| Parasitic nematodes can cause high morbidity and mortality in animal populations worldwide.Among them,Haemonchus contortus,with high prevalence rate in both developing and developed countries,is one of the economically significant small ruminant parasitic nematodes of the world.Haemonchosis caused by this worm infection can lead to growth and development obstacle,low productivity and even death of the host,which greatly threatens the health of ruminants.In addition,the high costs of prevention and control also restrict the sustainable development of animal husbandry.Fortunately,there are still several broad-spectrum antihelminthic drugs that have good therapeutic effects on H.contortus infection alone or in combination.However,longterm over-dependence on these drugs has led to different levels of drug resistance in H.contortus in different regions,which will result in exhaustion of effective drugs against this nematode.Additionally,there are no vaccines avaliable currently all over the world,which makes an extremely severe situation for haemonchosis prevention and control.Therefore,finding new key drug targets and vaccine candidate genes for developing new drugs free of pollution and residues,and safe and efficient vaccines are important research purposes of nematode disease control.Through long-term interaction with their host,parasitic nematodes have evolved a large number of serine protease inhibitors(SPIs),highly expressed during the parasitic stage,to protect their own survival needs and escape the killing mechanism of the host.Clarifying the role of SPIs in the process of parasite-host interaction could provide a new direction for the prevention and control of parasitic diseases.Based on these,the current study screened a kind of SPI,with two transcripts named Hc-spi-i8 a and Hc-spi-i8 b,highly expressed in H.contortus L4 stage,from the proteomics data obtained by our group previously,and their expression partterns were analyzed.The function of Hc-SPI-I8 A was verified by blood coagulation and activated partial thromboplastin time(APTT)experiments.Screening and verifying the interaction between Hc-SPI-I8 A and Oa TSP1 CP using yeast two-hybrid,Co IP,GST pull-down and co-localization techniques,and making a preliminary analysis of its mechanism.Combined with experiments such as ubiquitination,p65 nuclear entry and point mutations,the mechanism of Hc-SPI-I8 regulating the RACK1/IKKs/NF-κB/TNF-α pathway was preliminarily clarified.These results have laid a solid theoretical and realistic basis for the further study of the mechanism of H.contortus anti-coagulation and suppression of host immune response,and also provided a new direction for developing new vaccines and drugs.1.Analysis of the expression parttern of Hc-spi-i8 gene from Haemonchus contortusIn order to analyze the expression characteristics of Hc-spi-i8,a serine protease inhibitor of H.contortus,the c DNA sequence of Hc-spi-i8 was obtained through Blast.A reasonable speculation about the possible functions of Hc-SPI-I8 were made by the results of signal peptide and domain prediction and phylogenetic tree and homology analysis.Eukaryotic transfection technology was used to verify its signal peptide activity in HEK 293 T cells.Realtime fluorescence quantitative PCR(RT-q PCR)was performed to detect the transcription characteristics of the gene in different developmental stages of the parasite.Using immunofluorescence histochemistry(IHF)assay to present the location of Hc-SPI-I8 in the parasite.The results showed that Hcspi-i8 has two transcripts,Hc-spi-i8 a and Hc-spi-i8 b,both of which have an N-terminal signal peptide that might be active,and the former one also containing a TIL domain that is highly similar to blood-sucking parasites(such as hookworms).The transcription level of these two genes in the parasitic stage of H.contortus was significantly higher than that in the free-living stage,and Hc-SPI-I8 was mainly expressed in the intestines,gonads and hypodermis of the worm.To sum up,Hc-SPI-I8 plays an important role in the host’s blood coagulation system,which highlights the follow-up research content.2.The function and its mechanism of Hc-SPI-I8 A in inhibiting host coagulationTo clarify the anti-coagulant function of Hc-SPI-I8 and its molecular mechanism,sheep whole blood anticoagulation experiments were preformed.And different Hc-SPII8 truncated proteins were expressd to explore the importance of the TIL domain to the anticoagulant function of Hc-SPI-I8.Yeast two-hybrid system was performed to screen the coagulation-related factors in sheep whole blood that might interact with Hc-SPII8.Oa TSP1 CP was expressed by the insect baculovirus expression system,and its role in the anticoagulant function of Hc-SPI-I8 was analyzed.Rapid amplification of c DNA ends(RACE)was used to amplify the full length of Oa TSP1 CP and sheep’s main coagulation factors(II,VII,X and XI).The results have shown that Hc-SPI-I8 A could inhibit extrinsic and intrinsic pathway through the TIL domain.Hc-SPI-I8 might interact with four proteins in the sheep blood.Among them,Oa TSP1 CP,which could specifically bind to Hc-SPI-I8 A,may participate in the coagulation pathway and could help Hc-SPI-I8 A to perform its function on extrinsic pathway.The full-length CDS of Oa TSP1 CP and sheep coagulation factor were obtained by RACE.In summary,the mechanism of Hc-SPI-I8 A interfering extrinsic pathway,which could lay a solid foundation for comprehensively revealing the mechanism of Hc-SPI-I8 A inhibiting blood coagulation,has been preliminarily analyzed in this study.3.The function and its molecular mechanism of Hc-SPI-I8 in inhibiting host TNF-α-type inflammatory responseIn order to clarify whether Hc-SPI-I8 could regulate NF-κB activity by interacting with Oa RACK1 and thereby inhibit TNF-α-type inflammatory response,this study verified the interaction between Hc-SPI-I8,RACK1 and MKRN1,and analyzed the area of RACK1 binding to Hc-SPI-I8 and MKRN1.The conservation of the RACK1/IKKs/NF-κB pathway among different species was verified by phylogenetic tree analysis and p65 levels in the nucleus.The effects of Hc-SPI-I8 and MKRN1 on NF-κB activity were determined by multiple methods,and whether this effect was generated by interacting with RACK1 was also been determined.Ubiquitination experiments were performed to identify the effects of MKRN1 and Hc-SPI-I8 on RACK1 and MKRN1 on Hc-SPI-I8 and to reveal the effects of RACK1 ubiquitination and these two proteins on the TNF-α-type inflammatory response mediated by the RACK1/IKKs/NF-κB pathway.The results showed that Hc-SPI-I8,RACK1 and MKRN1 could bind to each other,and both Hc-SPI-I8 and MKRN1 bind to the same area of RACK1.The RACK1/IKKs/NF-κB pathway was conserved among species,and Hc-SPI-I8 and MKRN1 could affect NF-κB activity through interacting RACK1.The ubiquitination of RACK1 and Hc-SPI-I8 could be promoted by MKRN1,and Hc-SPII8 could inhibit the ubiquitination level of RACK1.Both effects of MKRN1 and HcSPI-I8 on the ubiquitination of RACK1 were related to K183 and K185,and the ubiquitination of these two sites affects the role of RACK1 in the TNF-α-type inflammatory response mediated by the RACK1/IKKs/NF-κB pathway.Taken together,Hc-SPI-I8 could inhibit the TNF-α-type inflammatory response via inhibiting the ubiquitination of RACK1 promoted by MKRN1.These results could lay the foundation for further in-depth exploration of the molecular mechanism of Hc-SPI-I8 inhibiting TNFα-type inflammatory response.Collectively,a TIL-type SPI was selected from H.contortus proteomics that was highly expressed in the parasitic stage,significantly in the intestine,gonads and subcutaneous tissues.It was clear that the inhibition of Hc-SPI-I8 A in extrinsic pathway of the host depends on its TIL domain and the interaction with Oa TSP1 CP.MKRN1,Hc-SPI-I8 and RACK1 bind to each other and the same areas of RACK1 bind to the former two.RACK1/IKKs/NF-κB pathway conserved among species,and the influence of MKRN1 and Hc-SPI-I8 on the function of RACK1 in the RACK1/IKKs/NF-κB pathway were contributed by affecting the two key ubiquitination sites of RACK1.These results not only enrich the understanding of the mechanism of H.contortus inhibiting host coagulation and inflammation,but also provide new targets for the development of new drugs and vaccines against this nematode. |
