Ssn6 Interacts with Polar Tube Protein 2 and Transcriptional Repressor for RNA Polymerase II: Insight into Its Involvement in the Biological Process of Microsporidium Nosema bombycis

Abstract

Nosema bombycis is a representative species of Microsporidia, and is the pathogen that causes pebrine disease in silkworms. In the process of infection, the polar tube of N. bombycis is injected into the host cells. During proliferation, N. bombycis recruits the mitochondria of host cells. The general transcriptional corepressor Ssn6 contains six tetratricopeptide repeats (TPR) and undertakes various important functions. In this study, we isolated and characterized Nbssn6 of the microsporidium N. bombycis. The Nbssn6 gene contains a complete ORF of 1182 bp in length that encodes a 393 amino acid polypeptide. Indirect immunofluorescence assay showed that the Ssn6 protein was mainly distributed in the cytoplasm and nucleus at the proliferative phase of N. bombycis. We revealed the interaction of Nbssn6 with polar tube protein 2 (Nbptp2) and the transcriptional repressor for RNA polymerase II (Nbtrrp2) by Co-IP and yeast two-hybrid assays. Results from RNA interference further confirmed that the transcriptional level of Nbptp2 and Nbtrrp2 was regulated by Nbssn6. These results suggest that Nbssn6 impacts the infection and proliferation of N. bombycis via interacting with the polar tube protein and transcriptional repressor for RNA polymerase II.

Keywords: general transcriptional corepressor; microsporidia; polar tube protein; transcriptional repressor for RNA polymerase II.

Figure 1

Cloning of Nbssn6 and Nbtrrp2 genes. (A) DNA agarose gel electrophoresis of Nbssn6. (B) DNA agarose gel electrophoresis of Nbtrrp2.

Figure 2

Sequence analysis of the Nbssn6 gene. (A) Three-dimensional model of Ssn6. (B) Amino acid alignment of Ssn6 from Nosema bombycis and other microsporidian species. Red box: TPR domains predicted by NCBI.

Figure 3

Sequence analysis of the Nbtrrp2 gene. (A) Domains of Nbtrrp2. (B) Three-dimensional models of Nbtrrp2. (C) Amino acid alignment of Trrp2 from Nosema bombycis and other microsporidian species. Red box: SCOP_d1xgra domain.

Figure 4

Expression, purification, and Western blot analysis of Nbssn6 protein. (A) SDS-PAGE analysis of expressed recombinant protein. Lane M: Protein molecular weight marker. Lane 1: Recombinant bacterial lysate (uninduced). Lane 2: Recombinant bacterial lysate induced with 0.5mM IPTG. Lane 3: Ultrasound supernatant. Lane 4: Ultrasonic precipitation. Black arrow: Ssn6 (B) SDS-PAGE analysis of protein purification. Lane M: Protein molecular weight marker. Lane 1: Urea solution for ultrasonic precipitation (sample to be purified). Lane 2: Flow through. Lane 3: NTAU-10 elution. Lane 4: NTAU-20 elution. Lane 5: NTAU-50 elution. Lane 6: NTAU-200 elution. Lane 7: NTAU-500 elution. (C) Western blot of Nbssn6 protein. Lane M: Protein molecular weight marker. Lane 1: Recombinant protein. Lane 2: Total protein of N. bombycis. (D) Pre-immune serum control. Lane M: Protein molecular weight marker. Lane 1: Recombinant protein. Lane 2: Total protein of N. bombycis.

Figure 5

Subcellular localization of Nbssn6 protein. The Nbssn6 antibody was coupled with Alexa Fluor 488 (green). DAPI (blue) was used to stain the nuclei of the host cells and N. bombycis. (A) White arrow indicated that Nbssn6 was mainly distributed in the perinuclear cytoplasm; (B) White arrow indicated that Nbssn6 was mainly distributed in the cytoplasm; (C) White arrow head indicated that Nbssn6 was distributed in the nuclei; (D) Negative control. Scale bars, 5 μm.

Figure 6

Colocalization of Nbssn6 with Nb-actin. The Nbssn6 antibody was coupled with Alexa Fluor 488 (green). The Nb-actin antibody coupled with Cy5 (red) were used to label the actin of N. bombycis. DAPI (blue) was used to stain the nuclei of the host cells and N. bombycis. (A) White arrow head indicated that Nbssn6 was distributed in the nuclei; (B) White arrow indicated that Nbssn6 was evenly distributed in N. bombycis; (C) White arrow indicated that Nbssn6 was distributed in both the nuclei and perinuclear cytoplasm, while the white arrow head indicated that Nbssn6 was distributed in the nuclei; (D) Negative control. Scale bars, 5 μm.

Figure 7

SDS-PAGE analysis of Co-IP results. Lane M: Protein molecular weight marker. Lane 1: Total protein of N. bombycis. Lane 2: Co-IP result using Nbssn6 antibody. Lane 3: Co-IP result using rabbit IgG antibody. Red arrow: specific band.

Figure 8

Interaction between Nbssn6 and Nbtrrp2. (A) Explanation for the 5 areas on the culture medium: the interaction of pGADT7-T and pGBKT7-P53 was used as the positive control, and the interaction of pGADT7-T and pGBKT7-lam was used as the negative control. (B) Yeast two-hybrid assay of the interaction between Nbssn6 and Nbtrrp2.

Figure 9

Effect of the knockdown of Nbssn6 on the transcription level of Nbtrrp2. (A) Effect of RNAi on the transcription level of the Nbssn6 gene. (B) Effect of RNAi on the transcription level of the Nbtrrp2 gene. Error bars represent the standard deviations of 3 independent replicates (n = 3, mean ± SE, * p < 0.05, ** p < 0.01, *** p < 0.001, NC, β-tubulin).

Figure 10

Interaction between Nbssn6 and Nbptp2. (A) Domains of Nbptp2. (B) Explanation for the 5 areas on the culture medium: the interaction of pGADT7-T and pGBKT7-P53 was used as the positive control, and the interaction of pGADT7-T and pGBKT7-lam was used as the negative control. (C) Yeast two-hybrid assay of the interaction between Nbssn6 and Nbptp2.

Figure 11

The effect of the knockdown of Nbssn6 on the transcription level of Nbptp2. Error bars represent the standard deviations of 3 independent replicates (n = 3, mean ± SE, *** p < 0.001, NC, β-tubulin).

Figure 12

Effect of down-regulating the Nbssn6 gene on N. bombycis proliferation via RNAi. The proliferation level is obtained by calculating the copy number of Nbβ-tubulin. Error bars represent the standard deviations of 3 independent replicates (n = 3, mean ± SE, * p < 0.05, ** p < 0.01, NC, β-tubulin).