Identification of a conserved glycolytic protein NbGAPDH interacting with NbMnSOD2 in Nosema bobycis

Two manganese superoxide dismutases, NbMnSOD1 and NbMnSOD2, were characterized previously in microsporidian Nosema bombycis. However, the functional information related to manganese superoxide dismutase in this obligate parasite is still limited. In this study, we further explored the potential proteins interacting with NbMnSOD2 using Co-immunoprecipitation and tandem mass spectrometry (LC-MS/MS) in N. bombycis, and the result indicated that a glyceraldehyde-3-phosphate dehydrogenase (NbGAPDH), which is a conserved glycolytic protein in microsporidia, may interact with NbMnSOD2. As following, we prepared the polyclonal antibody against NbGAPDH, and immunofluorescence analyses (IFA) revealed NbGAPDH colocalized with NbMnSOD2 on the spore wall of N. bombycis. Immuno-electron microscopy (IEM) further demonstrated that NbGAPDH could be detected on the spore wall of mature spores, consisting with the spore wall localization of NbMnSOD2 reported previously. The Co-immunoprecipitation, IFA and IEM results supported that NbGAPDH was a potential protein interacting with NbMnSOD2, suggesting it may display non-glycolytic functions in microsporidian N. bombycis, due to its special subcelluar localization. Moreover, the gene expression of NbGAPDH was detected in BmE cells during 1-4 days post-infection with qRT-PCR, and the result showed that the expression of NbGAPDH was present during 6h to 4 days post-infection, with the highest expression at day 2 post-infection, suggesting NbGAPDH may play essential roles during the development of N. bombycis. This study provides partial useful insights for the function of manganese superoxide dismutase in this obligate parasite.