Characterization Of Encephalitozoon Hellem Polar Tube Protein 4(EhPTP4)and Its Role In Host Cell Infection

Microsporidia are opportunistic zoonotic intracellular pathogens transmitted by food and water,they can infect a wide variety of animals ranging from invertebrate to vertebrate,including humans.The polar tube is a unique,highly specialized invasion organelle of Microsporidia.Before germination,the polar tube coils around the sporoplasm in the spore.Upon appropriate environmental stimulation the polar tube rapidly discharges out of the spore and pierces a cell membrane,serves as a conduit for sporoplasm passage into the new host cell.Despite its description as a morphologic structure over 100 years ago,however,we are still in lacking knowledge on the formation of polar tube,either the mechanism of infection and cell attachment.Proteomic and antibody studies have led to the identification of five different polar tube proteins(PTP1 through PTP5)in microsporidia,analysis of protein glycosylation has revealed that PTP1 contains many post translational O-linked mannosylation sites and could interact with unknown host cell mannose-binding molecule.Encephalitozoon hellem polar tube protein 4(Eh PTP4)was identified and studied in this work,and the localization,binding capacity to host cell and the cellular receptor was studied.This study analyzes the sequences of Ehhptp4 on the baisis of genomic data,the glycosylation sites and protein domain were analyzed according to the protein sequence.And we also expressed the recombinant protein of Eh HPTP4 and the monoclonal and polyclonal antibodies were prepared.Furthermore,indirect immunofluorescence assay(IFA),immunoelectron microscopy(IEM)and Correlated fluorescence and scanning electron microscopy(CLEM)were performed to determine the localization of Eh HPTP4.Subsequently,the experiments on the binding of Eh HPTP4 to host cell and deproteinated chitin spore coats(DCSCs)were analyzed,and a host cell receptor(Tf R-1)was identified by immunoprecipitation(Co-IP).Finally,antibody blocking and infection inhibition assays demonstrated that Eh PTP4 and Tf R-1 play key roles in polar tube infection to host cell.1.The bioinformatic characteristics of Ehhptp4 of E.hellem EhHPTP4 was predicted to have a signal peptide,chitin binding domain and Histidine-rich region(HRR).The first 16 amino acids were predicted to be the signal peptide of Eh HPTP4,and the signal peptide has high hydrophobicity.According to the multiple sequence alignment analysis,among the three key residues(Cys,Pro and Gly)in chitin binding proteins,Cys and Gly are highly conserved in PTP4 homologous,further confirmed the chitin binding function of PTP4.Additional,a Histidine-rich motif was found at the C-terminal tail of Eh HPTP4,which might help Eh HPTP4 bind to host cells.Sequence analysis revealed that Eh HPTP4 has 6 cysteines,most of them are conserved in PTP4 homologies.Multiple glycosylation modification sites and phosphorylation modification sites were predicted,revealed that Eh HPTP4 is a glycoprotein,might play important role on the process of polar tube binding to host cells.2.The localization of EhHPTP4 in E.hellem Recombinant Eh PTP4 was expressed in Escherichia coli as a fusion protein and this rec Eh PTP4 was used immunization of both mice and rabbits.An Eh PTP4 mouse monoclonal antibody was produced by screening a hybridoma library.Both the rabbit polyclonal antibody and mouse monoclonal antibody to Eh PTP4 react to the same antigenic band in E.hellem spore lysates.Indirect immunofluorescence assay(IFA),transmission electron microscopy(TEM)and Correlated fluorescence and scanning electron microscopy(CLEM)have been used to successfully assess the localization of microsporidian proteins.IFA and CLEM using rab-Pc Ab-Eh PTP4 demonstrated that the entire polar tube was labeled by this polyclonal serum,proving that Eh PTP4 is a polar tube protein;however,MAb-Eh PTP4 labeled only the tip of polar tube.The different localization pattern seen using the rab-Pc-Eh PTP4 Ab and MAb-Eh PTP4 suggests that there is an Eh PTP4 epitope which is only exposed at the tip of polar tube and that this epitope is specifically recognized by the MAb-Eh PTP4.Immunoelectron microscopy(Immuno EM)was utilized to examine the location of Eh PTP4 in E.hellem spores.Consistent with the labeling result using IFA,the gold particles were observed on the polar tube when using rab-Pc Ab-Eh PTP4.When using MAb-Eh PTP4,a more limited region of the polar tube was stained with gold with the majority of gold particles being found at an area where the polar tube appeared to be connected to the posterior vacuole.These combined IFA and TEM observations confirm that Eh PTP4 is a polar tube protein and the localization of Mab-EhPTP4 to the tip of the polar tube suggests that Eh PTP4 may be involved in an interaction with a host cell protein during invasion.3.Functional analysis of EhPTP4 in E.hellem Recombinant Eh PTP4 without signal peptide was expressed in 293 FT cells and together with the purified protein expressed in E.coli were used to study the function of Eh PTP4.Firstly,the epitopes of Eh PTP4 monoclonal antibody and polyclonal antibody were detected by Western blot,Native-SDS PAGE and Co-IP,both MAb and Pc Ab of Eh PTP4 could recognize same antigen in spore lysate,however,MAb could not reacted with Eh PTP4 which formed a complex with other unknown interaction proteins,which revealed that at the end of polar tube,Eh PTP4 might be monomer and some specific epitopes were exposed.According to our analysis of the protein sequence of Eh PTP4,a chitin-binding domain was found in Eh PTP4 sequence.We,therefore,investigated the ability of Eh PTP4 to bind chitin.The ability of Eh PTP4 to bind chitin was confirmed by incubating rec Eh PTP4 with deproteinated chitin spore coats(DCSCs)that have been previously demonstrated to contain mainly chitin.Our data demonstrated that after incubating rec Eh PTP4 with DCSCs a green fluorescence signal was observed on the spore coats indicating that Eh PTP4 binds to the chitin component present in DCSCs.The binding of Eh PTP4 to host cells was evaluated using ELISA,IFA and FACS,and the results revealed that Eh PTP4 could bind to host cell and interact with some unknown components on the host cell surface,which might play an very important role during the invasion of microsporidia.4.The identification and analysis of EhPTP4 cellular receptor Immunoprecipitation(IP)was utilized to identify the potential interacting targets of Eh PTP4 on its host cell.Co-IP demonstrated that an Oryctolagus cuniculus(rabbit)protein,Transferrin receptor 1(Tf R1)was identified by LC-ESI-MS/MS analysis.The same band was also identified using a Tf R1 monoclonal antibody.We repeated the IP using MAb-Eh PTP4(monoclonal anti-PTP4)and the same Tf R1 band was identified in this IP using MAb-Tf R1.A pull down assay using antisera to Eh PTP4(Mab Eh PTP4)demonstates that that rec Eh PTP4 can directly interact with rec Tf R-1 in vitro.IFA colocalization techniques using Eh PTP4 Alexa Fluor 488(green)and Tf R1 Alexa Fluor 594(red)staining on host cells demonstrated signal in the invasion synapse for both Eh PTP4 and Tf R1.During microsporidian infection we could also find that the tip of the polar tube co-localized with Tf R1 on host cell membrane at the site of the invasion synapse.Therefore,several lines of evidence suggest that Tf R1 is a host cell receptor that can interact with Eh PTP4.To further examine the role of Eh PTP4 and Tf R-1 during microsporidia infection,we performed antibody blocking,protein competition experiments and Tf R-1 knocking out experiments,the results demonstrated that when the interaction between Eh PTP4 and Tf R-1 was changed,the invasion of microsporidia to host cell will be inhibited as well,which further confirmed that the interaction of Eh PTP4 and Tf R-1 are important for the microsporidia invasion.As Tf R-1 enter cell by clathrin-mediate endocytosis(CME)pathway,then we use CME inhibitor Pitstop2 to pretreat with host cell and found the inhibition of endocytosis could also block the microsprodia invasion,revealed that during the infection of microsporidia,polar tube could enter host cell by endocytosis pathway through the interaction of Eh PTP4 and Tf R-1.In conclusion,we identified and characterized a new polar tube protein from E.hellem,and for the first time,we found a cellular receptor(Tf R-1)of microsporida polar tube,and the interaction between Eh PTP4 and Tf R-1 was also proved,meanwhile,the role of Eh PTP4 and Tf R-1 in microsporidia invasion was detected in our study,which proved that the tip of extruded polar tube entered host cell by endocytosis pathway during microsporidia invasion.The characterization and functional analysis of Eh PTP4 and Tf R-1 offered some clues to investigate the mechanism of infection to host cell.