Structural characterization of chia seed polysaccharides and evaluation of its immunomodulatory and antioxidant activities.

This study aims to extract an active heteropolysaccharide Chia seed polysaccharide (CSP-A) and further purified by DEAE Sepharose Fast Flow and Sepharose CL-6B chromatographic column, characterize its structure, and evaluate its antioxidant and immunomodulatory activities. Structural analysis revealed that CSP-A was composed of d-mannose, d-glucuronic acid and d-xylose in a molar ratio of 1:3:4 with molecular weight of 1.688 × 105 Da, owning 4 sugar residues of ß-d-Manp-(1→, →4)-α-d-GlcpA-(1→, →2,4)-ß-d-Xylp-(1→, and â†’ 4)-ß-d-Manp-(1 â†’. Congo red assay and microscopic characteristics showed that CSP-A in its solution may possess a helical conformation. In vitro experiments showed that CSP-A had moderate DPPH· and OH· scavenging activities. CSP-A also enhanced the phagocytosis ability of RAW 264.7 cells and prompted the release of NO, TNF-α, IL-6 and IL-1ß from RAW 264.7 cells, which indicated CSP-A had immune regulation effect. This experiment provides scientific basis for further utilization and development of chia seeds, a kind of functional food.

Kinetic Characterization and Inhibitor Screening of Pyruvate Kinase I From Babesia microti.

The apicomplexan Babesia microti is a main pathogenic parasite causing human babesiosis, which is one of the most widely distributed tick-borne diseases in humans. Pyruvate kinase (PYK) plays a central metabolic regulatory role in most living organisms and catalyzes the essentially irreversible step in glycolysis that converts phosphoenolpyruvate (PEP) to pyruvate. Hence, PYK is recognized as an attractive therapeutic target in cancer and human pathogens such as apicomplexans. In this study, we cloned, expressed, and purified B. microti PYK I (BmPYKI). Western blotting illustrated that anti-rBmPYKI antibody could specifically recognize the native BmPYKI protein in the lysate of B. microti with a 54-kDa band, which is consistent with the predicted size. In addition, the enzymatic activity of the purified recombinant PYKI (rPYKI) was tested under a range of pH values. The results showed that the maximum catalytic activity could be achieved at pH 7.0. The saturation curves for substrates demonstrated that the K m value for PEP was 0.655 ± 0.117 mM and that for ADP was 0.388 ± 0.087 mM. We further investigated the effect of 13 compounds on rBmPYKI. Kinetic analysis indicated that six inhibitors (tannic acid, shikonin, apigenin, PKM2 inhibitor, rosiglitazone, and pioglitazone) could significantly inhibit the catalytic activity of PYKI, among which tannic acid is the most efficient inhibitor with an IC50 value 0.49 µM. Besides, four inhibitors (tannic acid, apigenin, shikonin, and PKM2 inhibitor) could significantly decrease the growth of in vitro-cultured B. microti with IC50 values of 0.77, 2.10, 1.73, and 1.15 µM. Overall, the present study provides a theoretical basis for the design and development of new anti-Babesia drugs.

Identification and molecular characterization of a novel Babesia orientalis thrombospondin-related anonymous protein (BoTRAP1).

BACKGROUND: The thrombospondin-related anonymous protein (TRAP) family, a kind of transmembrane protein, is widely distributed with a conserved feature of structure in all apicomplexan parasites and plays a crucial role in the gliding motility and survival of parasites. METHODS: The Babesia orientalis TRAP1 gene (BoTRAP1) was truncated and cloned into a pET-42b expression vector and expressed as a GST-tag fusion protein with a TEV protease site. Rabbit anti-rBoTRAP1 antibody was produced and purified using a protein A chromatography column. Western blot analysis was performed to identify the native protein of BoTRAP1 and differentiate B. orientalis-infected positive from negative serum samples. The localization of BoTRAP1 on merozoites was identified by the indirect florescent antibody test (IFAT). RESULTS: The partial sequence of the TRAP1 gene was cloned from B. orientalis cDNA and identified to contain a von Willebrand factor A (vWFA) region and a thrombospondin type-1 (TSP-1) domain; it had a length of 762 bp, encoding a polypeptide of 254 amino acid residues with a predicted size of 28.2 kDa. The partial sequence was cloned into a pET-42b expression vector and expressed in E. coli as a GST fusion protein. Western blot indicated that rBoTRAP1 has a high immunogenicity and can differentiate B. orientalis-infected positive and negative serum samples collected from water buffaloes. IFAT showed that BoTRAP1 is mainly localized on the apical end of intracellular parasites by using polyclonal antibodies (PcAb) against rBoTRAP1. Meanwhile, the PcAb test also identified the native BoTRAP1 as a ~65 kDa band from B. orientalis lysates. The predicted 3D structure of BoTRAP1 contains a metalion-dependent adhesion site (MIDAS), which could be important for interaction with ligand on the surface of the host cells. CONCLUSIONS: Like all known protozoa, B. orientalis has a TRAP family, comprising TRAP1, TRAP2, TRAP3 and TRAP4. The newly identified and characterized BoTRAP1 may play a key role in the invasion of B. orientalis into water buffalo erythrocytes.

Characterization of a novel secretory spherical body protein in Babesia orientalis and Babesia orientalis-infected erythrocytes.

BACKGROUND: The spherical body, a membrane bound organelle localized in the apical organelle complex, is unique to Babesia and Theileria spp. The spherical body proteins (SBPs) secreted by spherical bodies include SBP1, SBP2, SBP3 and SBP4. Up to now, only SBP3 has been characterized in Babesia orientalis. METHODS: The BoSBP4 gene was amplified from cDNA and gDNA and cloned into the pGEX-6P-1 vector by homologous recombination, sequenced and analyzed by bioinformatics tools. The amino acid (aa) sequence of BoSBP4 was compared with that of Babesia bovis and Babesia bigemina as well as SBP3 of B. orientalis. The immunoreactivity was evaluated by incubating recombinant BoSBP4 (rBoSBP4) with the serum of B. orientalis-infected water buffalo. The native form of BoSBP4 was identified by incubating lysate of B. orientalis-infected water buffalo erythrocytes with the anti-rBoSBP4 mouse serum. The cellular localization of BoSBP4 was determined by indirect immunofluorescence assay. RESULTS: The full length of the BoSBP4 gene was estimated to be 945 bp without introns, encoding a 314 aa polypeptide with a predicted molecular weight of 37 kDa. The truncated recombinant protein was expressed from 70 to 945 bp as a GST fusion protein with a practical molecular weight of 70 kDa. BoSBP4 shared a 40% and 30% identity with B. bovis and B. bigemina, respectively. Furthermore, it was 31% identical to SBP3 of B. orientalis. BoSBP4 was identified in the lysate of B. orientalis-infected water buffalo erythrocytes with a molecular weight of 37 kDa, corresponding to the expected molecular mass of BoSBP4. The result of rBoSBP4 with positive serum revealed that BoSBP4 can elicit an immune response to B. orientalis-infected water buffalo. The cellular localization of BoSBP4 was detected to be adjacent to the merozoite nucleus in the intracellular phase, followed by the diffusion of the fluorescence of BoSBP4 into the cytoplasm of B. orientalis-infected erythrocytes as puncta-like specks and a gradual increase of the fluorescence. CONCLUSIONS: In this study, SBP4 in B. orientalis was characterized for the first time. It may play a key role in interaction with the host cell by being secreted into the cytoplasm of the B. orientalis-infected erythrocytes to facilitate parasite growth and reproduction.

A novel Babesia orientalis 135-kilodalton spherical body protein like: identification of its secretion into cytoplasm of infected erythrocytes.

BACKGROUND: The spherical body is a distinct organelle only existing in Babesia and Theileria. Spherical body proteins (SBPs) are secreted from spherical bodies and incorporated into the cytoplasm of infected erythrocytes during invasion and post-invasion stages. Four different SBP homologues (SBP1, SBP2, SBP3 and SBP4) have been identified in Babesia bovis and Babesia bigemina. So far, there has been no report available about the identification of SBPs in Babesia orientalis. METHODS: The SBP3-like in B. orientalis (BoSBP3-like) was cloned, sequenced, characterized and compared to the SBP3 sequences of B. bovis and B. bigemina by bioinformatics analyses. The BoSBP3-like gene was truncated into three fragments: BoSBP3-like-1 (915 bp), BoSBP3-like-2 (1311 bp) and BoSBP3-like-3 (1011 bp), which were amplified and cloned into the expression vector pET-28a and expressed as three truncated recombinant (His-fusion) proteins. The immunogenicity, native forms and localization of BoSBP3-like were identified by western blot and indirect immunofluorescence assay (IFA). RESULTS: The BoSBP3-like gene was intronless with an open reading frame (ORF) of 3237 bp, encoded a 1079 amino acid polypeptide with a predicted size of 135 kDa, and contained a cysteine-rich region, three dispersing FAINT domains and a signal peptide (1-16 aa) at the N-terminus. The amino acid sequence of BoSBP3-like was 61.6 and 35.0% identical to that of B. bovis and B. bigemina, respectively. BoSBP3-like was identified as 135 kDa in the parasite lysate by rabbit antiserum against the truncated recombinant BoSBP3-like-1 (rBoSBP3-like-1). Three specific bands corresponding to rBoSBP3-like-1 (1-305 aa, 43 kDa), rBoSBP3-like-2 (306-742 aa, 58 kDa) and rBoSBP3-like-3 (743-1079 aa, 52 kDa) were detected by reaction with serum from B. orientalis-infected buffalo. The BoSBP3-like was not only localized in the spherical body of B. orientalis but also in the cytoplasm of infected erythrocytes of buffalo as puncta-like protein specks at both single and paired parasite development stages. CONCLUSIONS: Through secretion into the cytoplasm of infected erythrocytes, BoSBP3-like may play a significant role in adaptation, interaction, and modification related to the host environment to benefit the growth and survival of Babesia. BoSBP3-like could react with the serum from B. orientalis-infected buffalo, but not healthy buffalo, implicating that BoSBP3-like is highly antigenic and may serve as a candidate diagnostic antigen for the detection of B. orientalis.