Characterization of a Novel OTU-like Deubiquitinase from Babesia microti: Implications for Babesiosis Treatment

Babesia microti is the primary agent of human babesiosis, an emerging tick-borne disease with limited treatment options and growing evidence of drug resistance. Deubiquitinases (DUBs) play critical roles in protein homeostasis and host–pathogen interactions, yet none have been characterized in B. microti. Here, we report the first molecular cloning, expression, and functional characterization of an OTU-like cysteine protease from B. microti (bm-OTU). The recombinant bm-OTU protein (~22 kDa) was expressed in E. coli, purified to high homogeneity, and exhibited ~93% solubility under native conditions. In vitro fluorogenic assays confirmed its deubiquitinase activity. Expression of bm-OTU in HEK293T cells was associated with reduced ubiquitination in cells and increased apoptosis in this overexpression model, as demonstrated by flow cytometry and Western blot analyses. Furthermore, transcriptomic analysis revealed that bm-OTU modulates host immune pathways, notably suppressing the expression of interferon-stimulated genes (APOBEC3G, G1P2) while upregulating the pro-apoptotic gene BAK and the inflammasome sensor AIM2. These findings establish bm-OTU as a functional deubiquitinase and a potential virulence factor that may contribute to immune evasion and pathogenesis in babesiosis and may represent a potential target, pending further validation.