Chen G et al. (2017), Functional characterization of an aquaporin fro...

XB-ART-53868

PLoS One 2017 Jul 10;127:e0181703. doi: 10.1371/journal.pone.0181703.

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Functional characterization of an aquaporin from a microsporidium, Nosema bombycis.

Chen G , Wang W , Chen H , Dai W , Peng X , Li X , Tang X , Xu L , Shen Z .

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Microsporidia are a diverse group of eukaryotic organisms, capable of causing parasitic infections in both vertebrates and invertebrates. During the germination process, there is an increase in the osmotic pressure of microsporidian spores. As part of this study, we cloned a homologous aquaporin gene in Nosema bombycis, and named it Nosema bombycis aquaporin (NbAQP). Sequence analysis revealed that the NbAQP contains an open reading frame with a length of 750 bp and encodes a polypeptide of 249 amino acids. Amino acid sequence homology was greater than 50% that of five aquaporins from other microsporidian species. Indirect immunofluorescence (IFA) and immunogold electron microscopy showed NbAQP to be located predominantly in the spore wall of N. bombycis spores. The results of qRT-PCR analysis revealed that NbAQP expression remained high 0 h after inoculation and decreased sharply to 24 h, increased gradually from 2 days and peaked at 6 days. After expression of NbAQP in Xenopus laevis oocytes, it was observed that NbAQP can promote rapid penetration of water into oocytes. The associated permeation rate was 2-3 times that of the water-injected and uninjected oocytes. Antibody blocking experiments showed that the inhibition rate of spore germination was approximately 28% after antibody blocking. The difference in germination rate between the control group and the NbAQP group was significant (P < 0.05). This study shows for the first time that N. bombycis contains functional water channel proteins and provides a platform suitable for further research into the mechanisms underlying the regulation of NbAQP protein expression. Further study of NbAQP and their inhibitors may have significance for prevention of microsporidiosis.

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Species referenced: Xenopus laevis
Genes referenced: aqp3 aqp7 aqp9

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	Fig 1. Amino acid sequence alignment of NbAQP and AQPs from other microsporidia.Eh: Encephalitozoon hellem (XP_003887579.1) homology with NbAQP was 51%; Ec: Encephalitozoon cuniculi (NP_586002.1) homology with NbAQP was 54%; Ei: Encephalitozoon intestinalis (XP_003073192.1) homology with NbAQP was 52%; Er: Encephalitozoon romaleae (XP_009264817.1) homology with NbAQP was 52%; Oc: Ordospora colligata (XP_014563378.1) homology with NbAQP was 56%; Nb: Nosema bombycis (API70721).
	Fig 2. Tertiary structure prediction of NbAQP.
	Fig 3. Schematic diagram of aquaporin structure.
	Fig 4. The phylogenetic tree of the aquaporins including NbAQP.Phylogenetic tree of NbAQP and other homologous protein sequences constructed using the Maximum Composite Likelihood method within the package MEGA 6.0. Encephalitozoon cuniculi (NP_586002), Dictyostelium discoideum (BAA85158), human AQPs 0–9 (NP_036196, NP_932766, NP_000477, NP_004916, P55087, NP_001642, Q13520, NP_001161, O94778, NP_066190, respectively), plant aquaporins of Arabidopsis thaliana (P25818) and Nicotiana tabacum (CAA69353), parasitic protist aquaporins of Leishmania major (AAS73184), Plasmodium falciparum (CAC88373), Toxoplasma gondii (CAE46485) and Trypanosoma cruzi (AAM76680), and AQP 2 of Saccharomyces cerevisiae (AAD10058). Human AQP3, AQP7, AQP9, and the aquaporins of Leishmania major, Plasmodium falciparum and Toxoplasma gondii are aquaglyceroporins.
	Fig 5. Western blotting analysis of His-NbAQP fusion protein.Lane M: Marker; Lane 1: His-NbAQP fusion protein after 15 h of induction; Lane 2: His-NbAQP fusion protein after 20 h induction; Lane 3: His-NbAQP fusion protein after 24 h induction.
	Fig 6. Specificity analysis of NbAQP antibody.Lane M: Prestained marker. Lane 1: Total protein from N. bombycis containing NbAQP.
	Fig 7. Indirect immunofluorescence.(A): Combination of (B) and (C). (E): Combination of F and G. (B): N. bombycis following incubation with primary antibody and secondary antibody. (C) and (G): N. bombycis cell nucleus stained with PI. (D) and (H): N. bombycis visualized using phase contrast microscope. (F): N. bombycis following incubation with negative serum and secondary antibody.
	Fig 8. Immunogold electron microscopy.Under immunogold electron microscopy, the N. bombycis spore wall appeared to be thick and relatively rough with non-uniform electron density (Fig 8). Colloidal gold particles were loosely distributed in the spore wall, and there was also limited localization of colloidal gold particles in the plasma membrane (Fig 8A). Conversely, no colloidal gold particles were detected in the spore walls or cytoplasm of the control group (Fig 8B).
	Fig 9. The relative expression level of NbAQP gene in the midgut of silkworm larvae at different time points after infection with N. bombycis.The y-axis indicates the relative expression level of NbAQP gene, and the x-axis indicates the time. Vertical bars represent the mean ± SE (n = 3).
	Fig 10. Resultant oocytes after expression of the fusion protein GFP and NbAQP.(A) Cells injected with T7Ts-EGFP; (B) cells injected with T7Ts-EGFP +T7Ts-NbAQP; (C) cells injected with T7Ts-EGFP-NbAQP; (D) cells injected with T7Ts-NbAQP-EGFP; (E) non-injected cells; (F) cells injected with DEPC water.
	Fig 11. Expansion experiment after NbAQP injection into Xenopus laevis oocytes.At 5 min after injection with pT7Ts-NbAQP-eGFP, pT7Ts-eGFP-NbAQP and pT7Ts-NbAQP + pT7Ts-eGFP.(the expansion rate was 7.1% ± 0.52%, 7.3% ± 0.49% and 7.1% ± 0.39%, respectively; the Pf was 24.7 μm/s, 24.4 μm/s and 25.6 μm/s, respectively, (p < 0.05)) were more than those groups injected with DEPC water, pT7Ts-eGFP and non-injected (the expansion rate was 1.9% ± 0.24%, 3.2% ± 0.5%, and 2.5% ± 0.3%, respectively; the Pf was 6.1 μm/s, 11 μm/s and 8.9 μm/s, respectively (p < 0.05)) (n = 10). (Vertical bars represent the mean ± SE; for clarity, only three error bars are displayed.)
	Fig 12. Spore germination rate.The experimental results showed that the average germination rate of spores was 23.05% in the blank control group and 16.58% in the group that was exposed to the blocking antibody. Vertical bars represent the mean ± SE (n = 10).

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