Studies On The Heparin-binding Characteristics Of Microneme Protein CpTSP4 Of Cryptosporidium Parvum And The Molecular Mechanism Of Its Secretion And Transport

Cryptosporidium parvum,as one of the pathogens of diarrhea in infants and young children worldwide,is also an important zoonotic pathogen that opportunistically infects individuals with low immune function,which has important public health significance.Cryptosporidium parvum possesses micronemes,rhoptries,and dense granules,among which micronemes play a vital role in parasite invasion.However,there are currently only a few reports on the microneme proteins and protein secretion pathway of Cryptosporidium parvum.Thrombospondin-related adhesion protein(TRAP),an adhesion-related type,exists in 1 to 2 in apicomplexa parasites(such as Plasmodium,Toxoplasma gondii,Eimeria,etc.),and it is found to play an extremely important role in parasite adhesion and invasion.However,in the genome of Cryptosporidium parvum,12 TRAP proteins(CpTSP1 to TSP12)are encoded.Because they all contain the Thrombospondin(TSP)repeat domain,they are also called TSP proteins.Among them,CpTSP1(also known as TRAP-C1)and CpTSP8(CpMIC1)are two micronemal proteins identified by immunofluorescence,but there is less research on their functions,and the localization and functions of other TSP proteins are unknown.Therefore,in this study,based on the CpTSP4 protein,which has a moderate protein size,a relatively simple domain,and is representative,the following in-depth studies were carried out.(1)Studies on adhesive properties and heparin-binding site of CpTSP4 protein: In an effort to explore the adhesive properties of the CpTSP4 protein,GST-tagged recombinant r CpTSP4 was expressed by a prokaryotic system.The adhesion properties of r CpTSP4 to host cells HCT-8 were investigated using FCM,IFA,and ELISA.It was discovered that r CpTSP4 can not only significantly bind to the HCT-8 cells,but also adhere to the HCT-8 cells at the nanomolar level,simultaneously demonstrating dose dependence and saturability.Through the analysis of the amino acid sequence of the CpTSP4 protein,it was found that there are certain basic amino acid clusters within its amino acid sequence,and this basic amino acid cluster exhibits the characteristic of binding heparin.Concurrently,heparin/heparan sulfate has been more extensively studied as the receptor for a multitude of pathogens invading host cells.Hence,it is hypothesized that heparin is one of the receptors for CpTSP4 binding to HCT-8.To verify this hypothesis,firstly,tests such as the heparin agarose bead pull-down,heparin salt competition,and heparin ELISA binding were adopted to initially verify that one of the main receptors for r CpTSP4 binding to HCT-8 cells is heparin.Then,tests including the potential heparin-binding peptide competition,peptide fluorescence labeling,heparinase digestion of the heparan sulfate on the surface of HCT-8 cells,amino acid site-directed mutation,and molecular simulation docking were employed to determine the novel heparin-binding motif(HBM)of r CpTSP4 in binding to the host cell,broadening the types of heparin-binding proteins,and simultaneously confirming that this motif participates in approximately 50% of the cell adhesive properties of r CpTSP4 towards the host cell.(2)Studies on the distribution and secretion of CpTSP4 protein in Cryptosporidium parvum: In an effort to explore the distribution and secretion status of the CpTSP4 protein at various developmental stages of the parasite,initially,a monoclonal antibody targeting the CpTSP4 protein polypeptide was fabricated,and the specificity of the antibody was identified through Western blot.Secondly,by employing the immunofluorescence labeling technique,an exploration was conducted on the distribution and secretion of the CpTSP4 protein on the sporozoites.The findings demonstrated that the CpTSP4 protein was colocalized with the microneme protein GP900 on the sporozoites within the oocysts,suggesting that the CpTSP4 protein is a microneme protein.Eventually,the IFA and ELISA were utilized to investigate the secretion,gliding track,and distribution at various intracellular stages during the excystation process of the CpTSP4 protein.The results indicated that the CpTSP4 protein was secreted abundantly during the excystation process and participated in adhesion and cell invasion.After the invasion,it developed into immature schizonts,and this protein was not expressed.When it evolved into mature schizonts,it was expressed once again in a significant quantity and stored in the micronemes of the merozoites.After the merozoites were released and successfully invaded cells,it advanced to the sexual stage,and the CpTSP4 protein was not expressed again,fully suggesting that the CpTSP4 protein is mainly a related molecule implicated in adhesion and invasion.(3)Studies on the transport mechanism of CpTSP4 protein on microtubules: To explore the transport mechanism of CpTSP4,it was initially discovered that the CpTSP4 protein was colocalized with β-tubulin on the sporozoites,suggesting that tubulin participates in the transport of the CpTSP4 protein.Subsequently,by employing the ELISA and targeting the motor proteins transported on the microtubules,the corresponding inhibitors capable of suppressing the secretion of the CpTSP4 protein were screened.The findings showed that two inhibitors of human selective kinesin-5(Eg5),namely SB-743921 and SB-715992,could block the secretion and distribution of the CpTSP4 protein on the microtubules without influencing the morphology and the excystation rate of Cryptosporidium parvum,and exhibited a dose-dependent manner,resulting in the accumulation of the CpTSP4 protein in the micronemes.There are seven types of kinesins in the genome of Cryptosporidium parvum.Through multiple sequence analysis and alignment of their motor domains,it was found that Cryptosporidium parvum kinesin-5(CpKin5)is the most distinctive,and has the highest homology with the human kinesin Eg5,which is much higher than the homology of the other six types of driving proteins in Cryptosporidium parvum.Concurrently,through Alpha Fold to predict the structure and conduct molecular simulation docking,it was revealed that CpKin5 and the human kinesin Eg5 share a conserved SB-743921 binding region.Finally,the colocalization of γ-tubulin and β-tubulin indicates that the head end of Cryptosporidium parvum is the negative pole of the microtubule,which is in line with the transport direction of the kinesin.In summary,this study identified and characterized the heparin-binding motif of CpTSP4,which contributes to ～50% of its cell adhesion.CpTSP4 is prestored in micronemes and transported and secreted via two unique central microtubules during excystation,gliding,and invasion,interacting with extracellular heparin/sulfated heparin.Finally,the kinesin-5(CpKin5)of Cryptosporidium was identified as a potential motor for CpTSP4 transport.These results provide a molecular insight into microneme protein secretion and host-cell interaction during Cryptosporidium sporozoite invasion,as well as the intracellular transport mechanism of microneme proteins.