Full length articleIdentification and functional study of a novel 2-cys peroxiredoxin (BmTPx-1) of Babesia microti
Graphical abstract
Introduction
Babesia microti is an intraerythrocytic apicomplexan parasite that is transmitted to mice and humans through the bite of an infected tick or blood transfusion. Since the first case of human babesia infection from a vole was reported in 1969 in Nantucket Island, which is off the coast of Massachusetts, there have been hundreds of reports of subsequent cases (Vannier and Krause, 2012). Moreover, there seems to be an increasing trend of Plasmodium falciparum co-infection (Zhou et al., 2013).
In general, Babesia infection can cause the mammalian immune system to attack the host red blood cells (RBCs) (Olliaro and Goldberg, 1995). Therefore, more efficient analytical approaches are needed to identify related antioxidant genes to elucidate the defense mechanism of B. microti and facilitate development of preventive measures against babesiosis.
Cysteine-based peroxidases, known as peroxiredoxins (Prxs) or thioredoxin peroxidases (TPx), are important antioxidant enzymes that prevent oxidative damage caused by reactive oxygen species (ROS). The thioredoxin-dependent peroxidase activity of Prxs was the first to be discovered and is probably the most relevant under physiological conditions in metazoan cells (Han et al., 2012), and are widely distributed among both eukaryotes and prokaryotes (Rhee et al., 2005). Prxs are the first-line defense against ROS and peroxynitrite (ONOO) (Klomsiri et al., 2011, Rhee and Woo, 2011). Recent studies have elucidated that typical 2-Cys-Prxs play multiple physiological roles, which include regulation of thiol-dependent peroxidase (detoxification of peroxides) and chaperone holdase (ATP-independent molecular chaperones) activities, regulation of H2O2 in the redox signaling cascade (Wood et al., 2003a, Wood et al., 2003b), and modulation of the immune response. Many parasites do not possess catalase and, therefore, are heavily dependent on Prx for peroxide scavenging (Gretes et al., 2012). However, there are indications of other relevant functions of Prxs as well.
A previous study described the structure of the Prx complex in mammalian cells (Wood et al., 2003a, Wood et al., 2003b). It has been reported that malaria parasites express and utilize Prx proteins during both the erythrocytic and insect stages to protect themselves against oxidative stress (Kawazu et al., 2003). The defense system of P. falciparum consists of a variety of antioxidant systems to maintain redox balance. Among these antioxidant systems, glutathione (GSH) and thioredoxin (Trx) are thought to play important roles in the Prx-anti-oxidation process in P. falciparum (Flohe et al., 1999, Jortzik and Becker, 2012). P. falciparum Trx reductase reduces Trx and a range of low molecular weight compounds, while GSH reductase is highly specific for its substrate GSH disulfide (Jortzik and Becker, 2012). P. falciparum employs a complex set of five Prxs with distinct subcellular localizations (cytosol, apicoplast, mitochondria, and nucleus) and different substrate specificity in order to defend against peroxides (Jortzik and Becker, 2012). However, whether the main physiological role of Prxs are same as in B. microti parasites remains unknown. Moreover, gene knock-out and RNA interference studies demonstrated that Prxs are essential for the survival and virulence of many parasites, including P. falciparum (Kimura et al., 2013), Plasmodium berghei (Usui et al., 2015), Leishmania infantum (Castro et al., 2011), and Schistosoma mansoni (Sayed et al., 2006). The PfTPx-1 disruption renders the parasite hypersensitive to heat stress (Kimura et al., 2013).
Recent research suggested that the peroxidase activity of Prx is irreversibly inhibited by conoidin A (2,3-bis(bromomethyl)-1,4-dioxide-quinoxaline) and the drug increased the sensitivity of the parasite to the antimalarial drug chloroquine (Brizuela et al., 2014). However, the multiplicity of roles played by Prxs constitutes an unexpected obstacle to drug development. Nonetheless, development of specific inhibitors of peroxidases that selectively inhibit the parasite enzyme, but not that of the human host, is needed. Therefore, Prx is a potential drug target for antiparasitic therapy in the foreseeable future for clinical use.
Previous studies have shown that Prxs are structurally homologous to the antioxidant proteins of yeast and have already been identified in all living organisms from Babesia spp. to humans (Masatani et al., 2014). Some Prx proteins reduce hydrogen peroxide (H2O2) and organic hydroperoxides with electrons through the Trx system (Chae et al., 1994b). According to the number of active cysteine residues, this family is divided into two groups: 1-Cys and 2-Cys. The former Prx protein contains a single catalytic cysteine residue (Cys-50), whereas the latter has an additional more conserved residue (Cys-170) (Nickel et al., 2005). Over the last few years, the 2-Cys Prx protein from the bovine parasite Babesia bovis (BbTPx-1) and the dog parasite Babesia gibsoni have been identified. Sequencing of the full genome of B. microti was completed in 2012 (Cornillot et al., 2012). However, until now, the 2-Cys Trx peroxidases-1 (TPx-1) of B. microti had not been characterized. Therefore, the main focus of the present study was to identify and characterize the antioxidant biological properties of TPx-1, a novel TPx of B. microti.
Section snippets
Parasite
B. microti strain ATCC® PRA-99TM was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in our laboratory (Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China) by serial passage in Kunming mice erythrocytes. The parasites were isolated upon infection of 30%–40% of RBCs, as determined by Giemsa staining of thin blood smears.
cDNA library construction and illumina sequencing
To characterize all transcriptional activity (coding and noncoding RNA), total RNA sequencing
Identification and characterization of the BmTPx-1 gene
Nucleotide sequences and the inferred amino acid sequences of the BmTPx-1 gene were compared with various nucleic acid and protein sequences retrieved from the GenBank database using the BLAST algorithm. The amino acid sequence of BmTPx-1 was deduced from the cDNA sequence (Supplementary Fig. S1). The full-length cDNA of BmTPx-1 was 720 bp in length (GenBank accession number KU670676) and included a single ORF of 591 bp, which encoded a polypeptide composed of 197 amino acid residues and
Discussion
Over the last few years, Prxs have been identified in all living cells (McGonigle et al., 1998) and the Prxs of B. bovis and B. gibsoni have already been sequenced and annotated (Tanaka et al., 2009, Masatani et al., 2014). In this study, we described the molecular cloning and biochemical characterization of the 2-Cys TPx-1 (BmTPx-1) from B. microti.
Sequence alignment and phylogenetic analysis showed that BmTPx-1 possessed significant sequence similarities (71%) with homologous sequences from
Acknowledgements
Project support was provided by the National Key Basic Research Program (973 Program) of China (grant no.: 2015CB150300).
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