4-NBT, a specific inhibitor of Babesia microti thioredoxin reductase, affects parasite biochemistry and proteomic properties

Abstract

Babesia microti is an emerging zoonotic pathogen that is transmitted by ticks and parasites and propagates in mammalian erythrocytes. Thioredoxin reductase (TrxR) plays a crucial role in B. microti survival by maintaining cellular redox homeostasis. In the present study, 4-nitro-2,1,3-benzothiadiazole (4-NBT) was selected as a specific B. microti TrxR inhibitor by comparing rat and parasite TrxR inhibition levels. Reactive oxygen species (ROS) levels were evaluated using flow cytometry, and in B. microti treated with 4-NBT, ROS levels increased with increasing inhibitor concentration. Furthermore, the inhibitor treatment increased lipid peroxidation and protein carbonyl levels, thus indicating a state of oxidative stress. While B. microti treated with 4-NBT appeared to lose the ability to multiply in mice, the fastigium of parasitemia between the treated and control groups was comparable. Furthermore, a TUNEL assay showed that 4-NBT induces apoptosis in B. microti. Proteomic analysis of B. microti treated with 4-NBT detected 960 proteins. Label-free quantitative proteomic analysis identified 118 proteins that were significantly up-regulated and 37 that were significantly down-regulated in the treatment group relative to the control. Of the differential proteins, proteasome and ribosomal subunit expression was up-regulated, thus suggesting that redundant proteins may be damaged by oxidation and waiting for degradation, while proteins for subsistence are waiting for de novo synthesis. Moreover, the findings obtained herein suggest that the DNA and lipids were also damaged and awaiting synthesis or repair. In conclusion, TrxR dysfunction in B. microti results in the breakdown of redox homeostasis and promotes apoptosis.