Identification And Functional Study Of The ER Membrane Protein NbYOP1 Of Nosema Bombycis

Microsporidia is a type of obligate intracellular parasitic single-cell eukaryote.Since the identification of Nosema bombycis in silkworms by Nageli in 1857,over 1500 species of microsporidia have been reported,belonging to more than 187 genera.Microsporidia has a wide host range and can infect most invertebrates and vertebrates,including insects,fish,animals,and humans.Nosema bombycis that infects silkworms,is a representative species of microsporidia and can be transmitted by ingestion or embryonic infection,causing pebrine disease,which can cause serious harm to sericulture.To adapt to intracellular parasitism,the organelles of microsporidia are highly simplified,but organelles related to protein synthesis and transport,such as the endoplasmic reticulum(ER),are still retained.The secretory proteins synthesized by microsporidia still need to be transported from the ER to other cell compartments.Therefore,the ER is an essential component in the process of protein synthesis and transport in microsporidia.However,the research on the ER structure or function in microsporidia is currently limited.The ER includes both sheet-like and tubular networks,which are connected to the cell membrane and nuclear membrane,forming a continuous membrane network with a common lumen.In mammalian cells and yeast cells,the morphology of the tubular ER is shaped by two evolutionarily conserved protein families,Reticulons and DP1/YOP1(DP1/REEPs in mammalian cells and YOP1(YIP one partner)in yeast).Whether the shaping of the tubular ER in microsporidia also depends on these two types of proteins is still unknown.In this study,we searched the genome database of Nosema bombycis by using the Reticulons or YOP1 protein sequences of mice or yeast for Blastp searching but only found the NbYOP1,a homologous protein of YOP1,but did not find homologs of Reticulons.Therefore,the Nbyop1 gene of Nosema bombycis was cloned,NbYOP1 protein was expressed and purified by prokaryotic expression,and a monoclonal antibody was prepared in order to identify the subcellular localization and function of the NbYOP1 protein.The gene sequencing results showed that the sequence length of the Nbyop1 gene was516 bp,encoding a protein with 171 amino acids,with a predicted protein molecular weight of 19.5 k D and an isoelectric point of 8.87.Bioinformatics analysis showed that NbYOP1 contained two transmembrane regions and no signal peptide.Multiple sequence alignment and phylogenetic tree results showed that Nosema bombycis clustered with Nosema granulosis,Nosema apis,and Nosema ceranae,with homologies of 74.2%,55.5%,and53.8%,respectively.In addition,the identities of the yop1 gene in four microsporidian species belonging to the Encephalitozoon genus were all above 50% when compared with that of Nosema bombycis,indicating that the yop1 gene is highly conserved in microsporidia.The Nbyop1 gene was inserted into the prokaryotic expression vector p ET28 a+,and the NbYOP1 protein was expressed and purified.Mice were immunized with the purified protein to prepare monoclonal antibody against NbYOP1.Western blot analysis showed that the NbYOP1 monoclonal antibody had good specificity.Further,the NbYOP1 monoclonal antibody was used for indirect immunofluorescence analysis,which showed that NbYOP1 protein was mainly distributed in the sporoplasm of mature spore cells,and was clustered near the cell membrane in a punctate manner.In germinating mature spore cells and various stages of intracellular parasites,NbYOP1 was mainly distributed in the cytoplasm.After infecting silkworms with Nosema bombycis,the relative expression levels of the Nbyop1 gene in the intestinal tissue of the silkworms at different time points(24 h,48 h,72 h,and96 h)post-infection was measured by using q RT-PCR.The results showed that the Nbyop1 gene was expressed at all time points following infection,with the highest transcription level observed at 48 h post-infection.These results indicate that NbYOP1 is expressed in various developmental stages of Nosema bombycis and mainly exists in the cytoplasm,suggesting that NbYOP1 plays an important role throughout the life cycle of Nosema bombycis.The expression of Nbyop1 was interfered by using si RNA and the standard curve method was used to detect the proliferation of Nosema bombycis(using the copy number of Nbβ-tubulin DNA to represent the copy number of the genome).The results showed that the expression of Nbyop1 was significantly inhibited after interference,and the copy number of Nbβ-tubulin was significantly reduced,indicating that inhibiting the expression of NbYOP1 protein can significantly affect the proliferation of Nosema bombycis.Finally,the prepared NbYOP1 monoclonal antibody was used for immunoprecipitation experiments and 56 candidate interacting proteins were identified by using mass spectrometry analysis.Four candidates were selected with high abundance for verification using the split-ubiquitin membrane yeast two-hybrid assay,but the results were negative.In summary,the Nbyop1 gene of Nosema bombycis was cloned,bioinformatics analysis of the NbYOP1 protein sequence was performed,monoclonal antibodies against the NbYOP1 protein was prepared,the expression levels of NbYOP1 at different stages of Nosema bombycis was detected by using Western blot and qRT-PCR,the subcellular localization of NbYOP1 was analyzed by using indirect immunofluorescence,NbYOP1 candidate interacting proteins were identified by using immunoprecipitation experiments,and the interactions with split-ubiquitin membrane yeast two-hybrid assay were verified.These preliminary studies on the function of the membrane protein NbYOP1 provide an experimental basis for identifying proteins related to endoplasmic reticulum structure or function in microsporidia.Furthermore,our findings may lead to the discovery of new potential targets for preventing and treating Nosema bombycis infection in sericulture.