Microsporidia are obligate intracellular eukaryotic parasites with a broad host spectrum characterized by a unique and highly sophisticated invasion apparatus, 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 several PTP2-like proteins are also present in other phylogenetically related insect microsporidia, including Anncaliia algerae and Paranosema grylli.
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 mammals (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 & Slamovits, 2011; Corradi & Selman, 2013).
Despite having considerably different host cell ranges and cell-type specificities, all microsporidian species possess 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 mammalian microsporidia Encephalitozoon cuniculi and E. intestinalis (Delbac et al., 2001; Peuvel et al., 2002; Corradi et al., 2010), in the bee parasite Nosema ceranae (Cornman et al., 2009) and in the Bombyx mori parasite Nosema bombycis (Wang et al., 2007). Genomic surveys have also allowed the identification of PTP1 and PTP2 orthologous proteins in the locust parasite Antonospora (formerly Nosema) locustae, as in the case of the cricket microsporidia Paranosema grylli, a parasite phylogenetically 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 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 identification 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 grasshoppers were commercially available from the M & R Durango, Inc. Insectary (Bayfield, CO).
DNA extraction and expression of recombinant AlPTP2b in Escherichia coli
Antonospora locustae genomic DNA was purified using the ELU-Quick DNA purification Kit (Schleicher and Schuell, Dassel, Germany). PCR primers (5′-CGGGATCCTCGTATTCGAGTAGTTGG-3′ and 5′-CGGAATTCTGCGGATGTATGTTGTTG-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 annealing step at 55 °C. The Alptp2b truncated form was cloned in the expression vector pGEX4T-1 modified to provide a C-terminal 8 × His-tag. The resulting recombinant vector was introduced into the E. coli BL21-codon+. After induction [2 mM isopropylthio-β-galactoside (IPTG), 4 h], the recombinant protein was purified under denaturing conditions on a Ni-NTA column according to the manufacturer'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 produced 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 Laemmli buffer (2.5% SDS, 100 mM dithiothreitol) by repeated freeze–thaw cycles in liquid nitrogen and separated 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 μL−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, 1 μL 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 Biosystems, Farmingham, MA) was used in the positive ion reflector mode for peptide mass fingerprinting. External calibration was performed with a standard peptide solution (Proteomix), while the internal calibration was performed using peptides from porcine trypsin autolysis. The protein database from A. locustae (http://forest.mbl.edu/cgi-bin/site/antonospora01) was explored using mascot software version 2.1 (http://www.matrixscience.com). Peptide masses were assumed to be monoisotopic.
The Alptp2, Alptp2b and Alptp2c nucleic acid sequences have been submitted under accession numbers GQ37125, GQ397126 and GQ397127, respectively. The AlPTP2, AlPTP2b and AlPTP2c protein sequence accession numbers are ACV20865, ACV20866 and ACV20867, respectively. Accession numbers for the three contigs containing A. algerae 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 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-kDa bands (Fig. 1a, lane 3). These data prompted us to characterize Al70 as a potential new component of the PT.
Two further genes coding for PTP2-related proteins are present in the A. locustae genome
Concurrently with our initial study, the A. locustae genome annotation progressed, leading to a final database containing ∼2600 CDSs (http://forest.mbl.edu/cgi-bin/site/antonospora01). A blastp 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 conserved (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).
Table 1. Major characteristics of the three PTP2-like proteins from Antonospora locustae
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.
Comparative analyses of the full-length amino acid sequences revealed three distinct regions: (1) an N-terminal serine- and glycine-rich extension that is absent in AlPTP2 (amino acids 20–310 for AlPTP2b 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 Supporting 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 (consensus 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 AlPTP2c, 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 AlPTP2b and 42 in AlPTP2c, amino acids). As in the previously 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. Secondary structure predictions suggest that both helix and coiled-coil domains are the predominant structural features present in the conserved region. Interestingly, analysis of the N-terminal regions of AlPTP2b/AlPTPT2c revealed that these regions formed mainly β-turn structures (Fig. 2). Structural analysis of already described elastomeric proteins suggested that coiled-coil and particularly β-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, suggest 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.
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 families 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 microsporidia are diploid organisms harbouring homologous chromosomes (Ironside, 2013; Pombert et al., 2013). Microsporidian sequence diversity may result from intragenomic and/or intergenomic 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 frequent 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 (AAIAQNAAASLPPDMAGSFLTNNPK, 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 (AAIAQNAAASLPPGMAGSFLTNNPK, 2412.7458 Da, Fig. 2). Our data suggest that 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.
Table 2. MALDI-TOF analysis of the Antonospora locustae 70 kDa band
Peptide mass (Da)
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.
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 present 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 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 labelling 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 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 analysis associated with the global structural sugar analysis that suggests the occurrence of O-mannosylation (Taupin et al., 2007).
PTP2b-like proteins are also present in other insect microsporidia phylogenetically related to A. locustae
Anti-AlPTP2b antibodies were then applied to the mammalian 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 associated 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 genomes (Katinka et al., 2001; Corradi et al., 2010). We also looked for PTP2 orthologous sequences in the available microsporidian genomes (Table 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 conserved (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 duplicated 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-terminal region containing cysteine residues in conserved positions, 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 genomic comparisons. Similarly, the development of genomic 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.
V.P. was supported by a grant from ‘Ministère de l'Education, de la recherche et de la technologie’.