Identification and subcellular localization analysis of membrane protein Ycf 1 in the microsporidian Nosema bombycis

Parasite and host

The silkworm strain SuRong × Xiyu, BmN cell line, N. bombycis, Nb-actin polyclonal antibody and SWP30 polyclonal antibody were provided by the Sericultural Research Institute of Chinese Academy of Agricultural Sciences. The BmN cell was isolated from silkworm and kept in our laboratory.

Cloning of Ycf 1 gene and analysis

The purified spore suspension of N. bombycis (10⁹ sopres/mL) was crushed in a bead grinder with 1:1 acid-washed glass beads for 1 min, cooled on ice for 5 min; this process was repeated six times. The genomic DNA (gDNA) of N. bombycis was extracted using fungal genomic DNA extraction kit (Sangong Bioengineering, Shanghai, China), and kept at −20 °C following concentration measurements.

The gene sequence of the putative membrane protein Ycf 1 (GenBank accession number:EOB13621.1) was searched from the NCBI database (https://www.ncbi.nlm.nih.gov) and the specific forward primer 5′-GCGCGGATCCATGAAATTTACTACTTTTTG-3′ (BamH I restriction site); reverse primer 5′-GCGCTCGAGTTATTTAGAAGCCATCAT-3′ (Xho I restriction site) were designed to amplify the target gene. The 50 µL PCR amplification reaction systems were as follows: PrimeSTAR HS DNA Polymerase (TaKaRa Biotechnology, Beijing, China) 25 µL, specific forward and reverse primers 2 µL (10 µM) respectively, gDNA 2 µL (100 ng), and ddH₂O 19 µL . The PCR products were separated on a 1% agarose gel and extracted with an Axyprep DNAGel Extraction Kit, then cloned into the pMD19-T-Vector (TaKaRa Biotechnology, Beijing, China) with poly(A) tail. The pMD19-T-Ycf 1 vector was transformed into E.coli TOP10 competent cells (Sangong Bioengineering, Shanghai, China), and then cultured on LB plates containing ampicillin at 37 °C. The recombinants were identified by PCR and sequenced by Sangong Bioengineering (Shanghai, China).

The molecular weight and isoelectric point of Ycf 1 protein were predicted by Compute pI/Mw tool (https://web.expasy.org/compute_pi/). Signal peptide was predicted by SignalP-5 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0). TMHMM Serverv.2.0 (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0) was used to predict transmembrane domains. The phosphorylation and glycosylation sites were predicted by DTU Health Tech (http://cello.life.nctu.edu.tw/) and its subcellular location was predicted by CELLO v.2.5 (http://cello.life.nctu.edu.tw/).

Expression of recombinant protein of Ycf 1

The correctly sequenced pMD19-T-Ycf 1 positive plasmid and pET-28a empty plasmid were identified by double enzyme digestion. The pET-28a-Ycf 1 was transformed into E.coli BL21 Star (DE3) (Sangong Bioengineering, Shanghai, China). The correctly identified positive colonies were used for prokaryotic expression.

The overnight cultured bacterial cells were added to LB liquid medium (Kanamycin, 100 mg/mL) at ratio of 1:100, then incubated at 37 °C in a shaker until the OD600 reached 0.6. One mL of the bacterial cells were collected as negative control before induction. The remaining bacterial cells were induced with different isopropyl β-D-thiogalactoside (IPTG) concentrations for 20 h at 20 °C. Ultrasonic disruption (250 W, 3 s, 5 s interval) was performed for about 10 min, the supernatant and precipitate were collected separately by centrifugation, and boiled with 1 × protein loading buffer for 10 min at 100 °C, respectively. SDS-PAGE was performed to confirm the expression of the recombinant protein, and the recombinant protein was tested by Western blot using Anti-His Tag antibody.

Preparation of Ycf 1 monoclonal antibody and Western blot analysis

The monoclonal antibody of Ycf 1 was prepared by Abmart Biopharmaceuticals in Shanghai, China referring to the method of previous report (Zhang et al., 2007).

The total protein of N.bombycis was extracted by the acid-washed glass beads (Dai et al., 2019). Western blot analysis was performed using Ycf 1 monoclonal antibody (overnight, 4 °C) as the first antibody and an HRP-conjugated goat anti-mouse IgG antibody (Sangong Bioengineering, Shanghai, China) as the second antibody (room temperature, 1 h). The mouse IgG was used as negative control. The PVDF membrane was incubated with Tanon™ High-sig ECL Western blot substrate (2 min), imaged with a Tanon 5200 Multi imaging system.

Immunolocalization of Ycf 1in N. bombycis

Dormant mature spore suspension was added to poly-lysine coated coverslips, airdried and fixed with 4% paraformaldehyde for 30 min. The monoclonal antibody of Ycf 1 (0.93 mg/mL) and the polyclonal antibody of spore wall protein of N. bombycis SWP30 (2.021 mg/mL) were visualized with Alexa Fluor 488 labeled Goat anti-mouse IgG (0.5 mg/mL, Sangong Bioengineering, Shanghai, China) or Cy5 labeled Goat anti-Rabbit IgG (0.5 mg/mL, Sangong Bioengineering, Shanghai, China), respectively. The immunolocalization was observed under a fluorescence inverted microscope (Olympus IX-71). The detailed steps of immunolocalization referred to the method of Qi et al. (2021).

The subcellular localization of Ycf 1 in the developmental stage of N. bombycis in BmN cells was investigated as the following procedure. The spore suspension of N. bombycis and 0.2 M KOH solution were pre-heated for 2 h at 27 °C, respectively, then mixed at the ratio of 1:1 (volume) and incubated for 40 min at 27 °C. The germinated spore suspensions were slowly dropped into flasks containing BmN cells at ratio of 10:1. After incubating for 1 h, the culture medium free of serum was replaced with TC-100 insect culture medium containing 10% serum (Gibco, Thermo Fisher Scientific, Australia), which was replaced with fresh culture medium after cultivation overnight. The BmN cell infected with N. bombycis at different days were added into the 6-well plate containing coverslips at different days post-infection. After washing three times with PBST, the infected BmN cell was fixed with 4% paraformaldehyde for 30 min. The next procedure is the same as IFA for dormant mature spores.

RNAi

Newly molted fifth instar silkworm larvae were fed on mulberry leaves for 6 h which were smeared with spores of N. bombycis (10⁸ spores/mL). 3 µL of siRNA (1 µg) and a nonsense fragment as the negative control were injected into the infected silkworm, while ddH₂O was used as blank control. The siRNA (Table 1) was synthesized by Sangong Bioengineering in Shanghai, China. The midgut tissues of the silkworm were collected at 24 h, 48 h, 72 h, 96 h, and 120 h post-infection, respectively, then washed with PBS and stored at −80 °C.

The midgut tissue was lysed with one mL of RNAiso plus lysate (TaKaRa Biotechnology, Beijing, China), then total RNA was extracted with Mini BEST Universal RNA Extraction Kit (TaKaRa Biotechnology, Beijing, China). The cDNA was synthesized with PrimerScript^® RT Master Mix (TaKaRa Biotechnology, Beijing, China). The qPCR was performed according to the TB GreenTM Premix ExTaqTM II (Tli RNase H Plus) kit (TaKaRa Biotechnology, Beijing, China) manufacturer’s instructions with the primers as shown in Table 1. The β-tubulin gene of N. bombycis was served as reference gene. The transcriptional levels were calculated by the 2^−ΔΔct values method with three biological replicates. The multiple t tests were conducted by using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, United States).

In order to analyze the effect of knockdown of Ycf 1 gene on the proliferation of N. bombycis, specific primers Nb β-tubulin-qF and Nb β-tubulin-qR (Table 1) were designed to detect the copy number of Nbβ-tubulin. The standard template was prepared according to the method of previous report (Huang et al., 2018a), and the standard curve covered five orders of magnitude (5.6 × 10²–10⁶). The multiple t tests were conducted by using GraphPad Prism 8.0 with three biological replicates.

Cloning and expression of Ycf 1and immunoblot analysis

The Ycf 1 gene contains a complete ORF of 969 bp that encodes a 322 amino acid polypeptide. PCR amplification and sequencing results showed that the similarity is 99.7% compared to the sequence of N. bombycis CQ1 in the NCBI database, there are two base differences (291 T-C 301 G-A), and one amino acid difference (100 E-K). Ycf 1 has one signal peptide and one transmembrane domain with a pI of 6.49. Secondary structure analysis showed that the alpha-helix, random coils, beta sheets and extended fragment accounts for 32.3%, 57.14%, 4.35% and 6.21%, respectively. The phosphorylation and glycosylation prediction showed that it has thirty-six phosphorylation sites and one O-glycosylation site.

The double digestion of recombinant plasmid resulted in a band corresponding to the target gene (Fig. 1A). Western blot showed a target band about 50 kDa that was larger than the predicted size, presumably due to phosphorylation or glycosylation modification. The recombinant protein was detected in the precipitate after sonication, indicating that the protein was expressed as inclusion body in the E. coli BL21 DE3 (Fig. 1B). The optimum induction conditions were 0.5 mM IPTG for 20 h at 20 °C.

Specific detection of monoclonal antibody

Western blot analysis showed that specific bands were detected both in the recombinant protein and extracted protein of N. bombycis while the mouse IgG was used as negative control (Fig. 2). These results indicated that the prepared monoclonal antibody has a specific antigen-antibody reaction and can be used in subsequent experiments.

Co-localization of Ycf 1 with SWP30 or Nb-actin in dormant spores of N. bombycis

In order to explore whether Ycf 1 is located on the plasma membrane in the mature spores of N. bombycis, we performed a co-localization analysis of Ycf 1 with SWP30 or Nb-actin. The Ycf 1 antibody and SWP30 or Nb-actin antibody were labeled with Alexa Fluor 488 (green) or Cy5 (red), respectively, while the nucleus was stained with DAPI (blue). SWP30 is a spore wall protein of N. bombycis and located on the endospore of mature spores (Wu et al., 2010). The IFA results showed that the green fluorescence was distributed on the inner side of mature spores, very close to the red fluorescence but not completely overlapped (Fig. 3A), the negative control has no green fluorescence (Fig. 3B). Nb-actin is a kind of multifunctional and indispensable protein of the cytoskeleton in the N. bombycis (Kühn & Mannherz, 2016). Co-localization results showed that the Ycf 1 protein was distributed on the inner side of the mature spore wall whereas the Nb-actin was distributed throughout the whole mature spore (Fig. 3C), the negative control has no green fluorescence (Fig. 3D). These results indicated that Ycf 1 protein is distributed on the plasma membrane of N. bombycis.

Subcellular localization of Ycf 1 in proliferative phase of N. bombycis

To further determine whether Ycf 1 protein is on the plasma membrane in the proliferative phases of N. bombycis, IFA was performed to investigate the subcellular localizations both individual Ycf 1 or with Nb-actin as reference in proliferative phase of N. bombycis. During infection, the N. bombycis invaded the host by injecting sporoplasm into the host cells. The Ycf 1 protein was mainly distributed on the plasma membrane of sporoplasm while the Nb-actin was distributed in the whole sporoplasm (Figs. 4A, 5A). Entering the proliferative phase, the meront is long fusiform and has multiple nuclei, Ycf 1 was distributed in the periphery, especially on the ends and connecting part of the dividing cells, whereas the Nb-actin appeared throughout the whole meront (Figs. 4B and 5B). In the sporogonic phase, the sporont has four nuclei, one sporont forms two sporoblasts through cytokinesis. The Nb-actin was present throughout the cell while Ycf 1 was mainly localized around the plasma membrane (Figs. 4C, 4D, 5C and 5D). There is no green fluorescence in the negative control (Figs. 4E and 5E). The above results further illustrated that Ycf 1 is a membrane protein of N. bombycis.

Knockdown of Ycf 1 gene inhibited the proliferation of N. bombycis

For understanding the interference effect on Ycf 1 gene by RNAi, the expression level of Ycf 1 gene was detected by qRT-PCR using the β-tubulin of N. bombycis as reference gene. The relative expression of Ycf 1 gene decreased remarkably at 48 h compared to 24 h, then reached the highest at 72 h in the control group (Fig. 6A, Fig. S1B). Expectedly, RNA interference down-regulated the transcriptional level of the Ycf 1 gene (Fig. 6A, Fig. S1A). The interference effect on Ycf 1 gene was significant at 24 h and 72 h ( P < 0.05) and extremely significant at 96 h ( P < 0.01) (Fig. 6A). After knockdown of the Ycf 1 gene, the copy number of Nbβ-tubulin was investigated to reflect the effect on proliferation of N. bombycis. It was shown that the copy number of Nbβ-tubulin increased gradually with the proceeding of infection time, reached the highest at 120 h both in the control group and RNAi group (Fig. S2). Compared to the control group, the copy number of Nb β-tubulin was reduced significantly at 24 h and extremely significantly at 72 h and 96 h (Fig. 6B). These results suggested that silencing of the Ycf 1 gene inhibited the proliferation of N. bombycis.