Characterization Of Three Proteins Of CpCDPK1 And MEDLE Family,and Screening Of Inhibitors Against Cryptosporidium

With the rapid development of national economy,more and more attention has been paid to the safety of water quality,especially the safety of drinking water.Thus reducing pathogenic microorganisms in water environment plays a particularly important role in water quality safety.Cryptosporidium spp.,as one of the necessary pathogenic microorganisms in drinking water quality standards,has caused a number of large-scale outbreaks of water borne diseases in the world,especially C.parvum and C.hominis.In order to solve the problem that no suitable target sites could be used in drug design of cryptosporidiosis,we conducted the following research:1.In this study,we assayed the functions of Cryptosporidium proteins.We chose CpCDPK1 protein（encoded by cgd3₉20）,a typical member with complete function domain in the family of calcium-dependent protein kinases（CDPKs）of Cryptosporidium,to conduct expression,protein purification,and functional assays.Accoding to qPCR analysis,target gene has peak expression at 12 h after the parasites invade the host cells.The enzymatic activity of recombinant CpCDPK1 was verified,and the Km value was 283 μmol/L.According to immunofluorescence microscopy,CpCDPK1 antibodies reacted with the entire surface of C.parvum sporozoites and merozoites.Polyclonal antibodies could inhibit Cryptosporidium parvum invasion by about 40%.We provided evidences for the hypothesis that CpCDPK1 protein play an important role in Cryptosporidium life cycle,and indicated that it may be related to the growth of Cryptosporidium in the host.We picked 50 small molecule inhibitors against CpCDPK1 using molecular docking prediction analysis,and examined the in vitro inhibition rate by high throughput assay.Ten of them showed significant inhibitory effects on Cryptosporidium infection,which would be potential drug against cryptosporidiosis.In order to determine the site of action of the inhibitor,the inhibition experiments of enzyme activity test were conducted.The inhibitor No.14 showed a significant inhibitory effect on CpCDPK1 activity,and its IC50 value was 4.315 μmol/L.It is suggested that the inhibition of the small molecule on Cryptosporidium infection may be due to the inhibition of CpCDPK1 protein activity of Cryptosporidium.This study provides evidence for the suggestion that CpCDPK1 plays an important role in lifecycle of Cryptosporidium.2.Through comparative genomics analysis,we found that Cryptosporidium-specific MEDLE family is one of the differences in gene content among different species of Cryptosporidium.In this study,we expressed in Escherichia coli the C.hominis-specific member of the MEDLE protein family,ChMEDLE-1 encoded by chro.50507,and C.parvumspecific member,CpMEDLE-3 encoded by cgd5₄600.Quantitative PCR,immunofluorescence staining and in vitro neutralization assay were conducted to assess their biologic characteristics.The expression of the cgd5₄600 gene was high during 12 h to 48 h of the in vitro culture.ChMEDLE-1 and CpMEDLE-3 proteins were mostly located in the anterior and mid-anterior region of sporozoites and merozoites.Polyclonal antibodies against MEDLE proteins had different neutralization efficiency,reaching approximately 50%for ChMEDLE-1 and 60%for CpMEDLE-3.The differences in protein and gene expression and neutralizing capacity indicated the MEDLE proteins may have different roles during Cryptosporidium invasion and growth,and contribute to differences in host specificity between C.parvum and C.hominis.CpMEDLE-3 and ChMEDLE-1 could be potential target of drug design against cryptosporidiosis.3.Monitoring of water quality is also important means to ensure the safety of drinking water quality.Luminescent bacteria is an indicator organism of biological toxicity detection method,which is often used to detect the comprehensive toxicity of water quality.However,due to the short survival time of bacteria and the difficulty of long-distance transportation and preservation,its application prospect in online detection of water quality is limited.In this study,we prepared lyophilized powder which could be preserved for a long time by vacuum freezedrying technology.We assayed the ability of the freeze-dried powder to detect Cryptosporidium in water and proved that this detection method had no effect on warning pathogenic microorganisms.However,the lyophilized powder was sensitive to standard toxic substances,and could serve as a tool for outdoor online detection of water quality.In conclusion,we provided potential therapeutic targets for drug design of cryptosporidiosis,and it is helpful for the development of prevention and treatment of cryptosporidiosis.