Study On The Potential Of Calcium-dependent Protein Kinases 3 And 5 As Drug Targets For Cryptosporidiosis

Cryptosporidium spp.and Enterocytozoon bieneusi are two kinds of emerging pathogens of infectious diseases,which can cause diarrhea,weight loss and growth retardation in humans and animals,and have a negative impact on human health and animal husbandry.Among Cryptosporidium,Cryptosporidium parvum can infect a variety of animals and has a wide range of hosts,which indicated a zoonotic significance.At present,the drug screening of cryptosporidiosis is still in progress,and there is still a lack of effective treatments.Calcium-dependent protein kinases?CDPKs?,which are widely found in plants,ciliates,and apicomplexan parasites,but have not been reported in fungi and animals,are considered as ideal drug targets.In apicomplexan parasites,changes in Ca2+concentration could affect many important life processes,such as invasion and migration.CDPKs,as protein molecules that receive and transmit Ca2+signals,may participate in many important life processes.At present,studies on the function of Cryptosporidium CDPKs?CpCDPKs?mainly focused on the CpCDPK1,while studies on other CpCDPKs,such as CpCDPK3 and CpCDPK5,are still scarce.In the meanwhile,the research on these two pathogens is mainly focused on beef cattle and dairy cows.As the main livestock in the Qinghai-Tibet Plateau in China,yaks share pastures and water sources with local wildlife,which have potential public health significance.In this study,two members of the calcium-dependent protein kinase family of Cryptosporidium,CpCDPK3 and CpCDPK5,were expressed,while the preliminary functional verification of these two enzymes were also performed.The corresponding inhibitors were screened and the feasibility of these two as drug targets was also evaluated.The recombinant CpCDPK3 protein was cloned and expressed in E.coli.The enzymatic activity of recombinant CpCDPK3was determined,and its polyclonal antibodies was prepared.The antibody cannot recognize CpCDPK1,which shares a similar structure with CpCDPK3,and therefore has a strong specificity.After infecting HCT-8 cells in vitro,the expression of cgd5₈20 gene,which encoding CpCDPK3 protein,maintained low expression at the initial stage and reached its peak at 12 h after infection,then decreased,and increased again at 48 h.Protein localization experiments showed that CpCDPK3 distributed on the entire sporozoite and merozoite,but not on the oocyst.Polyclonal antibodies against CpCDPK3 showed no significant inhibitory effects on host invasion by the parasites.Among the 50 candidate compounds docked with the CpCDPK3,one showed both inhibition on Cryptosporidium parvum development and CpCDPK3 enzyme activity,which was considered to be an effective inhibitor of CpCDPK3.The results indicate that CpCDPK3 may play an important role in the intracellular developmental stage of C.parvum.In addition,the recombinant CpCDPK5 protein was also cloned and expressed in E.coli.After infecting the cells,the cgd2₁300 gene,which encoding CpCDPK5 protein,maintained a low level of expression at first.The expression level suddenly increased to the apex at 12 h after infection,then it decreased again,and reached the peak again at 48 h after infection.The protein localization experiment showed that the CpCDPK5 protein expressed at a low level in the oocyst stage,located in the middle and posterior end in sporozoites,and distributed in spots on the surface of merozoites.CpCDPK5 polyclonal antibodies only had a significant inhibitory effect on Cryptosporidium invasion at the dilution of 1:100,the inhibition rate was 14.9%.Among the 50 candidate compounds docked with CpCDPK5 molecules,two compounds were screened to have a certain inhibitory effect on C.parvum.However,the recombinant CpCDPK5 did not have enzymatic activity in this study,so the inhibitors could not be verified.The loss of enzyme activity may be due to an incorrect protein folding process that disrupted the correct conformation and prevented it from producing activity.These results indicate that CpCDPK5 may participate in the host invasion process of C.parvum,but does not play an important role.In this study,881 yak specimens from Qinghai Province were collected,and the methods of PCR with sequencing based on SSU rRNA and internal transcription spacer?ITS?were used to identify the Cryptosporidium and E.bieneusi.The positive rate of Cryptosporidium in Qinghai yaks in China was 31.1%?274/881?,with six Cryptosporidium species identified.The positive rate of E.bieneusi in Qinghai yaks was 7.2%?63/881?,with 10 genotypes identified.Among these,C.bovis,C.andersoni and E.bieneusi genotypes BEB4,BEB6,and J have been reported in humans,suggesting that yaks may be a potential source of human infection with Cryptosporidium and E.bieneusi,which need to be prevented and controlled.In summary,this study provided potential drug targets for the treatment of cryptosporidiosis,which is helpful for the development of new drugs against cryptosporidiosis.It was also benefit to understand the distribution of these two pathogens in yaks,and provided basis data for the prevention of the spread of the two pathogens between humans and animals.