Abstract
Babesia microti is the primary causative agent of human babesiosis worldwide and associated with increased human health risks and the safety of blood supply. The parasite replicates in the host’s red blood cells, thus, in order to counteract the oxidative stress and toxic effects, parasites employ a thioredoxin (Trx) system to maintain a redox balance. Since thioredoxin reductase (TrxR) plays a critical role in the system, in this study, we report the cloning, expression, and functional characterization of a novel TrxR from B. microti (BmiTrxR). The complete gene BmiTrxR was obtained by amplifying the 5′ and 3′ regions of messenger RNA (mRNA) by RACE. The full-length complementary DNA (cDNA) of BmiTrxR was 1766 bp and contained an intact open reading frame with 1662 bp that encoded a polypeptide with 553 amino acids. Molecular weight of the predicted protein was 58.4 kDa with an isoelectric point of 6.95, similar to high molecular weight TrxR. The recombinant protein of BmiTrxR was expressed in a His-fused soluble form in Escherichia coli. The native protein BmiTrxR was identified with the mouse anti-BmiTrxR polyclonal serum by western blotting and IFAT. Moreover, the enzyme showed a disulfide reductase activity using DTNB as substrate and catalyzed the NADPH-dependent reduction of Trx. Auranofin, a known inhibitor of TrxR, completely abrogated the activity of the recombinant enzyme in vitro. These results not only contribute to the understanding of redox pathway in this parasite but also suggest that BmiTrxR could be a potential target for the development of novel strategies to control B. microti thus reducing the incidence of babesiosis.
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Introduction
Babesiosis, caused by Babesia, is an emerging tick-borne disease in humans. The parasite replicates in the host’s red blood cells, leading to myalgia, anemia, fatigue, and fever hematuria. Most severe infections are predominant in the elderly and in splenectomized or immunocompromised hosts (Holmgren and Lu 2010; Homer et al. 2000). Babesia microti, one of the severe pathogenic agents, undergoes a complex life cycle by infecting both arthropod ticks and mammalian hosts (rodents and human). In addition, the disease can be transmitted by blood transfusion and congenital transmission, which poses a risk to the blood supply. As blood transfusions are necessary for some illnesses, preventing the occurrence of babesiosis is based primarily on increasing the awareness of the public to the potential risks. Despite the diagnostic and preventive advances, the efficacy of currently used drugs to treat babesiosis is reported to vary in different strains of this parasite species. Therefore, new targets are needed to curb the emergence of drug-resistant Babesia parasites (Schnittger et al. 2012; Song et al. 2012).
In mammalian hosts, Babesia multiplies in erythrocytes. During the process, it has to counteract the toxic effects of reactive oxygen and nitrogen species (ROS, RNS) that could potentially lead to major oxidative DNA damage and lipid peroxidation (Gray et al. 2010; Regner et al. 2014). Thus, in order to counteract the oxidative stress and maintain a redox balance, parasites employ a thioredoxin (Trx) system, which includes the nicotinamide-adenine dinucleotide phosphate (NADPH), the Trx redox protein, and the NADPH-dependent disulfide oxidoreductase, namely Trx reductase (TrxR) (Fritz-Wolf et al. 2013). TrxR belongs to the dimeric flavoenzymes family and catalyzes the transfer of electrons from NADPH via FAD to the disulfide substrate. In protozoans, the C-terminal redox center is formed by two cysteines separated by four amino acids and this feature distinguishes it from human TrxR, which contain a cysteine-selenocysteine motif. The Trx system has been extensively studied in Plasmodium falciparum, while the enzyme system has not been studied in Babesia microti that causes babesiosis (Jortzik et al. 2010; Nickel et al. 2006; Peng et al. 2015).
The partial messenger RNA (mRNA) of TrxR gene is present in the genome of B. microti (NCBI Reference Sequence: XM_012793011.1). Then, we obtained the full-length gene BmiTrxR by amplifing the 5′ and 3′ regions of mRNA by RACE. Here, we report the protein on recombinant expression, purification, and biochemical characterization of its function in the redox metabolism of the parasite.
Materials and methods
Parasites and animals
The B. microti strain was obtained from the American Type Culture Collection (ATCCR PRA-99™) and was injected into Kunming mice. The parasites were isolated when 20–30 % of RBCs were infected, as determined by Giemsa-stained thin-blood films.
Bacteria and plasmids
Escherichia coli top 10 cells (Invitrogen) and E. coli BL21 (DE3) (Novagen) were used in routine plasmid construction and expression experiments, respectively. The vector pMD-18T (TaKaRa, Dalian, China) was used for cloning and sequencing purposes. The expression vector used was pET28a vector (Novagen).
Molecular cloning of TrxR from B. microti
RNA extraction from B. microti merozoites was performed using TRIzol reagent (Invitrogen). Complementary DNAs (cDNAs) were obtained by RT-PCR using RNA extracted from B. microti merozoites. The reactions were performed according to the instructions in the Reverse Transcription System Kit (TaKaRa, Dalian, China). The open reading frame (ORF) sequence of TrxR gene is present in the genome of B. microti (NCBI Reference Sequence: XM_012793011.1), but there is no complete sequence of BmiTrxR cDNA. Therefore, at first, we designed oligonucleotide primers from reported sequence to amplify the ORF of BmiTrxR (Cornillot et al. 2012). Their sequences are F: 5′-ATGATTCTTCGGGCTGGATT-3′ and R: 5′-TCAACCACACTTGCCCCCAC-3′.
The 5′ and 3′ regions of mRNA were amplified using a SMARTer RACE cDNA amplification kit (Clintech, San Jose, CA, USA) following the manufacturer’s instructions. The primers were designed based on the ORF sequence obtained from above. The primer sequences are GSP1: 5′-GTTTCTTAGGTACACAACCCACG-3′, GSP2: 5′-GCCTCCTCCAATCACAGCGAAGT-3′, and GSP3: 5′-GCTTAGTTTCGGTGAGTGATGCG-3′ for 5′ rapid amplification of cDNA ends (RACE); GSP1: 5′-TAGCACATAGGATGAAGACAAGGCA-3′ and GSP2: 5′-CAGGGAATGGCATTAGCTATGAAAC-3′ for 3′ RACE. Amplified PCR products were subsequently purified and ligated into the pMD-18T vector (TaKaRa, Dalian, China) and sequenced.
Expression and purification
Full-length BmiTrxR was excised from pMD-18T vector by digestion with Nco I and Xho I and subcloned into pET-28a (Novagen) expression vector. Accuracy of the sequences was confirmed by complete sequencing. The BmiTrxR gene was expressed as a His-tag fusion protein in E. coli BL21 (DE3) according to the manufacturer’s instructions. The recombinant proteins were induced with 1 mM IPTG, followed by incubation at 16 °C for 12 h. The cells were harvested and stored at −80 °C until further use. Purification of recombinant BmiTrxR was carried out by chromatography onto a Ni-NTA His · Bind Resin (Novagen). Then, the cell pellet was collected, resuspended in binding buffer (NI-NTA Buffer Kit, Novagen), disrupted by sonication and centrifuged at 12,000×g for 10 min at 4 °C. The soluble fraction was loaded onto a chromatographic column that was previously equilibrated with binding buffer. The column was washed two times with wash buffer and the recombinant protein was eluted in elution buffer (NI-NTA Buffer Kit, Novagen). Active fractions were pooled and quantified.
Production of antiserum against recombinant BmiTrxR and western blotting
The purified recombinant BmiTrxR protein was mixed with Freund’s complete adjuvant (Sigma) and 200 μg recombinant BmiTrxR was injected intraperitoneally into a mouse. Subsequently, booster injections were administered twice into the mouse at 2-week intervals each containing the same amount of Freund’s incomplete adjuvant and antigen. After the second boost, blood samples were collected via the orbital vein. Antibody titers of sera against recombinant BmiTrxR were measured by ELISA.
The lysates of B. microti-infected mouse erythrocytes or un-infected mouse erythrocytes were mixed with an equal volume of 2 × SDS gel-loading buffer. Then, the samples were boiled for 10 min and separated on a 12 % SDS-polyacrylamide gel by electrophoresis. The gel was transferred to a nitrocellulose (NC) membrane, blocked in 5 % milk solution overnight at 4 °C and the NC membrane was incubated with the recombinant BmiTrxR antiserum (1:200 dilution) for 1 h at room temperature, followed by five 10 min washes with PBST. Then, the membrane was incubated with HRP-conjugated goat anti-mouse IgG (1:2000 dilution) at room temperature for 1 h. After washing five times as above, positive signals were detected with the DAB substrate.
IFAT and confocal laser microscopic observation
Recognition of B. microti merozoites by anti-rBmiTrxR serum was tested by indirect immunofluorescence by confocal laser micrographs. Thin blood smears were fixed in a mixture of methanol and acetone (v:v/1:1) at −20 °C for 10 min. After three washes in PBS, blood cells were permeabilized with 0.1 % TritonX-100 for 20 min. Then, the fixed smears were incubated with the anti-rBmiTrxR-specific mouse serum (primary antibody) and incubated for 45 min followed by three 5 min washes in PBS. The slides were then incubated with goat anti-mouse immunoglobulin G conjugated with Alexa 488 (Molecular Probes Inc.) for 45 min and washed in PBS. Subsequently, the slides were incubated with 4′, 6′-diamidino-2-phenylindole (DAPI) (Thermo) for 20 min, washed in PBS and then covered with FluoromountTM Aqueous Mounting Medium (Sigma). Finally, the slides were examined under a confocal laser scanning microscope (Jia et al. 2009).
Recombinant BmiTrxR enzyme assays and inhibition test
The enzyme assays were performed at 25 °C using a Multiskan Spectrum (SpectraMax M5, Molecular Devices). TrxR activity in the recombinant BmiTrxR was determined by the 5, 5′-diothiobis (2-nitrobenzoic acid) (DTNB) reduction assay and the insulin reduction assay. Briefly, the DTNB reduction assay solution (200 μl) contained 100 mM potassium phosphate (pH 6.5), 2 mM DTNB and 2 mM EDTA. The reaction was initiated by the addition of BmiTrxR and the activity was monitored by the increase in absorbance at 412 nm during the first 3 min. Activity was calculated using the extinction coefficient of 13.6 mM−1 cm−1 for thionitrobenzoate (TNB), considering that 1 mol of NADPH yields 2 mol of TNB.
Trx reduction assay was carried out in the presence of 0.16 mM bovine insulin (Sigma), 10 μM recombinant BmiTrx, and 100 μM NADPH in 50 mM Tris-HCI (pH 6.5) containing 2 mM EDTA. Trx activity was determined by the decrease in the absorbance at 340 nm that was due to the consumption of NADPH. The extinction coefficient of 6.22 mM−1 cm−1 for NADPH was used in the calculations. For all enzyme assays, the controls did not have BmiTrxR or substrate in the reactions. The assays were repeated at least three times for each enzyme. In these TrxR activity assays, NADPH concentration ranged from 5 to 100 μM, DTNB concentrations ranged from 100 to 1000 μM, and Trx from 5 to 30 μM. The concentration of BmiTrxR was maintained at a constant 50 nM. In each reaction, the concentration of only one substrate was changed while keeping the others constant. All the kinetic parameters were acquired by fitting the experimental data with a nonlinear least-squares formula and Michaelis-Menten equation. Kinetic constants are represented as the mean of at least three independent data sets (Lothrop et al. 2014; Regner et al. 2014; Snider et al. 2014; Song et al. 2012).
Auranofin, a TrxR inhibitor, was used to monitor the reduction of DTNB at 412 nm. The assay medium contained 100 μM NADPH, 2 mM DTNB, and 50 nM BmiTrxR, in which the inhibitor concentrations were 0, 1.563, 3.125, 6.25, and 12.5 μM. The recombinant enzyme was preincubated with the inhibitor in the assay buffer for 15 min. Then, NADPH and DTNB were added to each well and residual enzyme activity was monitored (Song et al. 2012).
Results
Cloning and characterization of the BmiTrxR gene
Using the partial mRNA of TrxR gene present in the genome of B. microti (NCBI Reference Sequence: XM_012793011.1), we identified the ORF of BmiTrxR, which contains 5 introns, a single open reading frame with 1659 bp that encoded a polypeptide with 553 amino acids. The putative protein has a molecular mass of 58.4 kDa and an isoelectric point of 6.95, which is similar to other H-TrxRs members. The predicted signal peptide cleavage site was between 18 and 19 amino acids. Then, the cDNA was obtained by RT-PCR using the mRNA of B. microti merozoites. In order to obtain the complete cDNA of BmiTrxR, we amplified the 5′ and 3′ regions of mRNA by RACE and the full-length BmiTrxR cDNA was 1766 bp (GenBank accession KU845266). BLAST analysis of the predicted protein against all NCBI non-redundant databases revealed significant similarity scores with members of TrxR from other species. The amino acid sequence of BmiTrxR was 58 % identical to Babesia bovis TrxR (XP_001608869.1), 53 % identical to P. falciparum TrxR (XP_002808951.1), 54 % to Cryptosporidium hominis TrxR (XP_666572.1), 55 % to Theileria annulata strain Ankara (XP_954814.1), and 44 % to Human TrxR (3QFB_B) (Fig. 1). We found that the putative protein had a thiol-disulfide redox active center (-CVNVGC-) in its N-terminus and the C-terminus contained a conserved GCGGGKCG sequence, which is the catalytic domain of TrxR. A more detailed investigation of phylogram of the TrxR suggested a complex history for this gene family and an ancient origin and high degree of conservation during evolution. The phylogenetic analysis shows the close relativity between B. microti TrxR and T. annulata TrxR, which is based on their sequences high similarity (Fig. 2).
Identification of the native BmiTrxR in B. microti
The recombinant protein was successfully expressed and had a molecular weight of 58.4 kDa, which is in agreement with the expected size predicted from the amino acid sequence (Fig. 3). A mouse anti-BmiTrxR polyclonal serum was obtained after immunizing mice with the purified recombinant BmiTrxR. This antiserum was used to identify the native BmiTrxR in the lysate of B. microti. As shown in Fig. 4, a specific protein band with a molecular weight of ∼58.4 kDa was detected in B. microti-infected red blood cells but not in the un-infected cells by western blotting. The size of the recombinant BmiTrxR band was similar to that of the natural BmiTrxR. There were no other protein bands recognized by the antiserum. These results suggested that the recombinant BmiTrxR was immunoreactive, inducing antibodies specific to the native BmiTrxR protein. In order to determine the cellular localization of BmiTrxR, a thin blood smear was used to perform IFAT with the mouse anti-rBmiTrxR serum and observed under a confocal laser microscope. The specific fluorescence seemed to localize in the cytoplasm of B. microti merozoites (Fig. 5).
Activity and kinetics of recombinant BmiTrxR
Enzyme activity of the purified recombinant BmiTrxR was maximum at pH 6.5 in 100 mM phosphate buffer and 25 °C (Fig. 6). Moreover, the highest initial velocity of the enzyme was at the concentration of 100 nM (Fig. 7). The activity of BmiTrxR against NADPH-dependent disulfide reductase using DTNB as substrate was significantly higher than the TrxR of Rat (RatTrxR) at the same concentration (Fig. 8). BmiTrxR was also able to reduce both BmiTrx and the ortholog protein from E. coli (EcoTrx) (Fig. 9).
Inhibition assay of chemical compounds on BmiTrxR disulfide reductase activity
In addition, we investigated the efficiency of the auranofin inhibitor, which has been reported to inhibit P. falciparum TrxR. The experimental data showed that auranofin abrogated the enzyme activity of BmiTrxR completely at 12.5 μM (Fig. 10), suggesting that auranofin could be evaluated as a potential inhibitor of the BmiTrxR disulfide reductase activity.
Discussion
Infectious parasites are reported to be exposed to reactive oxygen and nitrogen species (ROS, RNS) created by the host immune response against the infection (Becker et al. 2004; Jortzik and Becker 2012). During the erythrocytic stages of apicomplexan protozoans, the thioredoxin (Trx)-thioredoxin reductase (TrxR) system plays a central role in the oxidative stress response (Regner et al. 2014). The NADPH-dependent disulfide reductase is the principal electron donor for the thioredoxin redox networks comprising, among others, thioredoxin, ribonucleotide reductase, and peroxiredoxins (Muller 2004). Previous work in the P. falciparum system had suggested that PfTrxR is vital for parasite growth under in vitro culture conditions (Krnajski et al. 2002) and the antioxidant defense in B. bovis is also supported by the BboTrxR (Regner et al., 2014). In this study, we describe the identification and biochemical properties of TrxR, which is likely involved in the Trx system of B. microti. To the best of our knowledge, this is the first identification and characterization of BmiTrxR activity and functionality in B. microti.
BLAST comparisons indicated the presence of only one TrxR gene in the B. microti genome. This gene belonged to the H-TrxR group with a redox-active center, which has a characteristic motif (CXXXXC) in the FAD-binding domain and a C-terminal domain (the interface domain) (Fig. 1). BmiTrxR shares more than 50 % sequence identity with homologous sequences from other protozoans, while with the host human TrxR the similarity was only 44 %. This is likely due to the presence of a characteristic cysteine-selenocysteine (Cys-SeCys) domain in the C-terminus of human TrxR. Because of the absence of a selenocysteine residue in the parasite’s TrxR site, the human and parasite enzymes have distinct substrate specificities, which validates the protein as an excellent drug target (Buchholz et al. 2010; Hirt et al. 2002; Jortzik and Becker 2012; Nepveu and Turrini 2013). In addition, Fig. 2 might provide a clearer picture of the evolution of this key detoxification TrxR. Phylogenetic analysis indicates that TrxR exists a broader range of species including apicomplexan parasites and their mammal hosts. Unexpectedly, the result shows a close homologous relationship between B. microti TrxR and T. annulata TrxR, indicating these enzymes may share common ancestry and function. Because the two parasites all belong to piroplasms multiplying in the host’s erythrocyte, transmitted by tick and similar life history (Li et al. 2014; Zanet et al. 2014). Western blotting result showed that the antiserum could recognize the native BmiTrxR in B. microti. Moreover, the previous study reported that P. falciparum TrxR were located in the cytosol and in mitochondria (Kehr et al. 2010); thus, this could also be the case for the occurrence of TrxR in B. microti.
Using the same methods previously published for production of recombinant SmTGR and conditions for enzyme kinetics analysis (Regner et al. 2014), the experimental data in the present study showed that the recombinant BmiTrxR was active as NADPH-dependent disulfide reductase, using DTNB as substrate (Fig. 8) and it was also able to reduce both BmiTrx and the ortholog protein from E. coli (EcoTrx) (Fig. 9). However, it is worth to point that we constructed the recombinant BmiTrxR-pET28a plasmid to express TrxR fused to N-term and C-term His-tags to reach high purification degree. On the one hand, we easily detected the enzyme activity with substrate DTNB. Unexpectedly, the recombinant protein exhibited no activity with EcoTrx, even when different experimental conditions were tested, which leads us to revisit the protein primary. By the analysis of three-dimensional structure of BmiTrxR in complex with its physiologic substrate BmiTrx (unpublished), we found the binding site in the C-term of the recombinant enzyme was affected by the C-term His-tag. After removing the C-term His-tag and remaining the N-term His-tag, the enzyme finally showed disulfide reductase activity with EcoTrx.
This reductase activity exhibited a maximum at pH 6.5 in 100 mM phosphate buffer and 25 °C, which is different with the pH 8.0 reported in previous study (Regner et al. 2014). In DTNB reductase activity assay, the initial velocity of reaction of Rat TrxR is higher than BmiTrxR (Fig. 8), which supported that the selenocysteine-cysteine (SeCys-Cys) motif of mammalian TrxR is highly reactive when compared to the Cys-xxxx-Cys motif in BmiTrxR. In addition, Table 1 show that the K cat/K m with NADPH (2.2 × 105 M−1 · s−1) was about five times than that with DTNB (4.04 × 104 M−1 · s−1). V max and K cat with NADPH were 20.08 ± 3.27 μM · min−1, 6.69 s−1, respectively, which was approximately three times than that with EcoTrx (6.37 ± 0.80 μM · min−1, 2.12 s−1). These values are practically in the same order of magnitude and similar to those determined for H-TrxRs from other species (Regner et al. 2014). The findings may suggest that NADPH, combined with BmiTrxR via FAD domain, tends to deliver electrons to the disulfide substrate DTNB and Trx. In P. falciparum, the electrons are shuttled from NADPH via FAD to the N-terminal active site. Subsequently, the electrons are transferred to the C-terminal redox motif that finally reduces the substrate Trx (Gilberger et al. 1997). Further study on the direction of electron transfer of BmiTrxR will be surveyed.
Interfering with redox metabolism represents a promising approach to antiparasitic drug development (Krauth-Siegel et al. 2005) prominent examples being P. falciparum thioredoxin reductase (Buchholz et al. 2010) and Schistosoma mansoni thioredoxin-glutathione reductase (Kuntz et al. 2007). Also Babesia TrxR was considered a leading drug target because (i) complete structural information, which permits computational inhibitor modeling (Sarma et al. 2003), (ii) some drugs have been proved to inhibit the TrxR of apicomplexan protozoa and mammal (Andricopulo et al. 2006), (iii) structural differences between the selenoprotein H-TrxR and Babesia TrxR occurring at the solvent exposed C-terminal end of the proteins were considered most attractive sites for directed inhibitor development (McMillan et al. 2006; Rahlfs and Becker 2006). Auranofin, a gold-based compound, was found to inhibit P. falciparum growth in vitro (Sannella et al. 2008). Auranofin-mediated inhibition of TrxR activity was competitive, which has a benzene ring structure (Angelucci et al. 2009) and is similar to DTNB with two benzene rings connected by a disulfide bond (Spiga et al. 2011). Our data also suggest that auranofin can inhibit the disulfide reductase activity of recombinant BmiTrxR in a low inhibition concentration (Fig. 10), which is similar to the findings in E. granulosus (Agorio et al. 2003) and S. mansoni (Angelucci et al., 2009). As mentioned above, auranofin could be a potential inhibitor of the native protein BmiTrxR in the parasite, which may be used as an anti-B. microti drug.
To our knowledge, this is the first report on the analysis, identification, molecular cloning, and functional characterization of the TrxR from B. microti. Sequences alignment and phylogenetic analysis shows that BmiTrxR shares 55 % sequence identity and most closely relationship with homologous sequences from T. annulata, suggesting these proteins may possess common ancestry and function. The recombinant enzyme exhibited NADPH-dependent disulfide reductase activity with the model DTNB and classic Trx, indicating that the enzyme may play a certain role in maintaining the redox balance in B. microti. These findings improve the knowledge of TrxR in B. microti and provide some useful reference for the further research.
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Acknowledgments
Funding for this study was provided by the “National Key Basic Research Program (973 program) of China” (Grant No. 2015CB150300).
Authors’ contributions
Prof. ZJL directed the project and participated in the design of the study and helped to draft the manuscript. ZSR performed the laboratory tests and the data analysis and wrote the manuscript. GHY and ZHS helped with various aspects of the experiments and interpretation of the data. ZYZ and CJ provided new analytical reagents and tools. All authors read and approved the final manuscript.
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The protocols were approved by the Institutional Animal Care and Use Committee of the Shanghai Veterinary Research Institute, and followed the misconduct policy of BMC Genomics, and authorized by the Animal Ethical Committee of Shanghai Veterinary Research Institute.
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Zhao, S., Gong, H., Zhou, Y. et al. Identification of a thioredoxin reductase from Babesia microti during mammalian infection. Parasitol Res 115, 3219–3227 (2016). https://doi.org/10.1007/s00436-016-5084-4
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DOI: https://doi.org/10.1007/s00436-016-5084-4