Identification and localization of SAS-6 in the microsporidium Nosema bombycis.

The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly. The evolutionarily conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. Microsporidia possess the spindle plaque instead of centriole as their MTOC to nucleate spindle assembly. However, little is known about the components of spindle plaques in microsporidia. In our present study, we identified a SAS-6 protein in the microsporidium Nosema bombycis and named it as NSAS-6. The NSAS-6 gene contains a complete ORF of 1104 bp in length that encodes a 367-amino acid polypeptide. NSAS-6 consists of a conserved N-terminal domain and a coiled-coil domain. The high identity of SAS-6 homologous sequences from microsporidia indicates that SAS-6 is a conserved protein in microsporidia. Immunolocalization in sporoplasms, intracellular stages and mature spores showed that NSAS-6 probably localizes to the nucleus of N. bombycis and exists throughout the life cycle of N. bombycis. These results suggest that NSAS-6 is required in cell morphogenesis and division in N. bombycis. The function and structure of NSAS-6 should be the focus for further studies, which is essential to elucidate the role of SAS-6 in spindle plaque assembly.

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

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.