Functional Exploration Of Phosphoenolpyruvate Carboxylase In Babesia Gibsoni

Babesiosis is an important blood parasitic protozoal disease.It caused by a variety of Babesia in the erythrocyte cells of different host animals.Babesia gibsoni mainly infects dogs,causing anemia,jaundice,hemoglobinuria and other symptoms in dogs and even death,which is very harmful to companion animal dogs worldwide.Faced with the dilemma of high incidence,wide distribution,poor drug treatment effect and frequent drug resistance,there are currently no commercial vaccines and drugs that can fundamentally treat or prevent B.gibsoni infection.Babesia belongs to the apicomplexan,and whole genome sequencing shows that most of the apicomplexan lacks the traditional tricarboxylic acid cycle essential enzymes,and may mainly produce ATP and lactic acid through glycolysis and anaerobic sugar metabolism.Phosphoenolpyruvate carboxylase（PEPC）catalyzes the reaction of phosphoenolpyruvate（PEP）and CO₂ to oxaloacetate acid（OAA）,which is an important way for B.gibsoni to produce OAA,fix CO₂,synthesize carbamoyl phosphate and pyrimidine,recover purines and generate glycolysis intermediates.PEPC does not exist in human and animal genomes,but genes encoding PEPC can be identified in the Babesia genome,suggesting that PEPC may have specific functions in parasites.The in-depth study of PEPC is helpful to elucidate the energy metabolism mechanism of parasites and provide basic data for the mining of drug targets.In this study,the biological information of PEPC was explored,and the expression of BgPEPC was destroyed by gene editing,thereby reducing the production of downstream metabolites,and exploring a series of effects of BgPEPC knockout on the growth of B.gibsoni.The main results are as follows:The PEPC gene was amplified using the g DNA and c DNA of B.gibsoni as the template,and the full length of the gene was 3255 bp,containing 8 introns and encoding958 amino acids.The protein has no transmembrane structure and signal peptide sequence,and has high similarity to protozoal proteins such as malaria and less similarity to species such as plants and bacteria.In this study,the BgPEPC-3x Flag localized strain was constructed,and the natural size of BgPEPC protein was 105 k Da by Western blot experiment,and the PEPC protein was localized in the cytoplasm of the B.gibsoni by IFA experiments.In this study,the knockout of PEPC was performed on the basis of BgPEPC-3x Flag strains and WT strains,and no knockout strains were obtained under routine medium condition after multiple attempts.Gene-deletional strain was obtained by adding downstream metabolite malate,a concentration of 5 m M that does not inhibit the growth of the B.gibsoni,to the medium.In this study,by comparing the growth rates of△BgPEPC strain,BgPEPC-3x Flag strain and WT strain,it was found that the growth rate of△BgPEPC strain was significantly lower than that of control strains,which proved that PEPC plays an important role in the normal growth of B.gibsoni and the production of downstream metabolites.The△BgPEPC strain can maintain a low growth rate in routine medium,which proves that there is an alternative way for the PEPC gene to function in the parasite,or they can obtain substances from erythrocytes of the host or culture medium to compensate for the adverse effects of gene deletion.The optimal concentration of malate was determined to be 1 m M,and although the growth rate of the control strain could not be completely reached after supplementation,its growth defect could be partially reversed.In addition,the knockout of PEPC gene also had a certain impact on the growth morphology of the parasite and the proportion of different parasites in erythrocytes,and the proportion of tetrad and single parasite in the control strain was significantly higher than that of the gene deletion strain.In order to further explore the effect of PEPC deletion on TCA cycling,six downstream metabolites were selected as exogenous additives,and it was found that only malate supplementation in culture medium could compensate for the growth of gene deletion strains.In order to explore the survival mechanism of gene deletion strains,the IC50 values of L-cycloserine and Diminazene Aceturate,metabolic inhibitors of aspartate aminotransferase and mitochondrial energy metabolism,were determined,and the results showed that the IC50 values of gene deletion strains for both drugs were significantly smaller than those of the control strains,and the sensitivity to L-cycloserine was reduced after malate supplementation,but had no significant effect on the sensitivity of Diminazene Aceturate,indicating that the deletion of PEPC increased the sensitivity of B.gibsoni to these two inhibitors.And only supplementation of malate can’t save the inhibitory effect of Diminazene Aceturate on B.gibsoni.In summary,in this study,the PEPC of Babesia gibsoni was identified through biological information analysis,the localization strain was determined to be localized in the cytoplasm,and the deletion of the gene was further constructed on the basis of the localized strain,and the effect of the deletion of the BgPEPC gene on the growth of B.gibsoni was analyzed through phenotypic experiments,replenishment experiments and inhibitor sensitivity experiments.This study explores the biological function of PEPC,a key enzyme in Babesia sugar metabolism,to provide a theoretical basis for the study of drug targets and the development of novel antiparasitic drugs against Babesia gibsoni.