Research On The Function Of Hc-hrg-1 Involved In The Heme Transport Of Haemonchus Contortus

Haemonchus contortus is a blood-feeding parasitic nematode which is living in the abomasum of ruminants such as cattle and sheep.Infection with H.contortus can cause digestive system syndrome and anemia in the host,which poses a great threat to the health of the host and the development of animal husbandry.Although broad-spectrum antihelmintics,have shown certain efficacy in the treatment of haemonchosis,the increasing drug resistance of H.contortus has led to new challenges in the prevention and control of haemonchosis.Heme,as a cofactor of a variety of proteins,is a necessary substance for most living organisms.However,most parasitic nematodes,including H.contortus,lack endogenous heme biosynthesis pathways and need to uptake and utilization of heme from the environment or host.Therefore,reasonable regulation of heme uptake,transport and metabolism is essential for the survival and reproduction of parasites,and the identification of this mechanism may provide a basis for the search of potential drug and vaccine targets.In this study,we have identified two hemeresponsive genes,named Hc-hrg-1 and Hc-hrg-2.The protein structures analysis,conserved-domain prediction and molecular evolution were performed and the hemerelated transcription manners of Hc-hrgs were also verified.The expression characteristics of Hc-HRG-1 were analyzed in H.contortus,Caenorhabditis elegans and Hela cell lines.The heme binding capacity of Hc-HRG-1 was analyzed and the function of Hc-HRG-1 in heme transport during the larva stage of H.contortus was investigated by RNA interference assay.Heme spot assay and GAPPIX toxic tolerance assay of yeast were used to verify Hc-HRG-1 protein was directly involved in heme uptake,and site-directed mutation was used to analyze the key amino acid sites of HcHRG-1 involved in heme uptake.The interacting proteins of Hc-HRG-1 were screened by yeast two-hybrid system and the interaction mode of Hc-HRG-1 and captured protein was preliminarily explored by RNA interference assay.This study laid a foundation for further understanding the mechanism of heme uptake and transport in H.contortus,and also provided a new idea for drug target screening.1.Analysis of heme-response characteristics of Hc-hrgsThe sequences of Hc-hrgs were obtained by PCR amplification and Genome walking techniques.Bioinformatics methods were used to systematically predict and analyze the gene structure,functional domains and evolutionary relationships of Hchrgs.Quantitative PCR was used to analyze the heme response characteristics of Hchrgs at different developmental stages.The results showed that the Hc-HRG-1 protein contained the HRG superfamily functional domain related to heme binding and transport,while Hc-HRG-2 protein contained a thioxoredoxin folding functional domain(GST-N)and a glutathione S-transferase-C-terminal functional domain(GSTC).Analysis of the heme response characteristics showed that the transcription level of Hc-HRG-1 showed a similar heme dose-dependent manner at larval and adult stages,while that of Hc-HRG-2 showed an obvious heme dose-dependent manner only at larval stage.2.Expression characteristics of Hc-HRG-1The transcriptional characteristics of Hc-HRG-1 in different developmental stages of H.contortus were analyzed by quantitative PCR.Immunofluorescence histochemistry was performed to analyze the localization of Hc-HRG-1 protein in adult H.contortus;Heterologous expression of Hc-HRG-1 was conducted to profile its expression characteristics in Caenorhabditis elegans.The subcellular localization was analyzed by the co-expression of Hc-HRG-1 with related organelle markers in He La cells.The results showed that the transcription level of Hc-HRG-1 was the highest at L3 stage,followed by the parasitic stages and the lowest at egg stage.In adults,HcHRG-1 was mainly expressed in the lateral membrane of the intestinal cells,the muscle surface(contact with pseudocoele),the reproductive tissues and the outer membrane of embryonic cells.In C.elegans,the expression of Hc-HRG-1 was obvious on the apical membrane of intestinal cells,and some dot-aggregated signals were also detected in the intracellular compartment.In He La cells,Hc-HRG-1 was expressed in both the cell membrane and endo-lysosomal organelles.This study clarified the expression characteristics of Hc-HRG-1 and laid a foundation for the follow-up functional research.3.The functional role of Hc-HRG-1 in the heme transport of H.contortusTo further explore the function of Hc-HRG-1 protein in heme transport,in vitro heme binding experiment was conducted to analyze whether Hc-HRG-1 could interact with heme.The role of Hc-HRG-1 in heme transport was further analyzed by RNA interference experiments in both C.elegans and H.contortus.The results showed that Hc-HRG-1 was a heme binding protein.The ds RNA of Hc-hrg-1 could partially inhibit the transcription of Ce-hrg-1 and lead to the obvious accumulation of Zn MP,a fluorescent heme analog,in the lysosome of intestine of C.elegans.RNA Interfering of Hc-hrg-1 in H.contortus could lead to larval growth and development defects,which could be rescued by exogenous heme to a certain extent.In addition,RNA Interfering of Hc-hrg-1 could also improve larval tolerance to toxic heme analog Gapp IX in H.contortus.In this study,RNA interference was used to clarify the involvement of HcHRG-1 in heme uptake and transport,which was proved to be important for the growth and development of larvae.4.Analysis of key residues of Hc-HRG-1 involved in heme transportTo directly verify the heme uptake function of Hc-HRG-1,the heme spot assay and GAPPIX toxic tolerance assay were performed to analyze whether the exogenous expression of Hc-HRG-1 protein could promote the heme uptake of Saccharomyces cerevisiae.Single mutations of amino acid residues related to heme transport were performed to identify the key residues of Hc-HRG-1 involved in heme transport.The results showed that exogenous expression of Hc-HRG-1 could promote the uptake and utilization of heme in S.cerevisiae,and rescue the growth of heme-deficient yeast.H52,H97,C2,T29 and T50 are the key residues of Hc-HRG-1 involved in heme uptake.In this study,S.cerevisiae was used as the model organism to verify that Hc-HRG-1protein can directly participate in heme uptake,and to identify the keyresidues of HcHRG-1 participating in heme uptake.These findings laid a foundation for studying the mechanism of Hc-HRG-1 protein in heme uptake.5.Identification of Hc-HRG-1 interacting proteins and preliminary exploration of their interaction patternsTo further explore the molecular mechanism of Hc-HRG-1 involved in heme transport,the yeast two-hybrid system was used to screen Hc-HRG-1 interacting proteins.The interaction between Hc-HRG-1 and capture protein was further verified by yeast rotation validation,cell co-localization,immunoprecipitation and pull-down analysis.The truncated expression of Hc-HRG-1 was performed to identify the key regions of interaction by immunoprecipitation.The interaction pattern was preliminarily explored by RNA interference in H.contortus.The results showed that there was an interaction relationship between Hc-HRG-1 and Hc-VHA-2,and the key functional domains of the interaction between which were the third and fourth transmembrane domains of Hc-HRG-1.RNA interference of Hc-vha-2 led to a developmental deficiency in H.contortus larvae and a significantly up-regulation of the transcription of Hc-HRG-1.It is speculated that the heme transport of Hc-HRG-1 may depend on the activity of Hc-VHA-2.These findings laid a foundation for revealing the molecular mechanism of Hc-HRG-1 involved in heme transport.In conclusion,two heme-responsive genes,named Hc-hrg-1 and Hc-hrg-2,were amplified and obtained in this study.The structure,functional domain and evolutionary relationship of Hc-hrgs were systematically analyzed by bioinformatics methods,and the heme response and expression characteristics were also identified in H.contortus.By in vitro heme binding assay and in vivo RNA interference assay,it was confirmed that Hc-HRG-1 was involved in heme uptake and transport in the intestine at the freeliving stages of H.contortus.Using S.cerevisiae as model organism,we verified that Hc-HRG-1 was able to directly participate in heme uptake,and H52,H97,C2,T29 and T50 were the key residues for Hc-HRG-1 to participate in heme uptake.The interacting proteins of Hc-HRG-1 were screened by yeast two-hybrid system,and the interaction pattern of Hc-HRG-1 and Hc-VHA-2 was preliminarily explored by RNA interference,and it was speculated that the heme transport of Hc-HRG-1 may depend on the activity of Hc-VHA-2.These results initially revealed the molecular mechanism of Hc-HRG-1involved in heme transport,and provided a new insight for the development of new drugs and vaccines against haemonchosis.