Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)

Abstract

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.

Interaction and Assembly of Two Novel Proteins in the Spore Wall of the Microsporidian Species Nosema bombycis and Their Roles in Adherence to and Infection of Host Cells

“…Previous studies showed that surface antigens of mature spores could be removed by SDS and alkali extraction (42,43,49). The reports showed that the SWP32 protein of N. bombycis is an exospore protein (21). Therefore, positive-control Western blotting was also performed using anti-SWP32 antibody to confirm that the protein isolation had been performed properly (Fig.…”

“…In recent years, several studies have found that the spores of N. bombycis not only infect the Bombyx mori ovarian cell line BmN-SWU1, but also adhere to and infect the B. mori embryonic cell line BmE-SWU1. SWP30 (SWP1), SWP25 (SWP2), and SWP32 (SWP3) are found in the spore walls of mature spores of N. bombycis cultured in BmE-SWU1 cells (21,25).Microsporidian spores contain a special invasion organelle …”

“…At present, several spore wall proteins that are localized to the exospore or endospore have been identified in the genus Encephalitozoonidae (14,(16)(17)(18)(19)(20). For N. bombycis, only two exosporal proteins (21-23), three endosporal proteins (21,24,25), and a BAR-2 spore wall protein (26) have been characterized. In addition, several spore wall proteins that adhere to host cells via a heparin-binding motif (HBM) and interact with sulfated glycosaminoglycans (GAGs) of the host cell surface have been identified (20,24,27,28).…”

“…Polyclonal antibody production and immunoblotting protocols to detect NbSWP9 and NbSWP7 in the total protein of N. bombycis have been previously described (21,22,24). In short, rabbits and mice were injected with recombinant rSWP9 -glutathione S-transferase (GST) and rSWP7-His proteins, respectively.…”

Expression and characterization of a novel spore wall protein from Nosema bombycis

“…To date, only a total of seven spore wall proteins have been reported in E. cuniculi (Bohne et al, 2000;Brosson et al, 2005;Peuvel-Fanget et al, 2006;Southern et al, 2007;Xu et al, 2006) and Encephalitozoon intestinalis (Southern et al, 2007;Hayman et al, 2001), and four spore wall proteins from N. bombycis including SWP25, SWP30, SWP26 and SWP32 were addressed on the location and function (Wu et al, 2008;Li et al, 2009). In our previous study, we identified an exosporal protein NbSWP5 that can protect spores from phagocytic uptaking by cultured insect cells (Cai et al, 2011).…”