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. 2017 Feb 23;85(3):e00872-16.
doi: 10.1128/IAI.00872-16. Print 2017 Mar.

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

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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

Donglin Yang et al. Infect Immun. .
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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.

Keywords: germination; interaction; microsporidia; polar tube protein; spore wall protein.

Figures

FIG 1
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.
FIG 2
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.
FIG 3
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
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.
FIG 5
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.
FIG 6
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).

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