This study was carried out in strict accordance with the recommendations of the guidelines for the Care and Use of Laboratory Animals of the Laboratory Animal Ethics Committee of Xuzhou Medical University. The protocol was approved by the Laboratory Animal Ethics Committee of Xuzhou Medical University (Permit Number: 201547).

The B. microti strain (ATCC^® PRA-99^TM) was provided by the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Shanghai, China), and maintained in our laboratory. The NOD-SCID (non-obese diabetic/severe combined immunodeficiency) mice were used as reservoirs for preserving the parasites and the percentage of parasitized RBCs was determined via Giemsa-stained thin blood films. Female Balb/c mice (4–6 weeks old) were intraperitoneally inoculated with 100 μl infected blood, which was collected, heparinized, and diluted with sterile saline to achieve a 30% RBC parasitic infection rate. Parasites were isolated once 50–60% of RBCs were infected. All mice were purchased from NBRI (Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China) and maintained at the Animal Center of Xuzhou Medical University.

All reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified.

Babesia microti enolase nucleotide sequence BLAST searches against published genomic sequences of B. microti were performed with the NCBI nucleotide database search program (Cornillot et al., 2012). The enolase signature sequence and other domains were defined using the ScanProsite online software¹, and the MotifScan online software². Multiple-sequence alignments were built with the ClustalX program³.

Total RNA was isolated from B. microti-infected RBCs with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The gene fragment encoding the entire BmEno coding sequence was obtained using RT-PCR amplified with a One Step PrimeScript RT-PCR Kit (Takara, Dalian, China), and the primers: BmEnoF (5′-CAGGGATCCATGACAAAGATAATTAGCGC-3′) and BmEnoR (5′-CCGCTCGAGTTACTTGTTAAAAACTTTGC-3′). Primers contained BamHI and XhoI restriction sites (underlined) to assist subsequent cloning into the expression vector pGEX-4T-2 (TransGen Biotech, Beijing, China), containing a GST tag to facilitate purification. The resulting plasmid was transformed into Escherichia coli BL21 Star (DE3) competent cells.

For recombinant expression, E. coli cells were grown to 0.5 OD₆₀₀ at 37°C, and then induced overnight with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25°C. Cell cultures were harvested by centrifugation and the resulting cell pellet was resuspended in sonication buffer (1 × PBS, 0.1% TritonX-100, 1 mg/mL lysosome, 5 mM DTT, 0.5 mM PMSF, pH 7.4) and lysed on ice with a sonicator. Debris was then pelleted by centrifugation, and the supernatant (containing recombinant BmEno, rBmEno) was purified by an affinity chromatography system on a GSTrap 4B column following the manufacturer’s instructions (GE Healthcare, Pittsburgh, PA, USA). Thrombin was used to cut the GST tag from the purified protein, which was then analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by Coomassie brilliant blue staining. The purified protein was quantitated, divided into aliquots, and stored at -80°C until used.

To determine whether anti-B. microti antibody recognizes rBmEno, Western blot was performed. In brief, 2 μg of purified rBmEno were separated by SDS–PAGE, and then transferred to activated polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with Tris-buffered saline-Tween 20 (TBST; 20 mM Tris-base, 150 mM NaCl, 0.05% (v/v) Tween 20, pH 7.5) containing 5% (w/v) dry non-fat milk powder for 3 h at room temperature. The membrane was then either incubated with mouse anti-B. microti serum or normal mouse serum (1:500 dilution) overnight at 4°C. Following three washes in TBST, the membrane was incubated with enhanced chemiluminescence (ECL)-goat alkaline phosphatase (AP)-conjugated anti-mouse IgG (1:2000 dilution) for 2 h at room temperature. Following additional washings, the membrane was stained with ECL substrate solution and reactions were detected by exposure to X-ray film.

To generate polyclonal antibodies against rBmEno, five female Balb/c mice (4–6 weeks old) were immunized three times at two-weekly intervals by subcutaneous injection with purified rBmEno protein (50 μg/mouse), mixed with MONTANIDE^TM ISA 70 VG adjuvant (Seppic, Puteaux, France). One week after the last immunization, mouse blood samples were isolated to generate anti-serum by centrifuging. Antibody titer was measured using an indirect enzyme-linked immunosorbent assay (ELISA), where each well of a 96-well plate was coated with 2 μg rBmEno diluted in 100 μl coating buffer (1.5 g/L Na₂CO₃, 2.93 g/L NaHCO₃, pH 9.6). Serum aliquots were then stored at -20°C until use.

To determine whether anti-rBmEno antibody recognizes native BmEno, immunoprecipitation was performed. Briefly, 2 mg of B. microti-infected or normal mouse RBC lysates were mixed with 10 μl anti-rBmEno antibody overnight at 4°C. Then 30 μl of a 1:2 A/G-Sepharose protein slurry was added for 2 h at 4°C. After three washes with PBS, the precipitate was mixed with SDS–PAGE sample buffer and boiled. Western blot was performed as described above.

Babesia microti-infected RBCs were smeared on coverslips, and fixed with 100% methanol for 30 min at -20°C. Samples were then permeabilized with PBS containing 0.1% (v/v) Triton X-100 for 15 min at room temperature. Following three washes with PBS, the samples were blocked in BSA-PBS containing 5% (w/v) BSA for 30 min at room temperature. Samples were then incubated overnight with polyclonal mouse anti-rBmEno serum (1:20 dilution) at room temperature. After three washes, samples were incubated with FITC-conjugated goat anti-mouse IgG (1:50 dilution) for 1 h at room temperature. After three washes, coverslips were briefly swirled in distilled water, mounted with 6-diamidino-2-phenylindole (DAPI), and then sealed with nail polish. Finally, images were acquired and analyzed using a ZEISS LSM 880 confocal microscope system (Zeiss, Munich, Germany).

The enzymatic activity of purified rBmEno was determined by direct monitoring of absorbance enhancement at 240 nm and 30°C using a Synergy HT spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA) as previously described (Figueiredo et al., 2015). Briefly, the assay was performed in a 30°C preheated reaction buffer (100 mM HEPES buffer, 7.7 mM KCl, 10 mM MgSO4, pH 7.0) with 1 mM 2-PGA and different concentrations of purified rBmEno (2.5, 5, 10, and 20 ng/μl). The total reaction volume was 200 μl. Continuous readings were performed every 3 min for a total of 66 min. BSA was used as a negative control, and reactions were performed in triplicate.

The Michaelis–Menten constant (K_m) for the glycolysis substrate 2-PGA was determined from initial reaction measurements at concentrations ranging from 1 to 10 mM. Reactions were initiated by the addition of purified rBmEno at 1 μg/reaction. K_m values were calculated using computerized non-linear regression analysis of the data fitted to the Michaelis–Menten equation using Graphpad Prism 5.0 (GraphPad Software, Inc., USA).

To determine the effects of pH and temperature on rBmEno activation, rBmEno activity was determined using a reaction buffer system covering a pH range of 2.0–12.0, and a temperature range of 4–90°C. Reactions were initiated by the addition of rBmEno (1 μg/reaction) diluted in the corresponding buffer. Continuous readings were carried out every 3 min for 66 min, and reactions were performed in triplicate.

Recombinant BmEno binding to plasminogen (PLG) was assayed by ELISA as previously described (Nogueira et al., 2012). In brief, each well of a 96-well plate was coated overnight at 4°C with 4 μg/well human PLG diluted in 100 μl PBS. Non-specific binding sites were blocked with 2% BSA in PBS for 2 h at 37°C. After three washes with PBST containing 0.05% (V/V) Tween 20, wells were incubated with 4 μg/well rBmEno for 2 h at 37°C; to analyze the role of lysine residues in enolase and PLG binding activity, 4 μg/well of rBmEno were added in the presence or absence of 0–100 mM ε-aminocaproic acid (ε-ACA, lysine analog). After three washes, wells were incubated with polyclonal mouse anti-rBmEno serum (1:200 dilution) for 1 h at 37°C. After another three washes, all wells were then incubated with goat AP-conjugated anti-mouse IgG (1:10,000 dilution) for 1 h at 37°C. Finally, tetramethylbenzidine substrate (TMB) was added, and reactions were stopped by the addition of 100 μl/well of 2 N H₂SO₄. Absorbance was read at 450 nm using a Bio-Tek plate reader (Bio-Tek Instruments, Winooski, VT, USA). BSA was used as a negative control, and each assay was performed in triplicate.

The assay was performed in 96-well plates as previously described (Floden et al., 2011). Briefly, each well was coated overnight with 4 μg rBmEno in 100 μl PBS at 4°C. After being blocked and washed as above, 4 μg/well of human PLG was added and incubated for 2 h at 37°C. Following three washes, 4 ng/well of human urokinase plasminogen activator (uPA) was added, and then the plasmin-specific substrate D-valyl-leucyl-lysine-p-nitroanilide dihydrochloride was added at a final concentration of 0.3 mM in PBS. Plates were incubated overnight at 37°C, and absorbance was read at 405 nm using a Bio-Tek plate reader. BSA was used as a negative control, and the assay was performed in triplicate.

Female Balb/c mice (4–6 weeks old) were immunized with adjuvant containing purified rBmEno protein (20 μg/mouse) as described above, and control group mice were vaccinated with PBS in adjuvant. Two weeks after the third boost, mice were intraperitoneally inoculated with B. microti as described above. Blood smears were performed every 2 days to detect the percentage of infected RBCs by visual examination and counting using a microscope. Mice were sacrificed 8 days after infection. Blood samples were collected and genomic DNA was isolated with the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). B. microti burden in mouse blood was evaluated via real time quantitative RT-PCR (qRT-PCR) with the isolated DNA.

qPCR was performed using the SYBR^® Premix Ex Taq Kit (Takara, Dalian, China) and run on the Roche LightCycler^® 480 System following manufacturer’s instructions. B. microti was quantified using primers targeting the small-subunit rRNA gene and qPCR results were analyzed followed as previously described (Bloch et al., 2013). qPCRs were performed in triplicate.

Statistical analysis of enzyme activity, plasminogen binding, and activation of rBmEno was performed using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests; statistical analysis of qPCR was performed using two-tailed Student’s t-tests. The values were presented as mean ± standard deviation (SD) and considered significant at p < 0.05, data analysis was performed with Graphpad Prism 5.0.

An online search for B. microti genomic sequences identified one full-length cDNA (accession number: XM_012794345) comprising 1317 nucleotides encoding a 438 amino acid protein. This protein was predicted to be a soluble 47.5 kDa polypeptide with a pI of 5.98, displaying an enolase signature motif spanning residues 349-362 (L³⁴⁹-S³⁶²). With subsequent protein sequence analysis, the 2-PGA binding residues of BmEno, specifically Ala⁴⁰, His¹⁶⁵, Gln¹⁷³, Lys³⁵², His³⁸⁰, Arg³⁸¹, Ser³⁸², and Lys⁴⁰³ were conserved, and both Glu¹⁷⁴ and Glu²¹⁷ shared a proton during dehydration. In addition, Asp²⁵², Glu³⁰⁰, and Asp³²⁷ were found to interact with an Mg²⁺ ion, and Ser⁴¹ bound the second Mg²⁺. Three catalytic loops of BmEno were predicted, loop1 (V³⁶∼R⁵⁷), loop2 (H¹⁶⁵∼Q¹⁷³), and loop3 (F²⁵⁸∼T²⁷⁹). BmEno includes pentapeptide (E¹⁰³W¹⁰⁴G¹⁰⁵F¹⁰⁶S¹⁰⁷) and dipeptide insertions (V²⁶³K²⁶⁴), which are commonly known as plant-like insertions. Two predicted plasminogen-binding motifs were identified at F²⁵⁸∼N²⁶⁷ and S²⁷³∼K²⁷⁸, suggesting that BmEno could bind plasminogen (Figure 1). The protein sequence also carried phosphorylation and N-glycosylation sites, but no trans-membrane domains or signal peptides.

Full-length enolase cDNA was amplified from the ATCC^® PRA-99^TM B. microti strain using specific primers, and then cloned in frame into the pGEX-4T-2 plasmid with a GST-tag domain as described in the “Materials and Methods.” rBmEno expression was induced in BL21 (DE3) E. coli with IPTG, and bacterial extracts were assessed by SDS–PAGE with Coomassie blue staining. Induced GST-tagged rBmEno protein migrated at approximately 73 kDa, with greater amounts observed in the supernatant than the sediment, whereas purified rBmEno produced a single 47.5 kDa band (Figure 2A).

To analyze whether anti-B. microti serum recognized purified recombinant BmEno, purified GST-tagged rBmEno was incubated with the mouse anti-B. microti serum. A strong band of approximately 73 kDa was observed in ECL-Western blot analysis (Figure 2B). Moreover, to determine whether anti-rBmEno antibody recognizes native BmEno, immunoprecipitation was performed, and native BmEno was immunoprecipitated with anti-rBmEno antibody (Figure 2C).

The localization of BmEno in intraerythrocytic B. microti parasites was determined by immunofluorescence analysis using a mouse anti-rBmEno antibody. Following incubation with the anti-rBmEno antibody, intraerythrocytic parasites were counterstained with an FITC-conjugated anti-mouse IgG and nuclei were stained with DAPI. In Figure 3, parasitic nuclei stained blue with DAPI and specific green anti-rBmEno antibody reactivity was also observed. BmEno appeared to localize both in the cytoplasm and on the surface of B. microti (based on the observed parasite shapes).

The classical enzymatic activity of purified rBmEno was tested via its ability to convert 2-PGA to PEP. The catalytic activity of rBmEno increased with increasing concentrations from 2.5 to 20 ng/μl (Figure 4A). The kinetics of the conversion reaction revealed an 8.849 mM Michaelis constant (K_m) and a maximum velocity (V_max) of 2.907 μmol/L/min (Figure 4B). rBmEno enzyme activity was measured over a range of pH values, and the enzyme’s optimal pH was determined to fall within a neutral range, with maximal activity at pH 7.0 (Figure 4C). rBmEno enzyme activity was also assessed at varying temperatures and was found to have maximal activity at 37°C (Figure 4D).

To investigate rBmEno’s ability to bind human PLG, microtiter plates were coated with human PLG and BSA (negative control), and ELISA binding assays were performed. rBmEno was found to bind human PLG compared to BSA (p < 0.0001) (Figure 5A). To identify whether lysine residues play a role in enolase and PLG binding, the lysine analog ε-ACA was used as a binding competitor. ε-ACA significantly reduced the interaction between rBmEno and PLG (p < 0.0001) (Figure 5B). Enolase has been reported to enhance plasminogen to plasmin activation, thus to determine whether rBmEno also has this ability, microtiter plates were coated with rBmEno, blocked, incubated with PLG and uPA, and proteolytic activity measured using a plasmin-specific chromogenic substrate. rBmEno dramatically enhanced plasmin activation in the presence of uPA compared to the negative control BSA (p < 0.0001) (Figure 5C).

We have demonstrated that BmEno is expressed at the surface of B. microti parasites, thus we hypothesized that immunization of mice with rBmEno could elicit protective immunity and influence any subsequent B. microti infection. Therefore Balb/c mice (four animals/group) were immunized with purified rBmEno, while control mice were injected with PBS mixed with similar volume of adjuvant. Two weeks after the final immunization, mice were intraperitoneally inoculated with B. microti. Blood smears performed every 2 days after infection demonstrated that the percentage of B. microti-infected RBCs from protein-immunized mice was significantly lower than that from control mice after 4 days following parasite (p < 0.001) (Figure 6A). Additionally, qPCR showed that the parasitic blood burden was significantly decreased 1.5-fold in immunized mice after 8 days following parasite infection, than in controls (p < 0.05) (Figure 6B).