Isolation And Identification Of Babesia Microti Exosomes And Functional Study Of Two Babesia Microti Antioxidant Enzymes

Babesia microti is one of the most important tick-borne causative agent that infects human and multiple animals.Babesia microti mainly survive in the red blood cells of corresponding hosts.At present,the prevention and control of Babesiosis still lacks efficient vaccines and drugs,hence it is urgent to understand the parasite-host interaction mechanism and develop new prevention and cure pathways.Exosomes are membrane-like vesicles that carry a variety of bioactive substances,such as proteins,RNA and lipids.Various functions of exosomes have been reported including immunomodulation,cell migration,and cell-to-cell communication.Presently,a variety of parasites exosomes have been identified to play an important role in the parasite-host interaction,however the exosomes of Babesia still remains unknown.Investigation in exosomes lays the foundation for the further investigation in Babesia parasite-host interaction mechanism.Moreover,Reactive oxygen species(ROS)and reactive nitrogen species(RNS)in the high oxygen environment of mammalian hosts' red blood cells(RBCs)are toxic to Babesia trophozoites and merozoites.Therefore,to survive in the host,the parasites must protect themselves from elimination by ROS.However,because these parasites lack antioxidant enzymes,such as catalase and glutathione peroxidase,the thioredoxin system plays a significant role in their resistance to host oxidative stress.The thioredoxin system consists of three major components,thioredoxin(Trx),thioredoxin reductase(TrxR)and NADPH,of which Trx plays a central role in redox regulation and antioxidation.Previous studies have demonstrated that Plasmodium falciparum Trx2 and TrxR both are effective potential drug targets,however studies on Babesia Trx have not been reported.Therefore,study on the molecular function of B.microti Trxs provides the basis for understanding the molecular mechanism of Babesia thioredoxin system and development of drug targets.1.Isolation and identification of Babesia microti exosomesThe present study was to isolate and identify the erythrocyte period Babesia microti exosomes(BmExo).A short-term culture in vitro system of B.microti infected red blood cells was established.After culturing for 12 h,the culture media was collected and subjected to ultracentrifugation for the isolation of BmExo.Transmission electron microscopy(TEM)and Nano-partial tracking analysis(NTA)were employed to identify BmExo.The results of TEM showed that typical cup-shaped vesicle structure with a diameter of about 110 nm was observed.The results of NTA showed that the size of the isolated exosomes was 127.7141.9 nm and the concentration was 1.62±0.17×109/ml.Western blot analysis showed that 15 specific bands were identified from the BmExo total proteins by the mice immune serum,indicating that BmExo carries a variety of highly immunogenic protein molecules.Then shotgun LC-MS/MS assay was performed.After comparing with the B.microti Uniprot protein database,A total of 207 proteins were identified.For exosomal markers(based on Exocarta database),proteins including Actin,Tubulin,and Heat Shock protein 20(HSP20),HSP70,GAPDH,Enolase,Aldolase,Lactate dehydrogenase(LDH),Elongation factors(EF),RAP protein,Rab protein,Phosphoglycerate kinase(PK),etc.were identified;For B.micorti secretory antigens,Surface Antigen(SA),Apical Membrane Antigen 1(AMA1)and several sero-reactive antigens were identified;For enzymes,Enolase,Aldolase,LDH,PK,etc.were identified;The other identified proteins were unannotated.Bioinformatics analysis of BmExo total protein were performed.For Biological Process analysis,the results showed that most proteins were involved in the metabolic process(30%),cellular process(30%),single-organism process(23%),localization(6%),biological regulation(6%),cellular component organization or biogenesis(3%),response to stimulus(3%)and other processes;For molecular function analysis,the results showed that the molecular functions of BmExo proteins mainly focused on binding(46%),catalytic activity(41%),transporter activity(6%),structural activity(5%);for cell component analysis,the results showed that 25%of BmExo proteins are compositions of cell,24%cell part,14%organelle,13%macromolecular complex,10%membrane,7%organelle part,7%membrane part.The exact functions of BmExo proteins still remain to be further studied.This study provides a basis for further analysis of the interaction mechanism between Babesia and host.2.The expression pattern and Functional analysis of Babesia microti thioredoxin 2In the current study,the obtained recombinant expression plasmid pET-3 0a(+)-Trx2 was used for the expression and purification of recombinant BmTrx2 protein and generation of polyclonal antibodies against rBmTrx2.Reverse transcription PCR,quantitative real-time PCR and Western blot were employed to detect the expression and native proteins of BmTrx2.Indirect immunofluorescence assay was used to localize BmTrx2 in B.microti.The results turned out that the expression of BmTrx2 was upregulated on both the third and eighth day post-infection in mice,whereas expression was downregulated during the beginning and later stages.The results of Western blot analysis showed the native BmTrx2 in parasite lysates could be detected by mouse anti-BmTrx2 serum and that the recombinant BmTrx2 protein could be recognized by serum of B.Wmicroti-infected mice.Immunofluorescence microscopy showed that BmTrx2 localized in the cell cytoplasm of B.microti merozoites in B.microti-infected red blood cells.Dihydroartemisinin and quinine,known anti-malaria drugs,and clindamycin,a known anti-babesiosis drug,induced significantly higher upregulation of BmTrx2 mRNA.These results indicate that BmTrx2 may be involved in the response of B.rmicroti to anti-parasite drugs and a potential drug target.3.The expression pattern and Functional analysis of Babesia microti thioredoxin 3In the present study,the obtained recombinant expression plasmid pET-3 0a(+)-Trx3 was used for the expression and purification of recombinant BmTrx3 protein and generation of polyclonal antibodies against rBmTrx3.Reverse transcription PCR,quantitative real-time PCR and Western blot were employed to detect the expression and native proteins of BmTrx3.Indirect immunofluorescence assay was used to localize BmTrx3 in B.microti.The results showed that the expression of BmTrx3 was upregulated on both the third and eighth day post-infection in mice,whereas expression was downregulated during the beginning and later stages.Western blot analysis showed that mouse anti-BmTrx3 serum could recognize the native BmTrx3 in parasite lysates and that the mouse anti-B.microti serum could recognize the recombinant BmTrx3 protein.Immunofluorescence microscopy showed that BmTrx3 localized in the cell cytoplasm of B.microti merozoites in B.microti-infected red blood cells.The anti-malaria drug chloroquine significantly inhibited the expression of BmTrx3,however,another anti-malaria drug qunine,and a known anti-babesiosis drug clindamycin,induced significantly higher upregulation of BmTrx3 mRNA.The results of the present study demonstrate that BmTrx3 may be involved in the response of B.microti to anti-parasite drugs a potential drug target.