| Cryptosporidium parvum is a major parasite causing diarrhea in humans and animals,and it has bringed more than 400 waterborne disease outbreaks around the world.There are currently no effective drugs and vaccines for C.parvum,so further pathogenic biological researches are needed to find the drug and vaccine targets.Results of comparative genomics analyses indicate that the genome of C.parvum is highly compat,over 98%genes are single copies,while C.parvum genome contains some multigene families,such as insulinase-like proteases（INS）family which has 22 insulinase-like proteases.Previous studies shown that INS20-19 and INS5 proteins seemed to be involved in the invasion of C.parvum.In this study,we selected two INS proteins containing the classic active functional motif "HXXEH" and having different numbers of domain,in order to further understand the functions of this family.At the same time,we optimized the genetic manipulation technology of C.parvum.The steps of CRISPR-Cas9 technology have been simplified,the protein labelling expriment based on CRISPR-Cas9 has been established,and the second selectable marker has been found.INS 15 encoded by cgd34260 gene has a classic structure of insulin-degrading enzyme,which contains four domains and belongs to the M16A metalloproteases subfamily.The full length of INS 15 and its first domain（domain I）were cloned,expressed and purified,respectively.Besdes,one specific polypeptide of INS 15 was artificially synthesized.The recombinant INS 15,the expected band with the predicted size of～130 kDa was seen.However,there were also several bands smaller than 130 kDa.suggesting INS 15 protein may be posttranslationally processed to perform its multiple biological activities.Further,polyclonal antibodies against INS 15-domain I and INS 15-polypeptide were prepared.It was found that the polyclonal antibodies had a good recognition to native proteins.The neutralization results showed that the inhibition efficiency of anti-INS15 polypeptide antibodies was low,while the anti-INS-15 domain I antibodies reduced the invasion of HCT-8 cells by over 40%.Furthermore,the INS 15 gene had peak expression during the early period of the life cycle of C.parvum,and the INS 15 protein was expressed in the mid-anterior region of sporozoites and merozoites,suggesting that INS 15 may play some roles in invasive stages of the parasite.INS23 encoded by cgd53400 gene contains two domains belongs to the M16B metalloproteases subfamily.INS23 was cloned,expressed and successfully purified.Then,the polyclonal antibodies against the recombinant INS23 were prepared in rabbits.The INS23 gene had a peak expression at early timepoint of in vitro C.parvum culture and it also have a relative low expression level at other timepoints of C.parvum culture,and the INS23 protein was expressed in the whole region of sporozoites and merozoites.Anti-INS23 antibodies reduced the invasion of C.parvum sporozoites by over 35%.Those results suggested that INS23 may be involved in the invasion mechanism of Cryptosporidium and also has specific functions in other developmental stages.In order to simplify the CRISPR-Cas9 technology and value the transfection efficiency in C.parvum,we optimized the CRISPR-Cas9 technology in C.parvum.First,Cas9 protein was fused with green fluorescent protein（GFP）,and the transfection efficiency of Cas9 protein in C.parvum was tested by transient transfection.In addition,we constructed a tagging plamid which 3HA epitope tag was added on C-terminus of INS1（cgd1680 gene）.The tagging plasmid and Cas9 plasmid were co-transfected into sporozoites,then transfected parasites were gavaged into Ifngr-/-mice.After drug selection,a stable INS 1-3HA transgenic strain was obtained.The INS1 gene began to expression at 36 h and had a peak expression at 48 h of in vitro C.parvum culture.Besides,INS1 protein was only expressed in macrogamont of C.parvum when staining INS1-3HA strain,and it was co-localized to a special dense filament during the maturation of marcogamont.There is only one selectable marker for genetic manipulation in C.parvum,which is the neomycin（Neo）gene selected by paromomycin,it limits applications of multigene knockout or conditional knockout in C.parvum.We tested nine drug candidates through the continuous culture（air-liquid interface system,ALI）system of C.parvum in vitro.The results shown two potential drugs can inhibit C.parvum growth in vitro,they were puromycin and bromodeoxyuridine（BrdU）.Then,the results showed that only BrdU can inhibit the reproduction of wild-type C.parvum in Ifngr-/-mice.BrdU is an analog of thymidine and it can be converted into bromodeoxyuridine 5’-monophosphate（BrdUMP）by thymidine kinase（TK）,which is fatal to C.parvum.On the other hand,Cryptosporidium has an alternative pathway to produce dTMP from thymidine,so TK gene isn’t an essential gene for parasite growth.Through this experiment,we successfully found a usable drug that can inhibit the growth of C.parvum both in vivo and in vitro.We can replace the TK gene with designed sequence using BrdU for selection and establish a new genetic manipulation system.Taken together,we briefly characterizied the INS family in C.parvum which may provide a basis for specific functions of this family and help to facilitate the development mechanisms of Cryptosporidium.At the same time,we improved the current CRISPR-Cas9 technology and found a usable screening marker in C.parvum which provided a new idea for further research on some essential genes of C.parvum. |
