The Interaction Between Cryptosporidium Parvum And HCT-8 Cells In Proteomics And The Biological Functions Of Related Proteins

Cryptosporidium parvum is an important zoonotic intestinal parasite with a wide range of hosts.Although cryptosporidiosis caused by the C.parvum is characterized by self-limited diarrhea but can evolve to chronic and fatal diarrhea in people with low immunity.At present,the molecules involved in the interaction between the C.parvum and host cells and the mechanisms involved in the pathologic processes of the disease are unknown.Therefore,this study is the first to conduct quantitative and bioinformatics analysis on the secreted proteins and related host cell membrane proteins during the interaction between C.parvum and HCT-8 cells using TMT labeled quantitative proteomics.Based on the results of omics analysis,three invasion related genes were selected for recombination expression,and their related biological functions in the process of invasion of host cells by C.parvum were studied,which laid a theoretical foundation for the study of the mechanism of Cryptosporidium invasion of host cells and the development of anti-Cryptosporidium drugs.(1)Purification of C.parvum oocysts and preparation of sporozoites.Excystation is considered as an important index to measure the viability and infectivity of C.parvum oocysts.In this study,a combination of monodensity sucrose centrifugation method and cesium chloride(Cs Cl)method was used to purify the oocysts,and a total of 7.2×1010 oocysts were obtained.By comparing 4 methods of excystation commonly used in laboratory,method 4 was determined to be the best method of excystation in vitro to release sporozoites.Percoll density gradient centrifugation method was used to purify C.parvum sporozoites.Based on PCR and time-course methods,C.parvum were determined to be in the gliding motility state when infecting HCT-8 cells for 0-3 h,and in the adhesion/invasion state at 3.5-6 h.(2)Secretome profiling of C.parvum sporozoites in the initial interaction processes with HCT-8 cells.Sporozoites secrete proteins which can help them adhere to/invade host cells during gliding motility on the surface of host cells.Currently,the composition of secreted proteins of C.parvum during adhesion/invasion of host cells is not fully understood.In this study,TMT labeled quantitative proteomics was used for the first time to compare and analyze the secreted proteins of C.parvum in thegliding motility state(3 hpi)and adhesion/invasion state(6 hpi).Bioinformatics analysis of the quantified differentially secreted proteins from C.parvum and HCT-8cells showed that a total of 626 and 2339 C.parvum and HCT-8 cells secreted proteins were quantified.Differentially secreted proteins of C.parvum are mainly modified after translation,which are related to protein degradation and kinase activity,and cap-domain proteins are highly expressed during sporozoite gliding motility.However,differentially secreted proteins of HCT-8 cells are mainly concentrated in the pathways related to extracellular matrix and cell adhesion molecules.This study identified some important molecules that may be involved in the interaction between C.parvum and HCT-8 cells,providing basic data for the screening of key functional genes and the study of invasion mechanism of C.parvum.(3)Membrane proteomics of HCT-8 cell after infection by C.parvum.In the process of invading host cells,C.parvum binds to a series of receptor proteins on the cell membrane,and then causes changes in receptor proteins on the cell membrane surface and mediates changes in related proteins in the signaling pathway to play relevant biological functions.At present,there have been no reports of related studies on membrane proteomics of HCT-8 cell after infection by C.parvum.In this study,TMT labeled quantitative proteomics was used for the first time to compare and analyze the membrane proteomics of HCT-8 cell after infection by C.parvum.A total of 741 differentially expressed proteins were identified,among which 625 were up-regulated and 116 were down-regulated.Through functional analysis of differentially expressed proteins and some membrane proteins,it was found that down-regulated differentially expressed proteins were mainly involved in the destruction of HCT-8 cell barrier function caused by C.parvum infection,while up-regulated differentially expressed proteins were mainly involved in the change of HCT-8 cytoskeletal proteins.The identification and functional analysis of related HCT-8 membrane proteins laid a theoretical foundation for the research on the interaction mechanism between C.parvum and host cells.(4)Biological function of Cp Trap/CpCrisp/CpCarp in C.parvum invasion of HCT-8 cells.In this study,the function of C.parvum proteins containing CAP domain were analyzed,and three proteins including CpCrisp,CpCarp and Cp Trap containing TSP domain were prokaryotic expressed and polyclonal antibodies were prepared.The biological functions of CpCrisp,CpCarp and Cp Trap were investigated from the aspects of gene expression patterns,endogenous protein localization and in vitroneutralization characteristics during C.parvum infection of HCT-8 cells.The results of q RT-PCR showed that CpCrisp,CpCarp and Cp Trap were all expressed during the detection time(0-72 h)with the peak at 72 hpi.Immunofluorescence results showed that Cp Trap was localized in the oocyst and on the surface of the sporozoite membrane.CpCrisp was localized in the oocyst,sporozoite and merozoite cytoplasm,while CpCarp was localized in the oocyst,sporozoite and merozoite cytoplasm.Compared with the control group,the anti-CpCrisp,CpCarp and Cp Trap polyclonal antibody has a certain blocking effect on C.parvum invading HCT-8 cells.In summary,this study is the first to identify and quantitatively analyze the Secretome and membrane proteomics of HCT-8 during the interaction between C.parvum and HCT-8 cells by using TMT labeled quantitative proteomics.The bioinformatics analysis of differentially expressed proteins laid a theoretical foundation for the screening of key functional genes that C.parvum invades and the study of the interaction mechanism between C.parvum and host cells.In addition,the biological functions of Cp Trap,CpCrisp and CpCarp proteins in C.parvum invasion of HCT-8 cells were investigated and confirmed as candidate targets for anti-Cryptosporidium vaccine.