Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall

ABSTRACT

All microsporidia possess a unique, highly specialized invasion mechanism that involves the polar tube and spore wall. The interaction between spore wall proteins (SWPs) and polar tube proteins (PTPs) in the formation, arrangement, orderly orientation, and function of the polar tube and spore wall remains to be determined. This study was undertaken to examine the protein interactions of Nosema bombycis SWP7 (NbSWP7), NbSWP9, and PTPs. Coimmunoprecipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and yeast two-hybrid data demonstrated that NbSWP9, but not NbSWP7, interacts with NbPTP1 and NbPTP2. Furthermore, immunoelectron microscopy (IEM) showed that NbSWP9 was localized mainly in the developing polar tube of sporoblasts, while NbSWP7 was found randomly in the cytoplasm. However, both NbSWP9 and NbSWP7 were located in the polar tube and spore wall of N. bombycis mature spores. The reason why NbSWP7 was localized to the polar tube may be due to the interaction between NbSWP9 and NbSWP7. Interestingly, the majority of NbSWP9, but not NbSWP7, accumulated in the beginning part of the extruded polar tube and the ruptured spore wall called the anchoring disk (AD) when the mature spores germinated under weak-alkaline environmental stimulation. Additionally, anti-NbSWP9 antibody reduced spore germination in a dose-dependent manner. In conclusion, our study further confirmed that NbSWP9 is a scaffolding protein that not only anchors and holds the polar tube but also tethers the polar tube to the spore wall.

INTRODUCTION

Microsporidia are obligate, spore-forming, intracellular and unicellular eukaryotic parasites that can infect a wide range of vertebrate and invertebrate species (1). Many species of microsporidia, which have been recognized as opportunistic pathogens, are found in immunocompromised individuals, particularly patients with AIDS (1, 2). Nosema bombycis, which was first recognized in 1857 by Nägeli (3), causes pebrine disease, which brings heavy economic losses to countries with silk production (1). However, the microsporidian cell exhibits high reduction at the cellular, organelle, biochemical, and genomic levels, which means that microsporidia underwent highly reductive evolution (4–6). The general life cycle in the development of microsporidian parasites comprises three phases: an infective or environmental phase and a proliferative phase, identified by some as merogony, followed by a sporogonic differentiation or spore-forming phase leading to the release of mature resistant spores by host cell disruption (7, 8).

The mature microsporidial spore is composed of a thick wall, a long thread-like polar filament, the polaroplast, a posterior vacuole, the anchoring disk (AD), the endoplasmic reticulum, the nucleus structure (uninucleate or binucleate), and so on (9, 10). The intact spore wall plays a key role in maintaining the spore's morphology, resisting the outer environment and adhering to host cells. The thick spore wall is usually comprised of an electron-dense proteinaceous outer layer (exospore) of 25 to 30 nm and an electron-transparent chitinous inner layer (endospore) of 30 to 35 nm (9, 11, 12). The major component of the endospore, chitin, possibly forms the fibril bridges across the endospore, which probably acts as a link between the exospore and the plasma membrane (12, 13). Microsporidial spore wall proteins (SWPs), the major component of the exospore, may function both as a protective screen for free-living spores and as a ligand for adherence to host cell. However, only a few spore wall proteins have been identified. Until now, 2 exosporal proteins and 4 endosporal proteins have been identified in the Encephalitozoonidae family. Encephalitozoon cuniculi SWP1 (EcSWP1)/Encephalitozoon intestinalis SWP1 (EiSWP1) and Encephalitozoon intestinalis SWP2 (EiSWP2) are exosporal proteins, and EcEnP1/EiEnp1, EcEnp2, EcSWP3, and EcCDA are endosporal proteins (14–19). In addition, 9 spore wall proteins have been identified in N. bombycis. N. bombycis SWP25 (NbSWP25), NbSWP26, NbSWP30, and NbSWP16 are endosporal proteins, and NbSWP32 and NbSWP5 are exosporal proteins (20–26). Furthermore, NbSWP7 and NbSWP9 are colocalized in both the exospore and the endospore of the spore wall (27). EcEnP1, NbSWP7, NbSWP9, and NbSWP26 play a catalytic role in spore adherence and infection processes (19, 22, 27). In addition, monoclonal antibody 2B10 could recognize EOB13320, which is an endospore protein of N. bombycis with a molecular mass of 50 kDa (28).

The microsporidian spore exhibits a number of unique features; however, the most typical basic characteristic is the special invasion organelle, the polar tube. It is coiled within the interior of the spore wall and attached to a cellular mushroom-shaped structure named the anchoring disk at the anterior end of the spore (29). The polar tube is composed of three distinct polar tube proteins (PTPs): PTP1, a proline-rich protein (30, 31); PTP2, a lysine-rich protein (32); and PTP3, a large protein with a molecular mass of >135 kDa (33, 34). Microsporidia infect host cells by a singularly unique invasion mechanism (34). Mature spores are activated under conditions of appropriate environmental stimulation, such as changes in pH and ion concentrations (35). This process finally leads to the explosive discharge of the hollow polar tube, followed by the transfer of the infectious sporoplasm into the host cell, a process known as germination. During this process, the spore wall allows an increase in hydrostatic pressure that causes spore discharge, implying that the spore wall is a critical part of the germination-and-invasion apparatus (36). While the spore wall is thought to mediate the sudden rise in osmotic pressure, the mechanism of spore wall and polar tube assembly and germination is not known.

This study was designed to describe the possible manner of assembly between the spore wall and polar tube, which are involved in the coiled polar tube arrangement and germination processes. The interaction between two spore wall proteins (NbSWP7 and NbSWP9) and two polar tube proteins (NbPTP1 and NbPTP2) was analyzed by methods of coimmunoprecipitation (co-IP), colocalization, transmission electron microscopy (TEM), and yeast two-hybrid analyses. To understand the role of NbSWP7 and NbSWP9 in polar tube formation, anchoring to the spore wall, and the germination process, the localization of NbSWP7 and NbSWP9 in early life cycle stages and germinant spores was studied for the first time. NbSWP9 in the endospore was also demonstrated to act as a scaffolding protein that may support the coiled polar tube within the mature spore and anchor the discharged polar tube to the spore wall. Furthermore, NbSWP9 may contribute to the germination process by enabling the coiled polar tube to attach to the spore wall.

RESULTS

NbSWP9 interacts with NbPTP1 and NbPTP2.

Our previous research found that NbSWP7 and NbSWP9 are mainly colocalized in the spore wall and polar tube of N. bombycis and presented the first examination of the interaction between them (27). In addition, NbSWP7 and NbSWP9 localized in the polar tube of mature N. bombycis spores germinating in 0.1 M K₂CO₃ (27). These interesting results enable us to infer interactions between NbSWP7 and NbSWP9 and NbPTP1 and NbPTP2. To determine whether NbSWP7 and NbSWP9 interact with NbPTP1 and NbPTP2 or not, co-IP and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses were employed. Total proteins from mature N. bombycis spores were immunoprecipitated with rabbit anti-NbSWP9 antibody. As shown in Fig. 1A, a band with an apparent molecular mass of 95 kDa was identified, which was significantly different from the band for the control. At the same time, Western blotting was used to detect NbSWP9, and it was displayed clearly (Fig. 1B). Subsequently, the differential band was excised and analyzed by LC-MS/MS. Sequence analysis was performed against the N. bombycis protein database, and five genes were identified. These genes encode NbPTP2, NbPTP3, the transitional endoplasmic reticulum ATPase TER94, M1 family aminopeptidase 1, and elongation factor 2 (Table 1). Co-IP and LC-MS/MS analyses showed that NbSWP9 probably interacts with NbPTP2 and NbPTP3. It was found that Encephalitozoon cuniculi PTP1 (EcPTP1), EcPTP2, and EcPTP3 interact with each other (37). Therefore, co-IP was further conducted with rabbit anti-NbSWP7 and anti-NbSWP9 antibodies, and immunoblotting was then performed with mouse antibodies against NbPTP1 and NbPTP2. As expected, NbSWP9 coprecipitated with NbPTP1 and NbPTP2; however, NbSWP7 did not coprecipitate with NbPTP1 and NbPTP2 (Fig. 1C). This result demonstrated that NbSWP9 interacts with NbPTP1 and NbPTP2 but not NbSWP7 in vitro. Furthermore, a yeast two-hybrid system was used to further confirm these interactions. NbSWP7 and NbSWP9 were used as the bait and NbPTP1 and NbPTP2 were used as the prey to determine if these proteins interacted in vivo. Yeast colonies containing the constructs pGBKT7-Nbswp9/pGADT7-Nbptp1 and pGBKT7-Nbswp9/pGADT7-Nbptp2 grew on synthetic dropout medium (SD) plates lacking Leu, Trp, His, and Ade and containing X-α-Gal (5-bromo-4-chloro-3-indoxyl-α-d-galactopyranoside) and turned blue by hydrolyzing X-α-Gal (Fig. 1D). However, the colonies including the constructs pGBKT7-Nbswp7/pGADT7-Nbptp1 and pGBKT7-Nbswp7/pGADT7-Nbptp2 grew on SD plates lacking Leu, Trp, His, and Ade and containing X-α-Gal and did not turn blue, indicating that α-galactosidase may not be synthesized and secreted in the medium. Therefore, the X-α-Gal is not hydrolyzed. The yeast colonies (including the pGBKT7-Nbswp7/pGADT7-Nbptp1 and pGBKT7-Nbswp7/pGADT7-Nbptp2 constructs) that grew on SD plates lacking Leu, Trp, His, and Ade and containing X-α-Gal plates may show false-positive results (Fig. 1E). The above-described data demonstrate that only NbSWP9 interacts with NbPTP1 and NbPTP2 in vitro and in vivo, while NbSWP7 does not.

FIG 1.

NbSWP9 interacts with polar tube proteins of N. bombycis, while NbSWP7 does not. (A) SDS-PAGE and silver nitrate staining were performed to identify the interacting proteins of NbSWP9. Total proteins of N. bombycis mature spores were extracted and immunoprecipitated by using an anti-NbSWP9 antibody. Lane M, prestained marker. The first two lanes in Fig. 1A (anti-NbSWP9 and negative sera) are co-IP positive, the third lane shows the detection of only rabbit anti-NbSWP9 antibody, and the last lane (Co-IP −) shows silver staining of the protein from the total spore protein. (B) Anti-NbSWP9 antibody precipitates NbSWP9 from total mature spore proteins. Immunoprecipitation was performed by using rabbit anti-NbSWP9 antibody. Immunoblotting of the NbSWP9 immunoprecipitation samples was then performed by using mouse anti-NbSWP9 antibody to detect the corresponding NbSWP9 protein. The total mature spore proteins contained NbSWP9 as a positive control. The negative-control antibody did not precipitate NbSWP9. Lane M, prestained marker. (C) Western blotting of coimmunoprecipitate samples was used to analyze the interaction between NbSWP7 and NbSWP9 and between NbPTP1 and NbPTP2. Total mature spore proteins were coimmunoprecipitated with rabbit anti-NbSWP9 and anti-NbSWP7 antibodies, respectively. Immunoprecipitates were treated with loading buffer, run on a 12% SDS-PAGE gel, transferred onto a polyvinylidene difluoride membrane, and probed with 10 μg mouse anti-NbPTP1 (1:6,000) or anti-NbPTP2 (1:6,000). The samples were incubated with HRP-conjugated anti-mouse IgG (1:8,000) and detected with enhanced chemiluminescence. The negative-control antibody did not precipitate NbPTP1 and NbPTP2. Lane M, EasySee Western marker. (D and E) A yeast two-hybrid assay was used to further determine the in vivo interactions of NbSWP7 and NbSWP9 with NbPTP1 and NbPTP2. Shown are interactions between NbSWP7 and NbSWP9, as bait, and NbPTP1 and NbPTP2, as prey. pGADT7-Nbptp1 (prey)/pGBKT7-Nbswp7 (bait), pGADT7-Nbptp2 (prey)/pGBKT7-Nbswp7 (bait), pGADT7-Nbptp1 (prey)/pGBKT7-Nbswp9 (bait), and pGADT7-Nbptp2 (prey)/pGBKT7-Nbswp9 (bait) constructs were transformed into competent yeast cells, respectively. A number of independent blue colonies including the pGADT7-Nbptp1 (prey)/pGBKT7-Nbswp9 (bait) and pGADT7-Nbptp2 (prey)/pGBKT7-Nbswp9 (bait) constructs grew on SD plates lacking Leu, Trp, His, and Ade and containing X-α-Gal. However, the colonies including the constructs pGADT7-Nbptp1 (prey)/pGBKT7-Nbswp7 (bait) and pGADT7-Nbptp2 (prey)/pGBKT7-Nbswp7 (bait) grew on SD plates lacking Leu, Trp, His, and Ade and containing X-α-Gal and did not turn blue. This demonstrates the interactions of pGBKT7-Nbswp9 (bait) with pGADT7-Nbptp1 (prey) and pGADT7-Nbptp2 (prey), while pGBKT7-Nbswp7 (bait) did not interact with pGADT7-Nbptp1 (prey) and pGADT7-Nbptp2 (prey). Positive-control pGBKT7-53/pGADT7-T (P) and negative-control pGBKT7-lam/pGADT7-T (N) reactions are provided for each group.

TABLE 1.

LC-MS/MS analysis of polar tube protein tryptic peptides of N. bombycis

GenBank accession no.	Gene locus	Annotation	Sequence coverage (%)	Isoelectric point	Molecular mass (kDa)	No. of unique peptides	No. of peptides	Unique peptidase sequence
EOB15197	NBO_7g0015	Polar tube protein 2	41.37	9.39	30.92	11	30	K.AKEAYVFNAIGEVLSTK.Q
								K.EAYVFNAIGEVLSTK.Q
								K.LLNELKNEPEYTVTGEENK.V
								K.LLNELKNEPEYTVTGEENKVNVF
								K.K
								K.NEPEYTVTGEENK.V
								K.NEPEYTVTGEENKVNVFK.K
								K.NYDLFVNILSNTTVSEPGPEEK.K
								K.STPQTAEGTPLINECK.Q
								R.AQQQMLPAVVDPR.K
								R.AVQTIEHIKQEPK.C
								R.QAQAIETAAR.A
EOB13243	NBO_95g0003	M1 family aminopeptidase 1	16.73	5.51	92.2	11	22	K.DVLTYNSLLVTNAIK.F
								K.EIIDGYYVFNTTPK.M
								K.FKVESLMNSWTGNK.G
								K.M*SIYNVAIVSGK.L
								K.MSIYNVAIVSGK.L
								K.NKDVLTYNSLLVTNAIK.F
								K.SPLNVEDR.Y
								R.DYIALSNMSVK.E
								R.EIILDFICDHFDDIR.A
								R.LCDSINNETDPEVLK.V
								R.YNLVSDMFALCNAK.M
EOB13741	NBO_61gi002	Transitional endoplasmic reticulum ATPase TER94	22.96	5.2	86.98	11	23	K.AVANETGAFIYLINGPEIMSK.M
								K.AVATECQANFISIK.G
								K.GILLYGPPGTGK.T
								K.GPELLTM*WVGESESNVR.E
								K.M*AGESENNLR.K
								K.TPLSPDVNLVQLAEATDR.F
								R.ISSGIGSVEYK.V
								R.IVSQLLTLMDGSK.S
								R.KTPLSPDVNLVQLAEATDR.F
								R.LDQLVYIPLPDLDSR.L
								R.SAAPCVLFFDEIDSVAK.S
EOB15062	NBO_10g0052	Polar tube protein 3	7.29	6.29	150.3	6	11	K.AGGSTDEAAAVR.V
								K.FIEDTFNSAYSSGQAQR.F
								K.TFIDEEVSNVGEAYVK.N
								R.ALADSLGMTEEDFIQFAR.K
								R.NALYDDIGKEPIEIK.S
								R.SDQVIAGPNGGTSALSQATAPR.T
EOB15403	NBO_4g0031	Elongation factor 2	6.67	7.03	93.07	4	6	K.GLVLAGCPVLYEPIFR.V
								K.SPFVVYVLNPIYK.V
								R.LYMTVEPLEEK.I
								R.VQEPGYSPASTTVANK.S

NbSWP9 contributes to polar tube arrangement, orderly orientation, and attachment to the spore wall during the spore maturation process.

Based on the interaction of NbSWP9 with NbPTP1 and NbPTP2, we sought to determine whether NbSWP9 mediated polar tube arrangement and maturation during the development process of N. bombycis. Our previous study showed that 60% and 75% Percoll gradient centrifugation fractions are rich in sporoblasts, while the 90% Percoll gradient fraction contains nearly mature spores (27). Therefore, sporoblasts (Fig. 2A1, A2, and B1) and mature spores (Fig. 2A3, A4, B2, and B3) were isolated by using Percoll gradient centrifugation. Notably, the polar tube was distributed in sporoblasts in disorder, but it was arranged in an orderly manner in mature spores (Fig. 2). Next, sporoblasts and mature spores were incubated with anti-NbSWP9 and anti-NbSWP7 antibodies. NbSWP9 and NbSWP7 are distributed differently during the sporoblast and mature spore life cycle stages of N. bombycis. An obvious concentration of colloidal gold particles of NbSWP9 was distributed mainly along the developing polar tube of sporoblasts (Fig. 2A1 and A2), while the vast majority of the gold particles of NbSWP7 were found randomly in the cytoplasm (Fig. 2B1). However, the colloidal gold particles representing both NbSWP9 (Fig. 2A3 and A4) and NbSWP7 (Fig. 2B2 and B3) were distributed in the polar tube and spore wall of N. bombycis mature spores. It is possible that the NbSWP7 localization in the polar tube was due to interactions between NbSWP7 and NbSWP9 and between NbSWP9 and NbPTPs. Furthermore, as shown in Fig. 2A4, NbSWP9 also localized along the junction of the spore wall and polar tube, implying that NbSWP9 tethers the polar tube to the spore wall. Overall, as demonstrated in Fig. 2, anti-NbSWP7 and anti-NbSWP9 antibodies clearly reacted with the polar tube and cytoplasm of sporoblasts, respectively, and both of them recognized the polar tube, spore wall, and junction of the polar tube and spore wall of mature spores, suggesting that NbSWP9 contributes to polar tube arrangement, tethering, and maturation during the spore maturation process.

FIG 2.

Immunoelectron microscopy localization and developmental expression of NbSWP7 and NbSWP9 in sporoblasts and mature spores of N. bombycis. (A1and 2) Images show that almost all of the NbSWP9 gold particles (18 nm) are presented along the developing polar tube of N. bombycis sporoblasts (Sb). Sporoblasts have a scattered developing polar tube (PT) and a “thick” surface coat. (A3 and 4) Immunolocalization of NbSWP9 in cross and longitudinal sections of mature spores (S). Colloidal gold particles of NbSWP9 are distributed primarily in the polar tube, the exospore (Ex), the endospore (En), and the junction of the spore wall and polar tube in mature N. bombycis spores. It is obvious that NbSWP9 tethers the polar tube in the images in panel A4. (B1) Image indicating that colloidal gold particles of NbSWP7 are dispersed randomly in the cytoplasm. (B2 and 3) The colloidal gold particles representing NbSWP7 are localized in the polar tube and spore wall of N. bombycis mature spores. N, nucleus; AD, anchoring disk.

To further confirm that NbSWP9 mediates the orderly orientation of the polar tube and tethering to the spore wall during the process of transformation of sporoblasts into mature spores, we performed an in vitro germination assay to observe the change in the localization of NbSWP9 during the process of spore germination. The discharged polar tubes were incubated with anti-NbPTP1, anti-NbPTP2, anti-NbSWP7, and anti-NbSWP9 antibodies. The antibody against NbSWP9 labeled mainly the end-terminal part of the extruded polar tube (Fig. 3A1 and 4A1), while the anti-NbSWP7 antibody barely recognized the polar tube (Fig. 3B1 and 4B1). The characteristics of NbSWP9 localized in the end-terminal part of the extruded polar tube suggest that NbSWP9 is discharged with polar tube eversion. Altogether, with the sporoblast being transformed into a mature spore, NbSWP9, serving as a scaffolding protein, may have enabled the disordered polar tube to turn in an orderly orientation, allowing the disordered polar tube to be pulled and tethered to the spore wall by NbSWP9 itself or other associated spore wall proteins, such as NbSWP5 (25).

FIG 3.

Analysis of colocalization of NbSWP7 and NbSWP9 with NbPTP1 in the extruded polar tube of N. bombycis. N. bombycis spore germination was induced with 0.1 mol K₂CO₃ for 30 min at 28°C, and spores were then incubated with mouse anti-NbPTP1 (A2 and B2), rabbit anti-NbSWP7 (A3), and anti-NbSWP9 (B3) antibodies. DAPI was used to stain nuclei (A1 and B1). Panels A4 and B4 are merged images. The red arrows represent the localization of NbSWP9 in the extruded polar tube. Note the presence of both red and green signals at the anchoring disk and the end-terminal part of the extruded polar tube with a yellow signal where they overlap (yellow arrows). Anti-NbSWP7, anti-NbSWP9, anti-NbPTP1, and anti-NbPTP2 antibodies were diluted 1:50. The secondary antibodies were FITC-conjugated goat anti-mouse IgG and anti-rabbit Alexa Fluor 647 at 1:64 and 1:2,000 dilutions, respectively. All images are shown at a ×1,000 magnification. Bar, 10 μm.

FIG 4.

NbSWP9 accumulates primarily in the end-terminal part of the extruded polar tube and ruptured spore wall called the anchoring disk, while NbSWP7 is not present in the discharged polar tube of germinated N. bombycis spores. The germinated spores were incubated with rabbit anti-NbSWP7 (A3) and anti-NbSWP9 (B3) antibodies and mouse anti-NbPTP2 (A4 and B4) antibodies, and then the spores were further incubated with anti-rabbit secondary antibody labeled with FITC (green) and anti-mouse secondary antibody labeled with Alexa Fluor 647 (red). In panels A1, B1, and C1, spores were visualized by using a differential interference contrast (DIC) microscope and stained with DAPI (A2, B2, and C2). At the same time, a negative control was designed by using negative rabbit (C3) and mouse (C4) sera. Panels A5, B5, and C5 are merged images. Note the presence of green NbSWP9 signals concentrating at the end-terminal part of the extruded polar tube and anchoring disk (green and yellow arrows). All images are shown at a ×1,000 magnification. Bar, 10 μm.

NbSWP9 plays a role in attachment of the discharged polar tube to the anchoring disk.

Next, we continued to determine whether NbSWP9 mediated the anchoring and attachment of the discharged polar tube to the spore wall. To avoid the impact of different fluorescence labeling, fluorescein isothiocyanate (FITC) and Alexa Fluor 647 were used to label NbSWP9 and NbSWP7, respectively. As shown in Fig. 3 and 4, anti-NbPTP1 antibody (Alexa Fluor 647 labeling) recognized the extruded polar tube of N. bombycis well, while anti-NbPTP2 antibody (FITC labeling) stained only the region of the polar tube next to the anchoring disk but not in the terminal end. In order to prove whether the localization of NbPTP2 in Nosema bombycis is different from PTP2 staining in other microsporidia, where PTP2 is found on the entire polar tube, indirect immunofluorescence staining was conducted by using only anti-NbPTP2 but not anti-NbSWP9 and anti-NbPTP2 antibodies. To our surprise, the data obtained from the indirect immunofluorescence assay (IFA) showed that NbPTP2 was located in the entire polar tube when only the anti-NbPTP2 antibody was used. However, NbPTP2 stained mainly the region of the polar tube next to the anchoring disk but not in the terminal end when both anti-NbSWP9 and anti-NbPTP2 antibodies were used. We thought that the reason for the localization of NbPTP2 may be due to interference during labeling with both anti-NbSWP9 and anti-NbPTP2 antibodies. Interestingly, the majority of NbSWP9 (regardless of labeling) accumulated in the ruptured spore wall called the AD, except for localization in the polar tube, suggesting that NbSWP9 in the area of the AD may support, tether, and hold the discharged polar tube in place to help the sporoplasm flow through the polar tube into the host cell. In addition, the germination supernatant contained NbSWP9 when mature spores germinated under K₂CO₃ induction. NbSWP9 was discharged and was located in the extruded polar tube of N. bombycis (Fig. 5B). Therefore, our data demonstrate that the discharged polar tube was anchored, held, and attached to the anchoring disk, mediated by the scaffolding protein NbSWP9.

FIG 5.

NbSWP9 contributes to the germination process of N. bombycis. Purified mature N. bombycis spores were induced to germinate in 0.1 mol K₂CO₃ for 30 min at 28°C. (A and B) DAPI was used to distinguish ungerminated spores (A) and germinated spores (B). The nuclei of ungerminated spores were stained with DAPI as a control. The nuclei of germinated spores are not stained with DAPI. This was evaluated as germinated spores of N. bombycis in this paper. Bar, 10 μm. (C and D) SDS-PAGE and Western blotting were performed to validate whether the supernatant of germinated spores contained NbSWP9. (C) Silver staining was used to detect the supernatant of germinated spores. (D) Western blotting using the anti-NbSWP9 mouse antibody as a primary antibody to detect NbSWP9. (E) Anti-NbSWP9 antibody inhibited spore germination in a dose-dependent manner. In the germination assay, NbSWP9-specific or negative-control antibodies were incubated with N. bombycis spores at different doses (2.5, 5, 7.5, and 10 μg) of IgG that had been purified from sera of rabbits immunized with the recombinant NbSWP9 protein or immunized with PBS (negative-control antibody). Additionally, data are shown as percentages of germinated spores relative to the number of spores of a control sample in which spores were incubated with only 0.1 mol/liter K₂CO₃ (pH 8.0) at 28°C for 30 min. The percent germination of the control sample was considered to be 100%. “*” represents a significant difference; “**” represents a highly significant difference. The above-described experiments were repeated three times and produced similar results each time. Results from at least 40 random fields were calculated for each data point.

NbSWP9 helps germination of N. bombycis spores in vitro.

Given our finding that NbSWP9 mediates the orderly attachment of the polar tube to the spore wall of mature spores and anchors the discharged polar tube to the AD of germinated spores, we reasoned that NbSWP9 may contribute to the germination of N. bombycis spores and then infect host cells. To test the function of NbSWP9 in the process of spore germination, an assay of the inhibition of spore germination with anti-SWP9 antibody was performed to determine whether anti-SWP9 antibody inhibits spore germination in vitro. As shown in Fig. 5A and B, 4′,6-diamidino-2-phenylindole (DAPI) staining demonstrated that mature spores were induced to germinate with a 0.1 M K₂CO₃ solution. At the same time, as shown in Fig. 5C and D, some NbSWP9 was released accompanying the complete discharge of the polar tube, suggesting that NbSWP9 may play a role in the process of spore germination. As expected, anti-NbSWP9 antibody reduced spore germination in a dose-dependent manner compared with negative-control serum (Fig. 5E). In the range of test concentrations (2.5, 5, 7.5, and 10 μg) of anti-NbSWP9 antibody, the maximum inhibitory rate for spore germination was >60% at a dose of 10 μg (Fig. 5E). These results showed that NbSWP9 interacts with NbPTPs to contribute to the germination of N. bombycis spores.

DISCUSSION

The microsporidial invasion mechanism involves the polar tube and spore wall. At present, researchers have identified three distinct polar tube proteins of the Encephalitozoon genus: PTP1, a proline-rich protein (30, 31); PTP2, a lysine-rich protein (32); and PTP3, a large protein with a molecular mass of >135 kDa (33). Moreover, full-length EcPTP1, EcPTP2, and EcPTP3 interact with each other in vivo through both the N and C termini of EcPTP1 but not the central region of this protein, which contains a repetitive motif (37). The microsporidian species N. bombycis, which causes the silkworm infection pebrine, is an obligate unicellular eukaryotic parasite. Until now, only a few spore wall proteins of N. bombycis have been reported. A previous study showed that NbSWP5 localized in the exospore wall and polar tube and may interact with PTP2 and PTP3 of N. bombycis (25). Furthermore, our recent research identified two spore wall proteins, NbSWP7 and NbSWP9, which localized to the exospore, endospore, and polar tube of mature spores (27). These data suggest that both of these proteins may interact with polar tube proteins of N. bombycis. Therefore, immunoprecipitation, LC-MS/MS, immunoelectron microscopy (IEM), and yeast two-hybrid analyses were performed to confirm this hypothesis. The results demonstrated that NbSWP9 interacts with NbPTP1, NbPTP2, and NbPTP3, while NbSWP7 did not interact with these NbPTPs. However, the interaction mechanism of NbSWP9 and NbPTPs is still unknown.

A previous study found that the synthesis of the majority of the microsporidial polar tube begins in the early sporoblast with the appearance of an oval body of membranes and dense material (38). Following this, a Golgi-like structure with cisternae, small vesicles, and sacs appears. Vacuole-like structures transform into tubules forming hollow rings that pinch off, eventually forming the polar tube (8). As far as is known, the polar tube coils surround the sporoplasm in an orderly manner after it has been completely formed in mature spores. Furthermore, our TEM data showed that the polar tube is synthesized and randomly dispersed in the sporoplasm of N. bombycis sporoblasts. This finding is in line with data from a previous study showing that the synthesis of the polar tube begins in the sporoblast. With sporoblasts changing into mature spores, how the polar tube forms the orderly coils that surround the sporoplasm during the development process is still unknown. At the same time, which spore wall proteins contribute to the anchoring of the polar tube to the spore wall during the development process are as yet unknown. This study indicated that NbSWP9 not only localized to the polar tube of N. bombycis sporoblasts and mature spores but also was distributed along the junction of the polar tube and spore wall. Furthermore, NbSWP9 also interacts with NbPTPs in vitro and in vivo. These results suggest that NbSWP9 may pull the polar tube to the spore wall and enable the unordered polar tube to turn into an orderly orientation and then be anchored to the spore wall by NbSWP9 itself and other spore wall proteins, such as NbSWP5 (25).

The spore includes a specific invasion apparatus, called the polar tube, connected to the anterior end and spore wall of the spore (11). The polar tube is divided into two regions: the straight portion attached inside the anterior end of the spore by a mushroom-shaped AD and the posterior coiled region that forms coils around the sporoplasm in the spore (8, 11). The spore germination process is usually believed to occur in four stages: spore activation upon appropriate environmental stimulation, increase of intrasporal osmotic pressure, eversion of the polar tube, and passage of the sporoplasm through the polar tube. The polar tube completely discharges from the anterior pole of the spore, which occurs in <2 s, and is thought to form a hollow tube by a process of eversion, similar to “everting the finger of a glove” (39). Our results showed that NbSWP9 interacts with NbPTPs and localizes mostly to the spore wall, the polar tube, and the junction between them in dormant mature spores of N. bombycis. In addition, the posterior coiled region of the polar tube may be anchored to the spore wall by NbSWP9 itself according to IEM results (Fig. 2). However, after the complete discharge of the polar tube, NbSWP9 accumulated mainly in the end-terminal part of the extruded polar tube in germinated spores. The different localizations of NbSWP9 before and after spore germination further demonstrate that NbSWP9 is a scaffolding protein that tethers the polar tube to the spore wall. This change may be due to the spore wall architecture that formed during the germination process (40, 41). Therefore, the parts of NbSWP9 that interact with NbPTPs and act as a support protein localizing in spore wall were discharged from the posterior coiled region of polar tube to the end-terminal part of the extruded polar tube during this germination process. Notably, the majority of NbSWP9 accumulated in the anchoring disk, except for concentrating in the end-terminal part of the extruded polar tube. Altogether, these results suggested that NbSWP9 in the ruptured place of the AD may anchor and hold the discharged polar tube in place to help the sporoplasm flow through the polar tube into the host cell. These discoveries are more evidence for the point that NbSWP9 can serve as a scaffolding and structural protein to support NbSWP7and NbPTPs and then tether the polar tube to the spore wall (27). There are hints that NbSWP9 is involved in the initiation of the formation of the polar tube and the germination process. Next, a spore germination assay was performed and showed that anti-NbSWP9 antibody reduced spore germination in a dose-dependent manner compared with negative-control sera. These results implied that NbSWP9 helps and participates in the germination of N. bombycis spores. However, the cause of this association is not known and warrants further investigation. Perhaps, NbSWP9, which localized to the exospore of N. bombycis spores, prefers NbSWP5 for acting as a receptor responsible for stimulation signals utilized during the germination process (25).

In conclusion, this study presents an examination of the interaction between two spore wall proteins (NbSWP9 and NbSWP7) and three polar tube proteins (NbPTP1, NbPTP2, and NbPTP3) of N. bombycis. This study provides necessary evidence for the involvement of NbSWP9 in the processes of the orderly orientation, arrangement, anchoring, and germination of the polar tube. In sporoblasts, NbSWP9 is localized mostly to the polar tube, which was newly formed and dispersed in the sporoplasm of early sporoblasts, while NbSWP7 was distributed randomly in the sporoplasm (Fig. 6A). However, both NbSWP9 and NbSWP7 were localized to the spore wall, the polar tube, and the junction of the polar tube and spore wall of mature spores. As maturation proceeds during the process of transformation of sporoblasts into mature spores, NbSWP9 may drag the scattered polar tube into the spore wall and enable the orderly anchoring of coiled polar tube coils to the spore wall around the sporoplasm of N. bombycis (Fig. 6B). With spore germination and polar tube discharge, the majority of NbSWP9 accumulated at and attached to the ruptured place of the anchoring disk and the end-terminal part of the extruded polar tube (Fig. 6C). Furthermore, NbSWP9 is also involved in the process of spore germination. Moreover, NbSWP9 is present and is conserved in other species of microsporidia (27). Therefore, research regarding NbSWP9 has profound implications for future studies of microsporidia. Finally, this study further confirmed that NbSWP9, as a scaffolding protein, contributes to the anchoring of the polar tube to the spore wall and AD before and after spore germination.

FIG 6.

Schematic model of NbSWP9 functions in polar tube anchoring to the spore wall and during the process of N. bombycis germination. (A) NbSWP9 interacts with polar tube proteins and is localized primarily in the polar tube and spore wall of N. bombycis. The polar tube is formed and scattered in the sporoplasm. Moreover, NbSWP7 is distributed mainly in the interior of the sporoblast (see also Fig. 1 and 2). (B) Subsequently, with sporoblasts being transformed into mature spores, the disordered polar tube turns in an orderly orientation and is anchored to the spore wall by the scaffolding protein NbSWP9 (see also Fig. 2). (C) During the spore germination process, NbSWP9 and the polar tube are discharged, and the sporoplasm was released under a suitable stimulus. The majority of NbSWP9 accumulated in the end-terminal part of the extruded polar tube and anchoring disk to mediate the anchoring and attachment of the discharged polar tube to the spore wall. Therefore, NbSWP9 is involved in the process of spore germination (see also Fig. 3 to 5).

MATERIALS AND METHODS

Ethics statement.

All animal experiments, animal care, and procedures were conducted in accordance with the guidelines of the China Council on Animal Care. This study was approved by the Laboratory Animal Welfare and Ethic Committee of the Third Military Medical University, animal utilization protocol number SYXK-PLA-2007035.

N. bombycis spore production and purification.

Mature spores of N. bombycis isolate CQ1 (CVCC 102059) were purified from infected silkworms and conserved in the China Veterinary Culture Collection Center, Chongqing, China. At the same time, N. bombycis spores at early stages were purified and harvested from laboratory-reared silkworm larvae, as previously described (7, 24, 27, 42, 43). The purified spores were washed and stored in sterile phosphate-buffered saline (PBS) with antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin) at 4°C for later use.

Protein extraction, antiserum production, and Western blotting.

Total mature N. bombycis protein was extracted from mature spores as previously reported (27). Briefly, 10⁹ mature spores were broken in 400 μl PBS (pH 7.3) by vibration with glass beads (150 to 212 μm; Sigma) at 4°C for 6 h. The supernatant protein was collected as the total spore protein by centrifugation at 13,200 × g for 10 min and conserved at −20°C for Western blotting and coimmunoprecipitation of NbSWP7, NbSWP9, and NbPTPs. In addition, the above-mentioned purified spores (10⁹) were germinated in 0.1 mol/liter K₂CO₃ (pH 8.0) at 28°C for 30 min to obtain the germinant supernatant protein. Western blotting was then performed to detect whether the germinant supernatant proteins contain NbSWP9 and to prove whether NbSWP9 drops from mature spores during the germination process. Finally, we assessed the role of NbSWP9 in the germination of mature N. bombycis spores. Polyclonal antibody production and immunoblotting protocols to detect NbSWP9, NbSWP7, NbPTP1, and NbPTP2 in the total protein of mature spores were described previously (27, 44). In brief, rabbits and mice were injected with recombinant proteins to obtain antibodies against NbSWP9, NbSWP7, NbPTP1, and NbPTP2, respectively. As negative sera, one control rabbit and one control mouse were injected subcutaneously with only PBS, and these sera were then obtained from the control rabbit and mouse. Subsequently, serum was purified by the hexylacetic acid-saturated ammonium sulfate method. For Western blotting, total mature N. bombycis protein and co-IP samples were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (pore diameter, 0.22 μm). The membranes were blocked overnight at 4°C in Tris-buffered saline–Tween (TBST) with 5% (wt/vol) nonfat dried milk and incubated with 10 μg anti-NbSWP7, anti-NbPTP1, anti-NbPTP2, or anti-NbSWP9 polyclonal antibodies diluted at 1:6,000 or negative-control serum, separately. After being washed in TBST, the membranes were then incubated at room temperature for 1 h with horseradish peroxidase (HRP)-labeled goat anti-mouse or anti-rabbit IgG antiserum (Sigma-Aldrich, St. Louis, MO), diluted 1:8,000, washed in TBST, and developed with the ECL Western blot detection kit (Thermo Fisher Scientific, Rockford, IL).

Indirect immunofluorescence assay.

For the IFA, in spores (10⁵), germination was induced with 0.1 mol/liter K₂CO₃ (pH 8.0) at 28°C for 30 min on a cover glass. A colocalization assay was then performed to detect the localization of NbPTP1, NbPTP2, NbSWP7, and NbSWP9 in the germinant spores and discharged polar tubes. In brief, the germinant spores were fixed with 4% paraformaldehyde for 20 min at room temperature and then blocked with a blocking solution consisting of 5% (wt/vol) bovine serum albumin (BSA) and 10% (vol/vol) goat serum in PBS for 2 h at 37°C. The samples were incubated with a 1:50 dilution of anti-NbPTP1, anti-NbPTP2, anti-NbSWP7, anti-NbSWP9, and negative-control antibodies for 1 h and then washed and incubated for an additional 1 h with a 1:64 dilution of FITC-conjugated IgG (Sigma) and a 1:2,000 dilution of Alexa Fluor 647 (Invitrogen, Carlsbad, CA) in a moist chamber at 37°C. DAPI (Sigma) was used to detect the existence of nuclear material of spores at 37°C for 10 min.

Transmission electron microscopy immunolabeling.

N. bombycis spores at different life cycle stages were fixed in 3% paraformaldehyde (freshly prepared) in 0.1 M sodium cacodylate buffer (pH 7.2 to 7.4) containing 0.1% glutaraldehyde and 4% sucrose overnight at 4°C. The fixed pellets were rinsed with 0.1 M sodium cacodylate buffer containing 4% sucrose (pH 7.4) at 4°C four times for 15 min and then sequentially dehydrated with graded methanol. The dehydrated spores were then imbedded and photopolymerized in Lowicryl K4M (Zhongjingkeyi Technology Co., Beijing, China) and under UV light (360 nm) and placed 20 to 30 cm away from the UV source with resin for 72 h at −35°C. Ultrathin sections were placed onto 200-mesh nickel grids coated with Formvar and carbon. Nickel grids were incubated in blocking buffer (1% BSA [Sigma], 0.05% Triton X-100, and 0.05% Tween 20) at room temperature for 1 h, followed by incubation with 1:300 dilutions of primary rabbit anti-SWP9, anti-SWP7, and negative-control rabbit antibodies overnight at 4°C, rinsing 6 times in PBS, and then incubation with a 1:70 dilution of goat anti-rabbit IgG conjugated to 18-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 h. Grids were rinsed with PBS, dried, stained with 3% uranyl acetate, and then examined and photographed with a Hitachi H-7650 transmission electron microscope at an accelerating voltage of 80 kV.

Coimmunoprecipitation and yeast two-hybrid assay.

Immunoprecipitation, yeast two-hybrid, and colocalization assays were used to investigate the interaction of NbSWP7 and NbSWP9 with polar tube proteins of N. bombycis. To demonstrate whether NbSWP7 and NbSWP9 coimmunoprecipitate with PTPs of N. bombycis, co-IP assays was performed on total mature spore proteins. The details of this experiment were described previously (25, 27, 45). Briefly, rabbit anti-NbSWP7 or anti-NbSWP9 antibody (20 μg) was incubated with protein A+G agarose beads (Beyotime, Chongqing, China) and then agitated with total mature spore proteins overnight at 4°C. The agarose beads binding antibodies and proteins were washed six times in 1× immunoprecipitation buffer and one time in 0.1× immunoprecipitation buffer. The samples were then eluted from the beads with 5× SDS loading buffer and boiled for 10 min. Subsequently, the samples were subjected to SDS-PAGE and analyzed by LC-MS/MS. At the same time, Western blotting was performed by using anti-NbPTP1 and anti-NbPTP2 antibodies to demonstrate the interaction of NbSWP7, NbSWP9, and NbPTPs. Furthermore, the yeast two-hybrid assay was used to confirm the interaction between NbSWP7 or NbSWP9 and NbPTP1 or NbPTP2 in vivo. The detailed procedure was described previously (27, 37). In short, Nbswp7 and Nbswp9 were constructed in the yeast two-hybrid vector pGBKT7, and Nbptp1 and Nbptp2 were constructed in pGADT7. The bait and prey constructs pGBKT7-Nbswp9/pGADT7-Nbptp1, pGBKT7-Nbswp9/pGADT7-Nbptp2, pGBKT7-Nbswp7/pGADT7-Nbptp1, and pGBKT7-Nbswp7/pGADT7-Nbptp2 were transformed simultaneously into competent yeast cells. Moreover, yeast cells were plated onto SD plates that do not contain leucine, tryptophan, histidine, and adenine but contain X-α-Gal (Biosciences Clontech, San Jose, CA).

Spore germination assay.

To further analyze the role of NbSWP9 in spore germination, a spore germination assay was designed. Mature N. bombycis spores were preincubated with negative IgG or anti-NbSWP9 antibody (2.5, 5, 7.5, and 10 μg/ml) for 2 h at 28°C prior to placement of the spores in K₂CO₃. The spores were then induced with 0.1 mol/liter K₂CO₃ (pH 8.0) at 28°C for 30 min. Subsequently, spores were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with DAPI to detect germination. This was evaluated as spores of N. bombycis germination in this paper. Germinant spores were quantified in at least 40 fields at a ×1,000 magnification. The results are expressed as the percentages of germinant spores relative to those in control samples, which contained spores incubated with 0.1 mol/liter K₂CO₃ only. The experiment was performed in triplicate and repeated at least three times, which always showed similar results. The significance of the differences among the control and experimental assays was measured by using the Student t test in the Statistical Package for Social Science (SPSS version 17.0; SPSS Inc., Chicago, IL). P values of 0.05 or lower were considered statistically significant; P values of 0.01 or lower were considered highly significant. Error bars signify standard deviations.