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Complete Molecular and Immunoprotective Characterization of Babesia microti Enolase.

ABSTRACT: The apicomplexan Babesia microti is the primary causative agent of human babesiosis, one of the most broadly distributed tick-borne diseases worldwide. B. microti undergoes a complex lifecycle within both the mammalian host and the tick vector, and employs several different specific molecular mechanisms to enter host cells. Enolase, the key glycolytic enzyme in intracellular glucose metabolism, can also be expressed on the parasite's outer surface, binds to human plasminogen, and coordinates apicomplexan parasite invasion of host cells, however, it lacks sorting sequences or lipoprotein anchor sites. In the present study, we isolated the coding gene of B. microti enolase (BmEno), expressed it within E. coli and purified the recombinant BmEno protein (rBmEno). Consequently, we confirmed cytoplasmic and surface localization of BmEno via immunofluorescence, and demonstrated that rBmEno catalyzes the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. Moreover, our results showed that rBmEno binds to human plasminogen, and that the lysine analog ?-aminocaproic acid significantly inhibited this binding. Furthermore plasminogen bound to rBmEno converts to active plasmin. Additionally, actively immunizing mice with rBmEno could evoke a partial protective immunity against B. microti infection following challenge. In conclusion, B. microti enolase is a multifunctional cytoplasmic protein which is also expressed at the parasitic outer surface, facilitates binding to host plasminogen, and could partially protect hosts against parasite infection.

SUBMITTER: Liu X

PROVIDER: S-EPMC5387042 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Complete Molecular and Immunoprotective Characterization of Babesia microti Enolase.

Liu Xiangye X Zheng Chen C Gao Xiaoge X Chen Jiaxu J Zheng Kuiyang K

Frontiers in microbiology 20170411

The apicomplexan Babesia microti is the primary causative agent of human babesiosis, one of the most broadly distributed tick-borne diseases worldwide. B. microti undergoes a complex lifecycle within both the mammalian host and the tick vector, and employs several different specific molecular mechanisms to enter host cells. Enolase, the key glycolytic enzyme in intracellular glucose metabolism, can also be expressed on the parasite's outer surface, binds to human plasminogen, and c ...[more]

PMID: 28443086

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Project description:BackgroundHuman 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.MethodsThe 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.ResultsThe 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.ConclusionsOur 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.

Dataset Information

Complete Molecular and Immunoprotective Characterization of Babesia microti Enolase.

Publications

Complete Molecular and Immunoprotective Characterization of <i>Babesia microti</i> Enolase.

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