Identification Of Two Proteases Of The Babesia Microti And Its Interaction With Tick Molecules

Babesiosis is a parasitic disease caused by Babesia,mainly transmission between humans and animals through ticks,which can cause fever,anemia,hematuria,and even death.Babesia microti is one of the most important zoonotic parasites,which is a serious threatened to human health.Therefore,the prevention and treatment of Babesia is particularly important.Cysteine protease(CP)is a kind of proteolytic enzymes with cysteine residues in the enzyme active center.CP belongs to papain-family,which is widespread from the virus to vertebrate organisms.The cysteine proteases in parasites mostly belong to papain-like protease Clan CA family C1(such as cathepsin B and cathepsin L)and family C2(calpain).These proteases play an important role in the process of development and survival of parasites.Cathepsin E is the major intracellular aspartic acid protease,and mainly exists in the immune system mediated by MHC II.Researches have shown that cathepsin E helped the removal of parasite in the late stage of infection.These proteases have been shown to play a variety of important roles in the development and survival of parasites and may be important drug targets.In order to screen for anti-Babesia drug target and understand the mechanism of pathogens transmission via ticks,we tried to explore the functional role of cysteine protease and cathepsin E,and the interaction between cysteine protease and cystatin molecules isolated from ticks in this study.The transcriptome sequences of B.microti were analyzed and an important papain-like cysteine protease and cathepsin E were screend.A novel cysteine protease was designated BmCYP.The amplificated cDNA of BmCYP is 1418 bp including the open reading frame of 1338 bp without a signal peptide and encodes protein of 445 amino acids with a predicted protein molecular weight of 49.7 kDa and an isoelectric point of 8.48.The putative protein had a enzyme active center(-QGACGSCWAFAT and SMHAVLLVGYG-),which is the catalytic domain of BmCYP.The amplificated cathepsinE,designated BmCATEB,has the open reading frame of 1440 bp without a signal peptide and encodes an expected protein of 479 amino acids with a predicted protein molecular weight of 53.4 kDa and an isoelectric point of 6.11.The putative protein had a enzyme active center(-AAVDTGSSLITT-).BmCYP and BmCATEB were subcloned into pET-28 a expression vector,expressed in E.coli BL21,induced with IPTG and purified using Ni-NTA His·Bind Resin.Western Blot indicated thatHis antibodies could recognize the recombinant protein.The results of proteinase assays showed that both BmCYP and BmCATEB had enzyme activities.An investigation of the RHcyst-1 and RHcyst-2 genes expression profile showed that RHcyst-1 and RHcyst-2 genes showed a significant up-regulattion in the partial fed nymphs,while a significant down-regulation in the engorged nymphs and molt adults.The results of proteinase inhibition assays showed that rRHcyst-1 and rRHcyst-2effectively inhibited the BmCYP' enzyme activities and also inhibited the BmCATEB'enzyme activities to a certain extent.The results showed that the recombinant protein BmCYP and BmCATEB had protease activity.The expression of RHcyst-1 gene and RHcyst-2 gene from Rhipicephalus Haemaphysaloides significantly changed in the ticks infected with B.microti.In addition,ticks protease inhibitors inhibited the activity of B.microti cysteine protease in vitro experiments,providing the evidence of protozoa interaction with ticks.These findings are important for elucidating the mechanism of interaction between tick and protozoa.