Screening And Identification Of The Proteins That Interact With Cryptosporidium Parvum Microneme Protein

Cryptosporidium parvum, a zoonotic intracellular parasite, is one of the most important pathogens that cause diarrhea, which has a wide host range and a high rate of human and animal infection. In immunocompetent hosts, diarrhea is generally self-limited; In immunocompromised hosts(such as AIDS patients), diarrhea is always chronic sustained watery diarrhoea which can be life-threatening in severe cases. So far, there is little understanding of the pathogenic mechanism of Cryptosporidium parvum, and there is no effective prevention and treatment measures for cryptosporidiosis.Adhesion and invasion of apicomplexan parasites to host cells is the pathogenic key of the parasite. A series of proteins are released from the top secretory organelles of parasites, and the interaction of these proteins result in the invasion of the host cells. The microneme protein is a kind of adhesion-related protein secreted by the microneme, which plays an important role in the invasion of parasites. Rhomboid protease is a intramembrane serine protease that exist in a variety of apicomplexans. It has been shown that the proteolysis of Rhomboid protease to the substrate microneme protein is an essential process of host cells invasion in Toxoplasma gondii. In C. parvum, the Rhomboid proteins that could process the microneme protein has not been reported. In addition, the adhesion and interaction with host cells of microneme proteins are important steps in the process of infection. Thus, the identification of host cell receptor proteins that interact with microneme protein is important on the study of molecular mechanism of C. parvum invasion. At present, the microneme proteins that have been reported are GP900,TRAP-C1(TRAP) and MIC1 in C. parvum. The GP900 and TRAP proteins contain the interaction site which can interact with Rhomboid protein. In this study,the adhesion and invasion function and immune characteristics of C. parvum microneme protein were studied. Then the yeast two-hybrid technology, co-immunoprecipitation and immunoprecipitation-mass spectrometry were used to screen and identify the rhomboid protein and host protein that interact with C. parvum microneme protein, which provide a theoretical basis for research on the C. parvum invasion mechanism.Studies on the adhesion and invasion function and immunological characteristics of C. parvum microneme protein TRAP. The recombinant expression plasmid p ET28a-TRAP was constructed, then expression and purification of recombinant protein TRAP were performed. The adhesion characteristic of r TRAP to host cells was detected by Western blot. Anti-r TRAP serum was obtained from immunized mice, then the inhibitory effect of sporozoites invasion was observed in vitro, and the humoral immunity and cellular immunity levels were detected. The results showed that the recombinant protein TRAP could specifically adhere to host cells, and anti-r TRAP serum could inhibit sporozoite invasion of host cells to a certain extent. The level of Ig G antibody in serum of immunized mice was increased, and the levels of cytokines IL-2, IL-12 and IFN-? were significantly increased, these showed that r TRAP could induce humoral immune response and Th1 cell immune response.Screening of Rhomboid protein that interact with C. parvum microneme protein. Using the yeast two-hybrid system, C. parvum Rhomboid 1/4, Toxoplasma gondii Rhomboid 2 were cloned into p GBKT7 to construct the recombinant bait plasmids, respectively. ?-galactosidase assay indicated that the baits have no self- activation. The C. parvum microneme protein TRAP and GP900 were cloned into p GADT7 to construct the recombinant prey plasmids. The preys were transformed into yeast competent cells AH109 with baits, respectively. Then the screening of Rhomboid protein that could interact with microneme protein was performed according to the growth condition of co-transformation groups on the selective medium and detected by ?-galactosidase assay. The recombinant eukaryotic plasmids of Rhomboid and microneme protein were constructed and co-transfected into Hela cells, then the interaction between the two proteins was further validated by immunoprecipitation. The results showed that C. parvum Rhomboid 1 had the activity in GP900 protein cleavage in vitro, and T. gondii Rhomboid 2 could cleave the TRAP protein in vitro. Inhibition of sporozoite invasion by serine protease inhibitor DCI was detected in vitro, and the results showed that the sporozoite invasion of host cells can be effectively inhibited by DCI at a concentration of 25?M.Screen the host cell proteins that interact with C. parvum microneme protein GP900. The recombinant protein GP900 was expressed and purified, then incubated with host cells lysates. The host proteins interacted with GP900 were screened by immunoprecipitation-mass spectrometry, and four host cell receptor proteins were abtained according to the MS analysis results. To further verify the interactions between GP900 and the receptor proteins, a yeast two-hybrid technology was performed and there are four host proteins were identified, namely annexin A2(ANXA2), Actin beta(ACTB), Heat shock 70 k Da protein 5(HSPA5) and glyceraldehyde-3-phosphate dehydrogenase(GAPDH). In this experiment, ANXA2 was expressed by prokaryotic expression system and recombinant protein r ANXA2 was purified. The mice were immunized with purified r ANXA2 to obtain anti-r ANXA2 serum. Antibody blocking test in vitro showed that anti-r ANXA2 serum have a certain inhibitory effect for sporozoite invasion of host cells.In conclusion, this experiment studied the adhesion function and immune characteristics of microneme protein TRAP. Yeast two-hybrid and co-immunoprecipitation methods were used to showed that C. parvum Rhomboid 1 had a activity in the cleavage of GP900 protein in vitro. The host cell proteins(ANXA2, ACTB, HSPA5, GAPDH) that interacted with microneme protein GP900 were screened by immunoprecipitation and mass spectrometry, then the interaction was further confirmed by yeast two-hybrid assay. This experiment laid a foundation for the molecular mechanism of C. parvum invasion, and provided a reference for develop new drugs and candidate vaccine for the prevention and treatment of cryptosporidiosis.