The Inhibitor Of Thioredoxin Reductase Affects The Growth And Metabolism Of Babesia Microti

Babesia microti is transmitted by ticks and parasites and propagates in mammalian erythrocytes,which causes emerging and zoonotic babesiosis.Currently,the most effective treatment is to utilize a combination of the drugs atovaquone and azithromycin,but these may cause adverse effects,such as fever,emesis,dizziness,arrhythmia,or anaphylaxis.However,with the presence of antibiotic resistance,it is imperative and urgent to identify new therapeutic targets that can aid in the development of a new potent drug to mitigate.Since B.microti parasitizes erythrocytes where is in a high iron concentration and hyperoxic environment,it should take positive coping strategies of facing abnormal oxidative stress.Thioredoxin reductase?TrxR?plays a crucial role in maintaining cellular redox homeostasis.Our lab has identified that there are only one type of TrxR in B.microti.Take it as research objects to study its molecular mechanisms and the possibility of drug targets,so that provides fundamental basis for development of new antiparasites drugs.The contents of this study included the following four areas:1.DTNB was used as a substrate to measure TrxR activity of parasite and host.And 4-NBT was selected as a specific B.microti TrxR inhibitor by comparing TrxR inhibition levels from four inhibitor,which was with small effects on host(IC₅₀=50.77?M)and large effects on parasites?IC50=11.70?M?.2.Establish a method of in vitro short-term cultivation of B.microti.The cytotoxicity of 4-NBT was evaluated with CCK8 assay and hemolytic test.Furthermore,in order to detect the anti effectiveness of 4-NBT on B.microti,we use SYBR GREEN method in vitro and reinject method in vivo to check parasites growing states.3.The suppression effect of 4-NBT on B.microti natural TrxR was confirmed after extracting treated parasites protein.ROS levels were evaluated using flow cytometry,and in B.microti treated with4-NBT,ROS levels increased obviously with increasing inhibitor concentration.Furthermore,the inhibitor treatment increased lipid peroxidation and protein carbonyl levels,thus indicating a degree of oxidative damage and a state of oxidative stress.4.B.microti was incubated with 4-NBT or DMSO after cultured in vitro.With the help of Label-free quantitative proteomics,we explored the differential expressed proteins between B.microti with or without TrxR activity.1113 proteins were dected in total.And there were 180 up-regulated proteins and 37 down-regulated proteins in B.microti that cultured in vitro two days,while 50up-regulated and 80 down-regulated in B.microti cultured five days.The proteins associated with Ribosome,proteasome and protein processing in endoplasmic reticulum were affected notably with TrxR inactivation according to GO functional annotation and KEGG analysis.Besides,from the perspective of oxidative stress to analyze,the metabolism about proteins,nucleic acid and lipid was in a state of disturbance.In conclusion,TrxR dysfunction in B.microti resulted in the breakdown of redox homeostasis and promoted apoptosis.And we found that the proteins associated with ribosome,proteasome and protein processing in endoplasmic reticulum were affected notably with TrxR inactivation,and B.microti was in a state of metabolic disorders.Those results revealed that the necessity of TrxR in B.microti survival,and provide theory reference for the development of effective vaccines and drugs.