spore and its extensible capacity during germination and sporoplasm transfer (Tatham & Shewry, 2002; Franzen, 2005). The high tensile strength of the PT could also be explained by the repeated proline-rich motifs of PTP1 (Delbac et al., 2001; Polonais et al., 2005), as observed for elastin or collagen.
Table 1. Major characteristics of the three PTP2-like proteins from Antonospora locustae
Protein
Length (aa number)
pI† Major aa (%)† Cysteine residue number† No. of potential O-glycosylation sites†
Precursor Mature protein* K G S
AlPTP2 287 268 9.1 12.3 7.1 9.3 8 1
AlPTP2b 568 549 8.4 6.7 22 16.4 8 43
AlPTP2c 599 580 8.7 5 25.3 16.9 9 42
The most abundant amino acid for each protein is underlined.
*The mature proteins correspond to proteins after removal of the predicted N-terminal signal sequence. †
The pI, amino acid percentages and number of potential O-glycosylation sites are deduced from the mature proteins. GenBank accession numbers: AlPTP2, ACV20865; AlPTP2b, ACV20866; AlPTP2c, ACV20867.
In conclusion, the A. locustae genome presents at least three potential ptp2 genes compared with the E. cuniculi genome. The presence of such PTP2-like multigenic fami-lies in the locust microsporidia could be explained by duplication events and/or by the diplokaryotic state of the nucleus. Recent studies support the idea that micros-poridia are diploid organisms harbouring homologous chromosomes (Ironside, 2013; Pombert et al., 2013). Microsporidian sequence diversity may result from intrag-enomic and/or intergintrag-enomic recombination between sequence variants (Ironside, 2013). As observed in Nematocida spp., high levels of heterozygosity could also result from a diploid nucleus and are associated with fre-quent recombination events resulting from meiosis and genetic exchanges (Cuomo et al., 2012). High sequence variability, most likely due to a polyploidy genome, was also observed in A. algerae (Peyretaillade et al., 2012).
MS analysis revealed the presence of AlPTP2b in the 70-kDa band
To characterize the Al70 antigen and to determine whether it corresponded to AlPTP2b and/or AlPTP2c, the 70-kDa band isolated from SDS-PAGE was submitted for MALDI-TOF MS analysis. The obtained peptides were compared with the A. locustae protein database using the
MASCOT software. The best scores were obtained for
AlPTP2b, AlPTP2c and AlPTP2 (Table 2). Among the generated tryptic peptides, one peptide (AAIAQNAAASL PPDMAGSFLTNNPK, 2470.2539 Da) is specific to AlPTP2b (Fig. 2 and Table 2). Indeed, this peptide is characterized by two variable amino acids in comparison with AlPTP2c (AAIAQNAAASLPPDMAGIFLTNNPK, 2496.8638 Da) and AlPTP2 (AAIAQNAAASLPPGMAG-SFLTNNPK, 2412.7458 Da, Fig. 2). Our data suggest that
AlPTP2 MKRLVSLVLLYSILEPVFALSHGYGSKYSVN--- 31 AlPTP2b MKGIIWYMLLISILQPVLSYSSSWSSSSRSSYGGGGGGYSGGGGYSGGGGYTGGGYTGGG 60 AlPTP2c MKGIIWYMLLISILQPVLSYSSSWSSSSRSSYGGGGGGYSGGGGYSGGGGYSG----GGG 56 AlPTP2 --- 31 AlPTP2b YTSGGYTSGGYTGGGYSGGSAKIMMGGHGTMSGTGSSAGTGTGAGT--- 106 AlPTP2c YTSGGYTSGGYTGGGYSGGSAKIMMGGHGTMSGTGSSAGTGTGAGTGSSAGTGTGAGTGS 116 AlPTP2 --- 31 AlPTP2b ---GSGAGTGSGSGTGS 120 AlPTP2c GAGTGSGSGTGSGAGTGSGSGMGSGAGTGSGSGTGSGAGTGSGSGTGSGAGTGSGSGTGS 176 AlPTP2 --- 31 AlPTP2b GAGTGSGSGTGSGAGTGSGSGTGSGAGTGSGSGTGSGAGTGSGSGTGSGAGTGSGSGTGS 180 AlPTP2c GAGTGSGSGTGSGAGTGSGSGTGSGAGTGSGSGTGSGAGTGSGSGTGSGAGTGSGSGTGS 236 AlPTP2 --- 31 AlPTP2b GAGTGSGSGTGSGAGTGSGSGTGSGVETGSGSGTGSGAETGSGSDMGSGKLGHDVVSSSY 240 AlPTP2c GAGTGSGSGTGSGVETGSGSGTGSGVETGSGSGTGSGAETGSGSDMGSGKLGHDVVSSSY 296 AlPTP2 --- 31 AlPTP2b GGGETSSSSNAVVSSTGVSHTGSTVSSPAHGGISSGVSIVPLPVMAQGVAATTAKPETSI 300 AlPTP2c GGGETSSSSNAVVSSTGVSHTGSTVSSPAHGGISSGVSIVPLPVMAQGVAATTAKPETSI 356 AlPTP2 ---AVSGSSGAVNNAAREKTLSLEQNRVLKAAIAQNAAASLPPGMAGSFLTNNPKCKSSS 88 AlPTP2b QQHTSAGTSAASRNATREQILSLEQNKVLKAAIAQNAAASLPPDMAGSFLTNNPK AlPTP2c QQHTSAGTSAASRNATREQILSLEQNKVLKAAIAQNAAASLPPDMAGIFLTNNPKCKSSS 416 CKSSS 360
AlPTP2 VDGNWSMLQTKLNQDCVEKVIEKNERAKKYAQILSKPSSKECIKKLNPNAMVCQARKVMS 148 AlPTP2b VDGNWSMLQTKLNQDCVEKVIEKNERAKKYAQILSKPPSKECIKKLNPNAMVCQARKVMS 420 AlPTP2c VDGNWSMLQTKLNQDCVEKVIEKNERAKKYAQILSKPPSKECIKKLNPNAMVCQARKVMS 476 AlPTP2 RVVEEPYYQIIMYGNVVQLVEDGRVKMMGVVEKIHYDVEKKNMRTPSPLKKPTALLEFNE 208 AlPTP2b RVVEEPYYQIIMYGNVVQLVEDGRVKMMGVVEKIHYDVEKKNMRTPSPLKKPTALLEFNE 480 AlPTP2c RVVEEPYYQIIMYGNVVQLVEDGRVKMMGVVEKIHYDVEKKNMRTPSPLKKPTALLEFNE 536 AlPTP2 LGGLLKQKGKIPAAKSPDPCLSSCIEALEKKAAVEGQNEGCGECESLMEITTVRPGIFKD 268 AlPTP2b LGGLLKQKGKIPAAKSPDPCLSSCIEALEKKAAVEGQNEGCGECESLMEITTVRPGIFKD 540 AlPTP2c LGGLLKQKGKIPAAKSPDPCLSSCIEALEKKAAVEGQNEGCGECESLMEITTVRPVCGAR 596
AlPTP2 ASKKKEETPSGENKEKSEG--- 287 AlPTP2b ASKKGKDSYSSENKKSEDQDNEKEKEES 568 AlPTP2c GHR--- 599 C-terminal tail Conserved Region N-terminal extension
Fig. 2. Alignment of the AlPTP2, AlPTP2b and AlPTP2c amino acid sequences. Residues that are identical between at least two sequences are shaded in grey. The peptide specific to AlPTP2b that was identified by MALDI-TOF is underlined (positions 331–355). The arrowhead indicates the predicted cleavage site of the peptide signal between S19 and Y20 for both AlPTP2b and AlPTP2c. The eight conserved cysteine residues are in black boxes. The red springs indicate theb-turn domains, the blue cylinders show the helix and the black lines indicate the coiled-coil regions. These secondary structures were predicted using the PSIPRED and BETATPRED2 servers. Three regions can be distinguished: an N-terminal extension specific to AlPTP2b and AlPTP2c that is rich in repeated motifs (see Fig. S1), a region that is conserved between the three PTP2 proteins and a divergent C-terminal tail. Amino acids are numbered on the right.
the 70-kDa band contains at least AlPTP2b. However, even though no specific peptide of AlPTP2c was identified, the presence of this protein in the 70-kDa band cannot be fully excluded.
Antibodies against AlPTP2b specifically labelled the PT
To better characterize the AlPTP2b product and assess its localization, specific antibodies were raised against the
N-terminal serine- and glycine-rich extension that is pres-ent in both AlPTP2b and AlPTP2c (amino acids 20–306, Fig. 2) but absent in AlPTP2. To check if the GST-AlPTP2b-His recombinant protein could be used as an AlPTP2b-specific antigen, anti-AlPTP2 and anti-Al70 antibodies were tested on GST-AlPTP2b-His. As expected, a 56-kDa band corresponding to the recombinant protein was detected using the antibodies produced against the Al70 band (Fig. 3a). In contrast, no reaction was obtained with anti-AlPTP2 (Fig. 3a), suggesting that the
Table 2. MALDI-TOF analysis of the Antonospora locustae 70 kDa band
Peptide sequence Peptide mass (Da) AlPTP2b AlPTP2c AlPTP2
EQILSLEQNK 1201.6530 + + LNPNAMVCQAR 1287.6379 + + + KPTALLEFNELGGLLK 1743.0106 + + + AAIAQNAAASLPPDMAGSFLTNNPK 2470.2539 + VVEEPYYQIIMYGNVVQLVEDGR 2713.3154 + + + AAVEGQNEGCGECESLMEITTVRPGIFK 3110.4084 + + KAAVEGQNEGCGECESLMEITTVRPGIFK 3228.4915 + + + Coverage 20% 14% 28%
For each protein, the identified peptides are compared with peptides obtained in silico. The presence (+) or the absence ( ) of each peptide is indicated for the three proteins. The 2470.2539-Da peptide in bold is specific of AlPTP2b.
+ – – – – + – – – – + – – – – + CB staining anti-AlPTP2 anti-Al70 anti-AlPTP2b purified recombinant AlPTP2b A. locustae extract + – – – – + – – – – + – – – – + 35 kDa 70 kDa (b) AlPTP2 AlPTP2b 56 kDa (a) GST-AlPTP2b-His PT (c) 4 µm
Fig. 3. Antibodies produced against the recombinant AlPTP2b protein only recognized the 70-kDa band and specifically stained the extruded PT. Immunoblotting with the anti-AlPTP2, anti-Al70 and anti-AlPTP2b antibodies against the purified recombinant protein AlPTP2b (a) and the Antonospora locustae sporal protein extract (b). (c) IFA with antibodies raised against the AlPTP2b recombinant protein showing the specific labelling of the A. locustae extruded PT.
recombinant protein could be used to generate specific antibodies to AlPTP2b. The AlPTP2b-specific antiserum was first applied to whole protein extracts from A. locustae spores. As expected, only the 70-kDa band was detected using the antibodies raised against the N-terminal extension, which is present in AlPTP2b but not in AlPTP2 (Fig. 3b). The size of the detected band is, however, higher than expected ( 53 kDa for the mature AlPTP2b protein, Table 2). Anti-AlPTP2b antibodies were
then used in IFAs on A. locustae spores. Strong PT label-ling was observed (Fig. 3c), confirming that (1) AlPTP2b is a new PT protein related to PTP2 and that (2) more than one PTP2-like protein is encoded in the A. locustae genome.
As previously described for PTP1 (Delbac et al., 2001), a migration defect was observed for AlPTP2b, which could be explained by post-translational modifications because of the occurrence of a high number of potential
CAIR01005461 ---GSG 3 CAIR01008152 ---GSG 3 CAIR01002286 ---SGGGSGGASMSGVSSGGSSMSFSGGSSGGASSGGSSSHGSGGSGSG 46 AlPTP2b MKGIIWYMLLISILQPVLSYSSSWSSSSRSSYGGGGGGYSGGGGYSGGGGYTGGGYTGGG 60 CAIR01005461 SGSG---HGSGSGGGAPGG---GSGHGSGSGTPGG-GSGSGPGSGSGHGSGSG--- 49 CAIR01008152 SGSG---SGSGHGSGSGGG---APGGGSGSGAPGG-GSGGGSGSGSGHGSGSG--- 49 CAIR01002286 SSSG---HGSGSGGGAPGK---SSGSESGSGAPGG-GSGSGSGSGSGHGSGSG--- 92 AlPTP2b YTSGGYTSGGYTGGGYSGGSAKIMMGGHGTMSGTGSSAGTGTGAGTGSGAGTGSGSGTGS 120 CAIR01005461 GGAPGGGSDSGSGAGSG---AQGGSS---AGE 75 CAIR01008152 GGAPGGGSGNGSGAGSGNGSGSG--APGGGSGSGPGSGSGHGSGSGGGAPGGGSDSGSGA 107 KI0APB10YG18AHM1 GGAPGGGSGNGSGSGSGHGSGSGGGAPGGGSGSGAPGG---GSGGGSGSGSGHGSGS 146 AlPTP2b GAGTGSGSGTGSGAGTGSGSGTG-SGAGTGSGSGTGSG---AGTGSGSGTGSGAGTGS 174
CAIR01005461 GSG-AKGSGS---AGEGSGAQGSGSAGEGSGAKGSGSAGEGSGAQGGSSAGEGSGAQG-- 129 CAIR01008152 GSG-AQGGSS---AGEGSGAKGSGSAGEGSGAQGGSSAGEGSGAKGGSSAGEGSGALG-- 161 CAIR01002286 GGG-APGGGSDSGSGAGSGAQGGSSAGEGSGAKGSGSAGEGSGAQGGSSAGEGSGAKG-- 203 AlPTP2b GSGTGSGAGTGSGSGTGSGAGTGSGSGTGSGVETGSGSGTGSGAETGSGSDMGSGKLGHD 234 CAIR01005461 --SGSAGEG---NAPEKGAEGSGQ---AQKPGIGQA 157 CAIR01008152 --SGSAGEG---NAPEKGAEGSGQ---AQKPGIGQA 189 CAIR01002286 --SGSAGEG---NAPEKGAEGSGQ---AQKPGIGQA 231 AlPTP2b VVSSSYGGGETSSSSNAVVSSTGVSHTGSTVSSPAHGGISSGVSIVPLPVMAQGVAATTA 294 CAIR01005461 GPGNAAAQGDSTG--AAN---DLVALETHKALKTALNQSMANSVSPQTANEFLAGNP 209 CAIR01008152 GPGNAAAQGDSTG--AAN---DLVALETHKALKTALNQSMANSVSPQTANEFLAGNP 241 CAIR01002286 GPGNAAAQGDSTG--AAN---DLVALETHKALKTALNQSMANSVSPQTANEFLAGNP 283 AlPTP2b KPETSIQQHTSAGTSAASRNATREQILSLEQNKVLKAAIAQNAAASLPPDMAGSFLTNNP 354 CAIR01005461 ECNQQAVEGNWKVINEKIKQECRNKVNERKAKAEQIAKFITKPTPDKCVKKLNPNALVCQ 269 CAIR01008152 ECNQQAVEGNWKVINEKIKQECRNKVNERKAKAEQIAKFITKPTPDKCVKKLNPNALVCQ 301 CAIR01002286 ECNQQAVEGNWKVINEKIKQECRNKMNERKAKAEQIAKFITKPTPDKCVKKLNPNALVCQ 343 AlPTP2b KCKSSSVDGNWSMLQTKLNQDCVEKVIEKNERAKKYAQILSKPPSKECIKKLNPNAMVCQ 414
* * * *
CAIR01005461 ALTVMNTIIKQPRFKIDLYGSNVVEIKKDGSPILLGVAESLNYNVTKMKRKRYNPLKNPT 329 CAIR01008152 ALTVMNTIIKQPRFKIDLYGSNVVEIKKDGSPILLGVAESLNYNVTKMKRKRYNPLKNPT 361 CAIR01002286 ALTVMNTIIKQPRFKIDLYGSNVVEIKKDGSPILLGVAESLNYNVTKMKRKRYNPLKNPT 403 AlPTP2b ARKVMSRVVEEPYYQIIMYG-NVVQLVEDGRVKMMGVVEKIHYDVEKKNMRTPSPLKKPT 473 CAIR01005461 ARLEFNELGSLLKQEGEIPKPKRLDPCG-SCLEKLSKKAQVEGEKDMCGGCDALMELNSL 388 CAIR01008152 ARLEFNELGSLLKQEGEIPKPKRLDPCG-SCLEKLSKKAQVEGEKDMCGGCDALMELNSL 420
CAIR01002286 ARLEFNELGSLLKQEGEIPKPKRLDPCG-SCLEKLSKKAQVEGEKDMCGGCDALMELNSL 462 AlPTP2b ALLEFNELGGLLKQKGKIPAAKSPDPCLSSCIEALEKKAAVEGQNEGCGECESLMEITTV 533
* * * *
CAIR01005461 KRGSFNNLQTENKKPEVTPTETTKAAEESK--- 418 CAIR01008152 KRGSFNNLQTENKKPEVTPTETTKAAEESK--- 450 CAIR01002286 KRGSFNNLQTENKKPEVTPTETTKAAEESK--- 492 AlPTP2b RPGIFKDASKKGKDSYSSENKKSEDQDNEKEKEES 568
(a) A. algerae anti-AlPTP2b P. grylli anti-AlPTP2b (b) PT PT
Fig. 4. AlPTP2b orthologous proteins in other microsporidia parasites of insects. (a) Indirect immunofluorescence labelling of the extruded PTs of both the Anncaliia algerae and Paranosema grylli spores using the anti-AlPTP2b antibodies. PT. Scale bar= 2 lm. (b) Amino acid sequence alignment between AlPTP2b and three partial orthologous sequences identified in the Anncaliia algerae genome. The three Anncaliia algerae sequences are incomplete, as the N-terminal sequence is missing (contig extremity). Residue numbers are indicated on the right of the sequences. Residues that are identical between two or three sequences are shaded in grey, and residues that are identical among all four sequences are shaded in black. Cysteine residues in conserved positions are indicated by an asterisk. GenBank accession numbers are: ACV20866 (AlPTP2b), CAIR01005461 (contig KI0AQA2YO04FM1), CAIR01008152 (contig KI0ALA25YH17FM1) and CAIR01002286 (contig KI0APB10YG18AHM1).
phosphorylation sites (44) and potential O-glycosylation sites (43, Table 1) as predicted by in silico analysis. These results are in accordance with the genome sequence anal-ysis associated with the global structural sugar analanal-ysis that suggests the occurrence of O-mannosylation (Taupin et al., 2007).
PTP2b-like proteins are also present in other insect microsporidia phylogenetically related toA. locustae
Anti-AlPTP2b antibodies were then applied to the mam-malian microsporidia E. cuniculi and to different insect microsporidian spores, including P. grylli, A. algerae and N. bombycis. Although no cross-reaction was observed with E. cuniculi PTs (not shown), specific labelling associ-ated with the extruded PT was obtained for P. grylli and A. algerae (Fig. 4a). These data are in agreement with the presence of a unique ptp2 gene in the Encephalitozoon ge-nomes (Katinka et al., 2001; Corradi et al., 2010). We also looked for PTP2 orthologous sequences in the avail-able microsporidian genomes (Tavail-able S1). Three PTP2b orthologous proteins were identified in the recently sequenced A. algerae genome (Peyretaillade et al., 2012), confirming our IFA data (Fig. 4b).
The identified incomplete sequences are highly con-served (at least 80% identity, Fig. 4b), with some amino acid variations in the N-terminal part that are probably due to the polyploidy of A. algerae nuclei or to dupli-cated genes that evolved independently because of the high structural and functional confines in the C-terminal part of the protein in contrast to the N-terminal end. Anncaliia algerae sequences displayed between 36.3% and 41.2% identity with AlPTP2b (Fig. 4b). Anncaliia algerae incomplete protein sequences displayed an N-terminal glycine- and serine-rich region associated with a C-termi-nal region containing cysteine residues in conserved posi-tions, suggesting an important role of these positions in the secondary structure.
We also were able to amplify a ptp2b orthologous gene in P. grylli (data not shown), demonstrating that this gene is also conserved in the cricket parasite. All of these data suggest that a PTP2 multigenic family is present in insect parasites that are phylogenetically related (Lee et al., 2008a) but is not present in mammalian microsporidia.
In conclusion, genome comparisons based on sequence and gene order conservation associated with MS (Wang et al., 2007) will improve our knowledge of the PT components and structure in microsporidia. Numerous sequencing projects are also underway to improve geno-mic comparisons. Similarly, the development of genogeno-mic approaches will be crucial to clarify the role of PTPs, to study PTP interactions and to determine if the presence
of multigenic families correlates with a broad spectrum of hosts or is separate from the immune system of the host.
Acknowledgement
V.P. was supported by a grant from ‘Ministere de l’Edu-cation, de la recherche et de la technologie’.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Fig. S1. Alignment of the N-terminal repeated amino acid motifs of both AlPTP2b and AlPTP2c.
Table S1. Identification of PTP2b and PTP2c orthologous sequences in microsporidian genomes.
