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The Structural Basis of Babesia orientalis Lactate Dehydrogenase.

ABSTRACT: Glycolytic enzymes play a crucial role in the anaerobic glycolysis of apicomplexan parasites for energy generation. Consequently, they are considered as potential targets for new drug development. Previous studies revealed that lactate dehydrogenase (LDH), a glycolytic enzyme, is a potential drug target in different parasites, such as Plasmodium, Toxoplasma, Cryptosporidium, and Piroplasma. Herein, in order to investigate the structural basis of LDH in Babesia spp., we determined the crystal structure of apo Babesia orientalis (Bo) LDH at 2.67-Å resolution in the space group P1. A five-peptide insertion appears in the active pocket loop of BoLDH to create a larger catalytic pocket, like other protozoa (except for Babesia microti LDH) and unlike its mammalian counterparts, and the absence of this extra insertion inactivates BoLDH. Without ligands, the apo BoLDH takes R-state (relaxed) with the active-site loop open. This feature is obviously different from that of allosteric LDHs in T-state (tense) with the active-site loop open. Compared with allosteric LDHs, the extra salt bridges and hydrogen bonds make the subunit interfaces of BoLDH more stable, and that results in the absence of T-state. Interestingly, BoLDH differs significantly from BmLDH, as it exhibits the ability to adapt quickly to the synthetic co-factor APAD⁺. In addition, the enzymatic activity of BoLDH was inhibited non-competitively by polyphenolic gossypol with a K _i value of 4.25 μM, indicating that BoLDH is sensitive to the inhibition of gossypol and possibly to its new derivative compounds. The current work provides the structural basis of BoLDH for the first time and suggests further investigation on the LDH structure of other Babesia spp. That knowledge would indeed facilitate the screening and designing of new LDH inhibitors to control the intracellular proliferation of Babesia spp.

SUBMITTER: Yu L

PROVIDER: S-EPMC8766848 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe 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.MethodsThe 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.ResultsThe 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.ConclusionsIn 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.

Project description:Babesia orientalis is an obligate intraerythrocytic protozoan parasite of the buffalo (Bubalus bubalis, Linnaeus, 1758) transmitted by the tick Rhipicephalus heamaphysaloides. It is the causative agent of water buffalo babesiosis, one of the most important pathogens of water buffalo in central and southern China. As a member of the phylum Apicomplexa, B. orientalis possesses a relatively independent and alga originated organelle the apicoplast. Apicoplasts in other apicomplexa parasites are involved in the biosynthesis of haem, fatty acids, iron-sulphur clusters and isoprenoids. Some of these metabolic pathways were shown to be essential for parasite survival, therefore can serve as potential drug targets.30 pairs of primers were designed based on the full genome sequence of B. orientalis (unpublished data) and by aligning reported apicoplast genomes of Babesia bovis and Theileria parva. Conventional PCRs was performed to obtain overlapped fragments to cover the whole apicoplast genome. Then the apicoplast genome of B.orientalis was sequenced, assembled and aligned with reported apicoplast genomes of B. bovis and T. parva. The obtained apicoplast genome was annotated by using Artemis and comparing with published apicomplexan apicoplast genomes. The SSU and LSU nucleotide sequences generated were used in a phylogenetic analysis using the maximum likelihood implemented in MAGE 6.0.We have obtained and analyzed the complete genome sequence of the B. orientalis apicoplast. It consisted of a 33.2 kb circular DNA (78.9 % A?+?T). The apicoplast genome unidirectionally encodes one large and one small subunit ribosomal RNAs, 24 tRNA genes, 4 DNA-dependent RNA polymerase beta subunits (rpoB, rpoC1, rpoC2a and rpoC2b), 17 ribosomal proteins, one EF-Tu elongation factor, 2 Clp protease chaperones, and 14 hypothetical proteins. In addition, it includes two copies of the clpC gene. The structure and organization of the B. orientalis apicoplast genome are most similar to those of the B. bovis apicoplast.This is the first report of the complete sequence of the B. orientalis apicoplast genome. This information should be useful in the development of safe and efficient treatment against buffalo babesiosis.

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The Structural Basis of Babesia orientalis Lactate Dehydrogenase.

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