Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design
Introduction
The Babesia genus comprises a group of hemoparasites transmitted by ticks [1] and since Babesia vectors are distributed worldwide, these piroplasmids are commonly found in mammals’ bloodstream including, but not limited to, humans, horses, sheep, dogs, cats and cattle [2,3]. Bovine babesiosis caused by B. bovis induces severe fever, hemolytic anemia, hemoglobinuria, and hypotensive shock leading to high mortality rates in susceptible cattle [4]. Since more than half of the world cattle population is considered to be at risk of babesiosis infections [5], the Rhipicephalus microplus - B. bovis complex represents an important association from the economical perspective.
Babesiosis control is currently based on three different strategies, (i) vector control, (ii) cattle immunization and (iii) anti-Babesia drugs [6,7]. Vector control is mainly carried out by acaricides, although tick strains resistant to different compounds has already been described [8,9]. Cattle immunization has been proposed for both vector and Babesia sp. control. Tick immunological control studies resulted in the development of two commercially available vaccines, although the levels of protection vary largely between different regions [[10], [11], [12]]. Cattle immunization against B. bovis has been mainly carried out with live vaccines using attenuated parasites [13,14]; however, the use of recombinant vaccines based on B. bovis proteins has been proposed and is a subject of active investigation [15,16]. Currently the most commonly used drug to treat Babesia infections is imidocarb [17], but several other drugs are in the development stage [6,18].
In apicomplexan parasites, cysteine peptidases from the C1A subfamily have been identified and shown to be crucial for parasite survival and proliferation [[19], [20], [21], [22]]. Therefore, it was proposed that such enzymes could be explored as targets for drug development, in which enzyme specificity plays an important role [[23], [24], [25]]. Notably, B. bovis treatment with cysteine peptidase inhibitors reduces parasite growth [26], showing the contribution of cysteine peptidases for Babesia sp. life cycle. So far, a handful of C1A cysteine peptidases have been characterized [[27], [28], [29]]. One of them, ovipain-2 from B. ovis, displays a high degree of conservation with P. falciparum falcipain-2; ovipain-2 was found in merozoite stages and appears to be secreted into the erythrocyte cytoplasm, moreover, treatment of B. ovis in vitro cultures with anti-ovipain-2 antibodies resulted in a significant decrease of parasite proliferation [30], indicating the participation of ovipain-2 in the process of invasion of or egress from host red blood cells (RBCs). Similarly, bovipain-2, the falcipain-2 ortholog from B. bovis, was also found in merozoite stages scattered in the erythrocyte cytoplasm, suggesting its role during parasite egress [31]. Although these studies reinforce the role of cysteine peptidases for Babesia survival there is no information regarding their proteolytic specificity. So far only the BbiCPL1 from B. bigemina has been biochemically characterized [29], in which a preference for Val > Leu > Phe at the P2 position was found.
B. bovis genome is available [32] and 17 putative cysteine peptidases are currently annotated, from which four belong to the C1A subfamily [33]. Since C1A cysteine peptidases play an important role in apicomplexan life cycle and represent potential targets for anti-parasitic drug development, we present here the biochemical characterization of a putative C1A cysteine peptidase from B. bovis and its possible role during RBC invasion.
Section snippets
Bioinformatic analysis
The complete mRNA sequence of XP_001612131 was obtained from the B. bovis T2Bo strain deposited genome (http://protists.ensembl.org/). Domain analysis was conducted using PFAM (https://pfam.xfam.org/) [34] and detection of putative signal peptides was carried out with SignalP 5.0 [35]. The theoretical molecular weight and isoelectric point were estimated using the Compute pI/MW tool [36] and the topology analysis was carried out with TMHMM V.2.0 (http://www.cbs.dtu.dk/services/TMHMM/). An amino
Analysis of B. bovis cysteine peptidase (BbCp) primary structure
The deposited sequence XP_001612131 comprises a 49.1 kDa protein with a theoretical isoelectric point of 5.39 and no predicted signal peptide. Domain analysis of XP_001612131 revealed the presence of an inhibitory pro-domain I29 (F120 – F177) and a catalytic domain from the C1A family (I230 – A435). Topology prediction shows a short cytoplasmic stretch (M1 to S35), followed by a transmembrane domain (A36 to G58) in the N-terminal, while the rest of the protein is predicted as extracellular (K59
Discussion
Cattle babesiosis, caused by B. bovis, is a major concern for livestock production worldwide [2,5], yet knowledge regarding the molecular mechanisms of parasite survival is still sparse. Since cysteine peptidases play an important role for apicomplexan survival and proliferation [47] it was proposed that such molecules could be used as targets for parasite control, in which enzyme specificity is determinant [49]. Therefore, in this study, we carried out a kinetic characterization of a novel
Conclusion
In this study, we describe the biochemical characterization of a putative cysteine peptidase from B. bovis named BbCp. The active recombinant BbCp was obtained in bacteria after the refolding process and presented increased proteolytic activity in the presence of DTT and an optimum pH between 6.5 and 7.0. The rBpCp was inhibited by R. microplus Bmcystatin-1 and Rmcystatin-3 but not by Rmcystatin-4. Moreover, substrate profiling of rBbCp revealed preference for Val > Leu > Phe residues. Finally,
Declaration of competing interest
The authors declare there is no conflict of interest.
Acknowledgments
We are grateful to Jacilene Barbosa of Laboratório multiusuário 3 - INFAR, UNIFESP for performing the DNA sequencing. We also thank Dr. Luís Fernando Parizi and Dr. Itabajara da Silva Vaz Jr. from Universidade Federal do Rio Grande do Sul (UFRGS) for supplying the ticks used in this work.
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