The Process And Mechanism Of Hemoglobin Digestion By Babesia Assisted With Pain2 And HDP

Babesia is an obligate erythrocyte protozoon that specializes in parasitizing inside red blood cells（RBCs）,where it can replicate.Mature RBCs cannot express or transport proteins due to the loss of their nucleus and organelles.However,Babesia outputs large amounts of proteins into the host RBCs for molecular transport,to perform the basic activities of life.Babesia lack many important pathways,and its limited biosynthetic pathways indicate that the need to obtain nutrients from host RBCs.As 95%of the RBC’s components are hemoglobin,the uptake and digestion of erythrocyte cytoplasm,especially hemoglobin,are essential for the survival of Babesia.In this study,bioinformatics and molecular biology were used to explore the interaction between recombinant proteins and hemoglobin,gene editing and cell biology techniques to investigate the localization and function of genes in the parasites,and transcriptomics and metabolomics to explore changes in the transcriptional and metabolic levels in gene knockout isolates,the mechanism by which Babesia digests host RBCs hemoglobin during its intra-erythrocytic period has been deciphered.The main results are as follows:（1）Establishment of continuous in vitro culture of Babesia gibsoni by using VP-SFM medium with low-concentration serum.The establishment of in vitro culture methods has greatly facilitated the research of Babesia.However,the current Babesia gibsoni in vitro culture medium requires high concentrations of canine serum,which intensively limits the culture and did not meet the demands of long-term studies.In this study,Albumax I（2 mg/m L）and 2.5%dog serum（v/v）were added to VP-SFM medium to develop a low-concentration serum culture medium named VP-SFMAD（2.5%）,and the effectiveness of this medium was assessed by the growth of B.gibsoni.Results showed that VP-SFMAD（2.5%）could support the continuous growth of the parasite,and the parasitemia has no difference with the cultivation in RPMI-1640 with 20%dog serum.In contrast,either a low concentration of dog serum or absence of Albumax I will significantly lower the parasite growth or failed to maintain B.gibsoni growth in the long term.The strategy of reducing the hematocrit was also evaluated,and VP-SFMAD（2.5%）improved the parasitemia to over 50%within five days.The high parasitemia is helpful for larger numbers of parasite collection which is valuable for studying the biology,pathogenesis,and virulence of Babesia and other intraerythrocytic parasites.In addition,VP-SFMAD（2.5%）medium was successfully used for monoclonal parasite screening,which is similar to RPMI-1640D（20%）medium that obtains monoclonal strains on the 18th day.Those results showed that VP-SFMAD can be applied to B.gibsoni continuous long-term,expansion culture,and subclone culture.The VP-SFM as a base medium supplemented with Albumax I and a low concentration of canine serum（2.5%）allowed the continuous in vitro culture of B.gibsoni at both small and large volumes,which was to meet different experimental needs,such as long-term culture,obtain high parasitemia and subclone culture.The establishment of in vitro culture systems allows researchers to better understand the metabolism and growth patterns of Babesia.（2）Characteristic and functional analysis of genes involved in hemoglobin metabolism in Babesia gibsoniThe rapid asexual proliferation of Babesia in erythrocyte requires a large amount of nutrients.Malaria parasites have the same parasitic location,use hemoglobin as its primary source of nutrition during intra-erythrocytic stage.However,there are currently no reports on the source of nutrition for Babesia.Specific inhibitors of the metabolic pathway of hemoglobin in malaria parasites were used to demonstrate the significant inhibitory effect on the growth of B.gibsoni with artesunate(IC₅₀=0.37μM)and chloroquine(IC₅₀=0.34μM)showing obvious parasiticidal effects.Transmission electron microscopy showed that B.gibsoni could form phagocytic vacuoles to engulf host cell cytoplasm,suggesting that hemoglobin could be one of the main sources of nutrition for Babesia.In this study,Babesia gibsoni was used as a model identify genes that may be related to hemoglobin degradation and hemozoin formation,such as Bgpain2 and Bg HDP.Recombinant Bgpain2 and Bg HDP were expressed and purified,and enzyme-catalyzed reactions showed that r Bgpain2 and r Bg HDP could effectively convert hemoglobin into hemozoin-like in vitro,Electron microscopy results showed that the degradation products had a clear hemozoin structure.However,how these proteases function during the process of Babesia hemoglobin degradation remains to be further explored.（3）Biological characteristics of Bgpain2 and its role in host metabolismBgpain2 is a cysteine protease with hydrolytic activity that has the potential to digest hemoglobin in vitro,but its function in vivo is not yet known.In this study,gene editing was used to locate the protein in the cytoplasm of the parasite.Overexpression of Bgpain2 also confirmed its cytoplasmic localization in the parasite,and did not cause lethal damage to the parasite.To characterize the specific role of Bgpain2 in the parasite,homologous recombination was used to replace the open reading frame（ORF）of Bgpain2 with m Cherry and PAC,and the effects of this genetic knockout on parasite growth,development,gene transcription,and metabolism were evaluated.PCR and Southern blot confirmed the disruption of the Bgpain2 gene.Compared to wild-type parasites,there were no significant changes in the phenotype of growth cycle,replication rate,or morphology in the Bgpain2 knockout strain,indicating that other enzymes may compensate for the loss of Bgpain2.In addition,the sensitivity of the knockout strain to cysteine protease inhibitors ALLN and E64d increased by 3.7 and 1.6 times,respectively.Transcriptome analysis showed that the differentially expressed genes between the Bgpain2 knockout and wild-type strains were mainly clustered in ribosome biogenesis,aminoacyl biosynthesis,and metabolism,and that the knockout strain upregulated transport proteins,aspartic acid hydrolases,papain-like phospholipases,and perforin-like proteins.Metabolomics analysis showed that multiple metabolic pathways,including central carbon metabolism and amino acid metabolism,were affected in the Bgpain2 knockout strain.The results suggest that although Bgpain2 is expressed in the parasite,its function in normal development or invasion of red blood cells at this stage can be supplemented by other homologs,and transcriptome and metabolome analysis reveal the flexibility of B.gibsoni in host amino acid uptake.