Preliminary Functional Studies Of Glutathione S-transferase In Cryptosporidium Parvunm

Cryptosporidium parvum is one of the main effective species causing infection in Cryptosporidium spp..It is a zoonotic protozoa that invades intestinal epithelial cells and is one of the important causes of diarrhea worldwide.At present,the interaction between Cryptosporidium parvum and host cells,the invasion mechanism and the pathological process of related diseases are still unclear,which restricts the research and development of anti-cryptosporidium drugs and vaccines.Glutathione S-Transferase(GST)is a kind of multifunctional superfamily protease,which is a "universal" carrier protein for intracellular substance transport,can clear and detoxify endogenous or exogenous electrophiles,participate in the biosynthesis of alkene compounds and sterols,and also have signal transduction,regulation of cell proliferation and death.Moreover,they have been shown to be good targets in a variety of parasite studies.However,There are no research reports on Cryptosporidium parvum GST,and combined with the upregulation of the expression of secreted proteins containing GST＿C domain in our laboratory proteomics data of 3 h after the invasion of Cryptosporidium parvum,this study intends to conduct bioinformatics analysis,prokaryotic expression and biological function of Cryptosporidium parvum GST and related proteins.In this study,three Cryptosporidium parvum GSTs were screened through Crypto DB alignment and previous proteomics studies,named CpGST1,CpGST2 and CpGSTC,respectively.The results of systematic bioinformatics analysis showed that CpGSTs had certain antigenicity,among which CpGST1 had the strongest antigenicity.CpGST1 has no signal peptide and transmembrane region,while CpGST2 and CpGSTC have signal peptide but no transmembrane region.The domain analysis found that CpGST1,CpGST2 and CpGSTC all had GST＿C domain,and CpGST1 contained an additional GST＿N domain.The three-dimensional structure model of three CpGSTs was predicted by Alpha Fold 2,and the prediction results were consistent with the prediction results of the secondary structure,and compared with the predicted antigen determinant start and end sequences,it was found that most of them were the joint structure of irregular coil and α-helix.Based on the Neighbor-joining algorithm,the phylogenetic tree of multi-species GSTs was constructed,and the results showed that CpGST1 was closely related to Arabidopsis GSTs,CpGST2 was closely related to Plasmodium GST,and CpGSTC was closely related to Toxoplasma GST.In order to further study the biological function of CpGSTs,pET-28a-CpGSTs recombinant expression plasmids were successfully constructed by molecular biology technology,and high-purity CpGSTs recombinant proteins were obtained by prokaryotic expression system.According to the characteristics of GST catalyzing the binding of GSH to CDNB,the recombinant protein had enzymatic activity,and the detection results showed that the three recombinant proteins had certain enzymatic activity,and their specific activities were 134.1 μM/min/mg,110 μM/min/mg,and 20μM/min/mg,respectively,and the active proteins laid the foundation for subsequent functional research.The purified target protein was used as an immune antigen,BALB/C mice were used as immune objects to prepare murine polyclonal antibodies,and serum was finally collected and serum was isolated after 4 immunizations,and the titer of polyclonal antibodies was determined by indirect ELISA,and the titers of CpGST1,CpGST2 and CpGSTC reached 1:512000,1:512000 and 1:8000,respectively.The prepared polyclonal antibody could specifically recognize the natural protein of sporozoites.The results showed that the transcription patterns of the three target genes were basically similar,reaching the highest level at 3 h after infection with HCT-8cells in Cryptosporidium parvum,while maintaining a lower level of transcription in other time periods.From this,we infer that CpGSTs mainly play an important role in the early stage of Cryptosporidium parvum infection,and may play a characteristic role as its carrier protein in the final stage.We further used the prepared polyclonal antibody to localize the subcells by indirect immunofluorescence,and found that the three CpGSTs were all localized to the cytoplasm of sporozoites,indicating that all three CpGSTs belonged to cytosolic GST.Among them,the fluorescence signal of CpGST1 in the endogenous development stage of Cryptosporidium parvum suggests that CpGST1 may be related to the formation of parasitophorous vacuole.The prepared polyclonal antibodies were diluted in 1:50,1:100 and 1:500 proportions to detect the effect of Cryptosporidium parvum invasion into host cells,and it was found that the polyclonal antibodies of three CpGSTs had certain inhibitory effects under different dilution ratios,and the inhibition effect was significant at the dilution ratio of1:50.In summary,bioinformatics analysis,prokaryotic expression purification and preliminary functional studies of three GST proteins of Cryptosporidium parvum were carried out,and CpGST1 protein with high immunogenicity was identified,which participated in the invasion of host cells and was related to the formation of parasitophorous vacuole in the endogenous development stage.The results lay a foundation for further exploration of the infection mechanism of Cryptosporidium parvum.