Recombinase polymerase amplification with lateral flow strip for detecting Babesia microti infections.

Babesia microti is one of the most important pathogens causing humans and rodents babesiosis-an emerging tick-borne disease that occurs worldwide. At present, the gold standard for the detection of Babesia is the microscopic examination of blood smears, but this diagnostic test has several limitations. The recombinase polymerase amplification with lateral flow (LF-RPA) assay targeting the mitochondrial cytochrome oxidase subunit I (cox I) gene of B. microti was developed in this study. The LF-RPA can be performed within 10-30 min, at a wide range of temperatures between 25 and 45 °C, which is much faster and easier to perform than conventional PCR. The results showed that the LF-RAP can detect 0.25 parasites/µl blood, which is 40 times more sensitive than the conventional PCR based on the V4 variable region of 18S rRNA. Specificity assay showed no cross-reactions with DNAs of related apicomplexan parasites and their host. The applicability of the LF-RPA method was further evaluated using two clinical human samples and six experimental mice samples, with seven samples were positively detected, while only three of them were defined as positive by conventional PCR. These results present the developed LF-RPA as a new simple, specific, sensitive, rapid and convenient method for diagnosing infection with B. microti. This novel assay was the potential to be used in field applications and large-scale sample screening.

Evaluation of enzymatic activity of Babesia microti thioredoxin reductase (Bmi TrxR)-mutants and screening of its potential inhibitors.

Babesia microti is a zoonotic pathogen that mainly parasitizes mammalian erythrocytes. Oxidative stress can induce gene mutation, protein denaturation and lipid peroxidation, such as reactive oxygen species (ROS) induced by hypoxic environment and the host immune system. An antioxidase, B. microti thioredoxin reductase (Bmi TrxR), has been identified in B. microti. We used a combination of homology modeling and domain prediction to explore the functional sites of Bmi TrxR and found that TrxR has three domains. Constructed a mutant pool which His-tag were at the N-terminus (TrxR-Nhis, C105-Nhis, C110-Nhis, C105110-Nhis, C547-Nhis, C552-Nhis, C547552-Nhis) and the His tag were at the N- and C-terminus (TrxR-NChis, C547-NChis, C552-NChis, C547552-NChis). The proteins were expressed as His-tagged fusion proteins in Escherichia coli. The His-tag of TrxR C-terminus were affected the reaction with Trx. The inhibitory efficiency of DNCB was decreased for mutant C547, compared with recombinant TrxR, indicating that the action site of DNCB might be cysteine at position 547. These results indicate that the N-terminal active site of Bmi TrxR plays an important role in accepting electrons and promotes electron transfer. The C-terminus His tag of Bmi TrxR affected the electron transfer and the reducing activity of Bmi TrxR. Reduce reactive oxygen produced in oxidative stress was reduced by Bmi TrxR, which is beneficial to Babesia survival. Therefore, reduction site of TrxR may become a potential target for Babesia microti treatment.

A cysteine protease of Babesia microti and its interaction with tick cystatins.

Babesiosis is a tick-borne protozoonosis caused by Babesia, which can cause fever, hemolytic anemia, hemoglobinuria, and even death. Babesia microti is a parasite found in rodents and can be pathogenic to humans. In this study, the full-length cDNA of a B. microti cysteine protease (BmCYP) was expressed and the recombinant rBmCYP protein analyzed and characterized. BmCYP is encoded by an ORF of 1.3 kb, with a predicted molecular weight of 50 kDa and a theoretical pI of 8.5. The amino acid sequence of BmCYP exhibits an identity of 32.9 to 35.2% with cysteine proteases of Babesia ovis, Babesia bovis, and Theileria, respectively. The results of the proteinase assays show that rBmCYP has cysteine protease enzymatic activity. In addition, we demonstrate that tick cystatins rRhcyst-1 and rRhcyst-2 were able to effectively inhibit the activity of rBmCYP; the inhibition rates were 57.2% and 30.9%, respectively. Tick cystatins Rhcyst-1 and Rhcyst-2 were differentially expressed in ticks that fed on Babesia-infected mice relative to non-infected control ticks. Our results suggest that BmCYP is a functional enzyme with cysteine protease enzymatic activity and may be involved in tick-B. microti interactions.

Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis.

The tick- and transfusion-transmitted human pathogen Babesia microti infects host erythrocytes to cause the pathologic symptoms associated with human babesiosis, an emerging disease with worldwide distribution and potentially fatal clinical outcome. Drugs currently recommended for the treatment of babesiosis are associated with a high failure rate and significant adverse events, highlighting the urgent need for more-effective and safer babesiosis therapies. Unlike other apicomplexan parasites, B. microti lacks a canonical lactate dehydrogenase (LDH) but instead expresses a unique enzyme, B. microti LDH (BmLDH), acquired through evolution by horizontal transfer from a mammalian host. Here, we report the crystal structures of BmLDH in apo state and ternary complex (enzyme-NADH-oxamate) solved at 2.79 and 1.89 Å. Analysis of these structures reveals that upon binding to the coenzyme and substrate, the active pocket of BmLDH undergoes a major conformational change from an opened and disordered to a closed and stabilized state. Biochemical assays using wild-type and mutant B. microti and human LDHs identified Arg99 as a critical residue for the catalytic activity of BmLDH but not its human counterpart. Interestingly, mutation of Arg99 to Ala had no impact on the overall structure and affinity of BmLDH to NADH but dramatically altered the closure of the enzyme's active pocket. Together, these structural and biochemical data highlight significant differences between B. microti and human LDH enzymes and suggest that BmLDH could be a suitable target for the development of selective antibabesial inhibitors.-Yu, L., Shen, Z., Liu, Q., Zhan, X., Luo, X., An, X., Sun, Y., Li, M., Wang, S., Nie, Z., Ao, Y., Zhao, Y., Peng, G., Ben Mamoun, C., He, L., Zhao, J. Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis.

Molecular characterization of Babesia microti thioredoxin (BmTrx2) and its expression patterns induced by antiprotozoal drugs.

BACKGROUND: Human babesiosis is an infectious disease that is epidemic in various regions all over the world. The predominant causative pathogen of this disease is the intra-erythrocytic parasite Babesia microti. The thioredoxin system is one of the major weapons that is used in the resistance to the reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host immune system. In other intra-erythrocytic apicomplexans like the malaria parasite Plasmodium falciparum, anti-oxidative proteins are promising targets for the development of anti-parasitic drugs. However, to date, the sequences and biological properties of thioredoxins and thioredoxin-like molecules of B. microti remain unknown. Understanding the molecular characterization and function of B. microti thioredoxins may help to develop anti-Babesia drugs and controlling babesiosis. METHODS: The full-length B. microti thioredoxin 2 (BmTrx2) gene was obtained using a rapid amplification of cDNA ends (RACE) method, and the deduced BmTrx2 amino acid sequence was analyzed using regular bioinformatics tools. Recombinant BmTrx2 protein was expressed in vitro and purified using His-tag protein affinity chromatography resins. Reverse transcription PCR, quantitative real-time PCR and Western blot were employed to detect the expression and native proteins of BmTrx2. Indirect immunofluorescence assay was used to localize BmTrx2 in B. microti. Bovine insulin reduction assays were used to determine the enzyme activity of the purified recombinant BmTrx2 protein. RESULTS: The full-length BmTrx2 was 564 bp with a 408 bp open reading frame encoding a protein of 135 amino acids. The predicted molecular weight of the protein was 15.5 kDa. A conserved thioredoxin-like family domain was found in BmTrx2. The expression of BmTrx2 was upregulated on both the third and eighth day post-infection in mice, whereas expression was downregulated during the beginning and later stages. The results of Western blot analysis showed the native BmTrx2 in parasite lysates could be detected by mouse anti-BmTrx2 serum and that the recombinant BmTrx2 protein could be recognized by serum of B. microti-infected mice. Immunofluorescence microscopy showed that BmTrx2 localized in the cell cytoplasm of B. microti merozoites in B. microti-infected red blood cells. The results of bovine insulin reduction assay indicated the purified recombinant BmTrx2 protein possesses antioxidant enzyme activity. Dihydroartemisinin and quinine, known anti-malaria drugs, and clindamycin, a known anti-babesiosis drug, induced significantly higher upregulation of BmTrx2 mRNA. CONCLUSIONS: Our results indicate that BmTrx2 is a functional enzyme with antioxidant activity and may be involved in the response of B. microti to anti-parasite drugs.

Identification and functional study of a novel 2-cys peroxiredoxin (BmTPx-1) of Babesia microti.

Babesia microti is an emerging human pathogen and the primary causative agent of human babesiosis in many regions of the world. Although the peroxiredoxins (Prxs) or thioredoxin peroxidases (TPx) enzymes of this parasite have been sequenced and annotated, their biological properties remain largely unknown. Prxs are a family of antioxidant enzymes that protect biological molecules against metabolically produced reactive oxygen species (ROS) and reduce hydrogen peroxide (H2O2) to water in both eukaryotes and prokaryotes. In this study, TPx-1 cDNA was cloned from B. microti (designated BmTPx-1). Recombinant BmTPx-1 (rBmTPx-1) was expressed in Escherichia coli as a histidine fusion protein and purified using Ni-NTA His bind resin. To test the defense capacity of enzymatic antioxidants against the effect of ROS, a mixed-function oxidation system was utilized with the recombinant BmTPx-1 protein. A decreased ability of rBmTPx-1 to donate electrons to the thioredoxin (Trx)/TrxR reductase system was clarified by reaction with H2O2. These results suggest that BmTPx-1 has a great impact on protecting parasites from oxidative stress in the erythrocytic stage.

Identification of a thioredoxin reductase from Babesia microti during mammalian infection.

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.

Inhibitory effects of pepstatin A and mefloquine on the growth of Babesia parasites.

We evaluated the inhibitory effects of pepstatin A and mefloquine on the in vitro and in vivo growths of Babesia parasites. The in vitro growth of Babesia bovis, B. bigemina, B. caballi, and B. equi was significantly inhibited (P < 0.05) by micromolar concentrations of pepstatin A (50% inhibitory concentrations = 38.5, 36.5, 17.6, and 18.1 µM, respectively) and mefloquine (50% inhibitory concentrations = 59.7, 56.7, 20.7, and 4 µM, respectively). Furthermore, both reagents either alone at a concentration of 5 mg/kg or in combinations (2.5/2.5 and 5/5 mg/kg) for 10 days significantly inhibited the in vivo growth of B. microti in mice. Mefloquine treatment was highly effective and the combination treatments were less effective than other treatments. Therefore, mefloquine may antagonize the actions of pepstatin A against babesiosis and aspartic proteases may play an important role in the asexual growth cycle of Babesia parasites.