Functional Characterization Of NbAQP And NPA Motifs From Nosema Bombycis

Nosema bombycis contains functional aquaporins(NbAQP)which is a key target for exploring the mechanism of infection of N.bombycis.Furthermore,although a lot of progress has been made in relation to research associated with N.bombycis infection,prevention and control,the mechanisms that underpin infection remain to be elucidated.During the germination process,there is an increase in the osmotic pressure of microsporidian spores.The prevailing belief is that transportation of water from the outer environment into the spores and/or metabolism of sugar may play an important role.Each of the homologous aquaporins contain six transmembrane domains which are linked to one another by five loop structures.The A,C,and E loops are located on the outside of the membrane,and the B and D loops are located on the inside of the membrane.The B and D loops contain two highly conserved NPA(Asn-Pro-Ala)motifs that are commonly found in aquaporins,forming a narrow channel that gives rise to an hourglass-structure.The two highly conserved NPA motifs play an important role in aquaporin biogenesis.1.The phylogenetic tree of NbAQPA phylogenetic tree displaying the aquaporins including NbAQP was generated using MEGA6,NbAQP and EcAQP clustered together in the resultant phylogenetic tree,,suggesting that these two proteins are functionally similar with a common origin.2.The location of NbAQP and NbAQP_NPAsIndirect immunofluorescence(IFA)was utilized to analyze the location of aquaporins in Nosema bombycis spores.We observed that aquaporins are predominantly located in the spore wall and protoplasm membrane of N.bombycis spores.We constructed a series of mutations of AQP in Nosema bombycis(NbAQP_NPA1,NbAQP_NPA2 and NbAQP_NPA1,2).Immunofluorescense studies indicated that the EGFP fusion proteins of wild-type NbAQP and NbAQP_NPAs are mainly distributed on the cell membrane and the nuclear membrane.Moreover,during cell division,EGFP fusion proteins has been present on the nuclear membrane of WT-GFP as well as NbAQP_NPAs-GFP.3.Water channel function of NbAQP and NbAQP_NPAsA specific band of about 54 kDa was detected in BmN cells transfected with NbAQP wt,NbAQP_NPAs(NbAQP_NPA1,NbAQP_NPA2,NbAQP_NPA1,2)by using EGFP rabbit polyclonal antibodies.There was no significant change in the bands ofNbAQP_NPAs and NbAQP wt,which indicated that either mutating an NPA motif or mutating two NPA motifs does not affect NbAQP expression in BmN cells.Following expression of NbAQP in Xenopus laevis oocytes,it was observed that NbAQP can promote rapid water penetration into oocytes.The associated permeation rate was 2-3 times that of the water-injected and non-injected oocytes,whereas the oocytes injected with NbAQP_NPAs have no significant change in size.The associated permeation rate of NbAQP_NPAs was significantly reduced 5–6 times compared to that of wild-type NbAQP.This study shows for the first time that N.bombycis contains functional water channel proteins and the NPA motif of NbAQP has an impact on water permeability of aquaporin in N.bombycisThis study provides a platform to conduct further research into the regulatory mechanisms underpinning NbAQP protein expression.Further study of NbAQP in N.bombycis and their inhibitors can have effective prevention and treatment of microsporidiosis.