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 chromatographytandem 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.

The humoral immune responses of grouper Epinephelus akaara to a natural infection with Glugea epinephelusis was studied by ELISA utilizing intact mature spores as the coated antigen. Results showed that a specific humoral immune response was elicited, but the intensity of infection (in terms of the number of cysts) was not related to the antibody level in naturally infected hosts. The differences in the antigenicity of intact mature spores and soluble spore proteins derived from cracked mature spores were also analyzed. Results suggested that similar antigen epitopes existed between the 2 groups. Additionally, antigen component patterns and the distribution of antigen with immunogenicity were investigated by using the western blot and the immunofluorescent antibody technique (IFAT). The new parasitic microsporidium has specific polypeptide patterns comparable to the reported fish microsporidians. The main antigenic substances are concentrated on the surface of spores, and are mostly located on the anterior and posterior end of the spore bodies. Most surface components of the G. epinephelusis spores are soluble. The potential role of the surface components in initiating infection was also discussed. KEY WORDS: Glugea epinephelusis · Microsporidia · Epinephelus akaara · Humoral immune responses · antigenicityResale or republication not permitted without written consent of the publisher

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Microsporidia are a big group of single-celled obligate intracellular organisms infecting most animals and some protozoans. These minimalist eukaryotes lack numerous genes in metabolism and vesicle trafficking. Here, we demonstrated that the spore wall protein NbSWP12 of microsporidium Nosema bombycis belongs to Bin/Amphiphysin/Rvs (BAR) protein family and can specifically bind with phosphatidylinositol 3-phosphate [Ptdlns(3)P]. Since Ptdlns(3)P is involved in endosomal vesicle biogenesis and trafficking, we heterologous expressed NbSWP12 in yeast Saccharomyces cerevisiae and proved that NbSWP12 can target the cell membrane and endocytic vesicles. Nbswp12 transformed into Gvp36 (a BAR protein of S. cerevisiae) deletion mutant rescued the defect phenotype of vesicular traffic. This study identified a BAR protein function in vesicle genesis and sorting and provided clues for further understanding of how microsporidia internalize nutrients and metabolites during proliferation.