Identification Of Polar Tube Protein 4(NbPTP4) And 5(NbPTP5) From Nosema Bombycis And The Role Of NbPTP4 In Host Cell Infection

Microsporidia are obligating intracellular and spore-forming single-cell parasites.They widely distributed among animal kingdom,and can infect the hosts of invertebrates to vertebrates,even human beings.Nosema bombycis is one of the earliest identified microsporidia,which causes the most important disease of silkworm,has brought huge economic loss to sericulture.Therefore,detection and prevention of N.bombycis is great significant on the production of sericulture.Microsporidia have a very special structure--polar tube,is the unique infective organ of microsporidia,which consists of a variety of polar tube proteins.Polar tube can not dissolve in SDS,1%Triton X-100,1-10%H₂O₂,Chloroform and so on,but can be soluble inβ-mercaptoethanol,DTT and other reducing agents.This feature is closely to the properties of the protein components of polar tube.At present,the mechanism of action of polar tube proteins in microsporidia infection are still unclear.In this study,the hypothetical polar tube protein 4（NbPTP4）and hypothetical polar tube protein 5（NbPTP5）from N.bombycis were analyzed their localization,host interaction target protein and the role in infection.These research laid a foundation for illuminating the molecular mechanism of microsporidia infection.In this study,firstly we analyzed the sequences of NbPTP4 and NbPTP5,expressed and purified the prokaryotic recombinant protein,then analyzed the localization of NbPTP4 and NbPTP5,screened and identified the host interaction target of NbPTP4.Finally,the role of NbPTP4 and its interaction target protein in the process of microsporidia infection was studied.The main results of this research as follows;1.The bioinformatic characteristics of NbPTP4 and NbPTP5Based on the homology sequence comparison between N.bombycis,E.hellem and E.cuniculi,two hypothetical polar tube protein genes（NbPTP4 and NbPTP5）was obtained.The bioinformatic analysis results showed that NbPTP4 encoded a protein of222 amino acids with a theoretical molecular weight of 25.3 kDa and pI 7.56.There was no signal peptide and no other known function domains.Meanwhile NbPTP5 encoded a protein of 409 amino acids with a theoretical molecular weight of 32.5 kDa and pI 8.68.There was no signal peptide and no other known function domain.Furthermore,there were five cysteine sites in NbPTP4 and eleven cysteine sites in NbPTP5.It was inferred that these cysteine sites might play an important role in formation of disulfide bonds and the interaction between polar tube proteins.2.The localization of NbPTP4 and NbPTP5 in N.bombycispET-32a（+）-NbPTP4 and pET-32a（+）-NbPTP5 prokaryotic expression vectors were successfully constructed,then the polyclonal antibody were prepared respectively.Western blot results showed that the antibodies could immunoreact with NbPTP4 and NbPTP5 expressed in E.coli and N.bombycis.Indirect immunofluorescence assay（IFA）results showed that the NbPTP4 and NbPTP5 antibodies could specifically bind to the polar tube of germinated spores.Immunoelectron microscopy（IEM）results suggested that the immunocolloidal gold particles of NbPTP4 antibody could localize on the polar tube of N.bombycis.These results indicate that NbPTP4 and NbPTP5 are the polar tube proteins of N.bombycis.However,unlike the localization characteristics of NbPTP1,NbPTP2 and NbPTP3,NbPTP4 and NbPTP5 are located at the front end of the polar tube and around the anchor disk,suggesting that NbPTP4 and NbPTP5 may play a vital role in interaction between polar tube and host cell.3.NbPTP4 binding to the host cell and identification of potential host proteins interaction with NbPTP4The polar tube of microsporidia is stable and coiled in spores before infect.When external environment changes,the spore can eject the polar tube and pierce into host cell membrane.Then,the sporoplasm transported to host cells through the hollow polar tube.The polar tube will contact directly with host cells during invasion,so there was a connection between them.The result of cell binding experiment showed that recombinant protein NbPTP4 could adhere to BmE cells.Co-IP used to search for the host target protein interacting with NbPTP4.The result of mass spectrometry suggested that the NbPTP4 interaction target protein might be Tubulin-like protein,HSC70-4 and so on.We analyzed the localization of Bmtubulinαand found that Bmtubulinαhad fluorescent signals on the cell membranes whether BmE cells permeabilized or not,which indicated that Bmtubulinαexpressed on the inner and outer membranes of host cells.Subsequently,yeast two-hybrid,Far-western and Co-IP methods used to confirm the interaction between NbPTP4 and Bmtubulinαprotein.It speculated that NbPTP4,which is located at the front end of the polar tube,could interact with Bmtubulinαon the surface of the host cell to promote microsporidia infection.4.Effects of NbPTP4 and its host interaction target proteins on the infection of N. bombycisIn order to demonstrate the role of NbPTP4 and Bmtubulinαin N.bombycis infection,we performed antibody blocking experiment and protein competition experiment,respectively.Microsporidia infecting rate was inhibition,when the interaction of NbPTP4 and Bmtubulinαwas influence.It further confirmed the importance of N.bombycis NbPTP4 and host Bmtubulinαin microsporidia invasion process.In conclusion,we have identified the polar tube protein 4（NbPTP4）and the polar tube protein 5（NbPTP5）from N.bombycis and analyzed the function of the polar tube protein 4（NbPTP4）.We found that the host protein Bmtubulinαinteracted with NbPTP4,which related with the infection of microsporidia.The role of two interaction proteins in microsporidia infection process studied.These results provide new clues and ideas for studying the infection mechanism of microsporidia.