Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design

C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.Instituto de PatobiologíaFil: Lu, Stephen. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Ascencio, Mariano E. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Torquato, Ricardo J.S. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; ArgentinaFil: Florin-Christensen, Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Tanaka, Aparecida S. Universidade Federal de São Paulo. Escola Paulista de Medicina. Department of Biochemistry; BrasilFil: Tanaka, Aparecida S. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular; Brasi

A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases

Rhipicephalus microplus is a cattle ectoparasite found in tropical and subtropical regions around the world with great impact on livestock production. R. microplus can also harbor pathogens, such as Babesia sp. and Anaplasma sp. which further compromise cattle production. Blood meal acquisition and digestion are key steps for tick development. In ticks, digestion takes place inside midgut cells and is mediated by aspartic and cysteine peptidases and, therefore, regulated by their inhibitors. Cystatins are a family of cysteine peptidases inhibitors found in several organisms and have been associated in ticks with blood acquisition, blood digestion, modulation of host immune response and tick immunity. In this work, we characterized a novel R. microplus type 1 cystatin, named Rmcystatin-1b. The inhibitor transcripts were found to be highly expressed in the midgut of partially and fully engorged females and they appear to be modulated at different days post-detachment. Purified recombinant Rmcystatin-1b displayed inhibitory activity towards typical cysteine peptidases with high affinity. Moreover, rRmcystatin-1b was able to inhibit native R. microplus cysteine peptidases and RNAi-mediated knockdown of the cystatin transcripts resulted in increased proteolytic activity. Moreover, rRmcystatin-1b was able to interfere with B. bovis growth in vitro. Taken together our data strongly suggest that Rmcystatin-1b is a regulator of blood digestion in R. microplus midgut.Fil: Lu, Stephen. Universidade Federal de Sao Paulo; BrasilFil: da Rocha, Leticia A.. Universidade Federal de Sao Paulo; BrasilFil: Torquato, Ricardo J.S.. Universidade Federal de Sao Paulo; BrasilFil: da Silva Vaz Junior, Itabajara. Universidade Federal do Rio Grande do Sul; BrasilFil: Jacobsen, Monica Ofelia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; ArgentinaFil: Tanaka, Aparecida S.. Universidade Federal de Sao Paulo; Brasi