The MEP pathway in Babesia orientalis apicoplast, a potential target for anti-babesiosis drug development

Lan He,Guan Zhu,Pei He,Long Yu,Muxiao Li,Xueyan Zhan,Junlong Zhao,Xiaoying Luo,Jiaying Guo

doi:10.1186/s13071-018-3038-7

Abstract

BackgroundThe apicomplexan parasite Babesia orientalis, the causative agent of water buffalo babesiosis in China, is widespread in central and south China, resulting in a huge economic loss annually. Currently, there is no effective vaccine or drug against this disease. Babesia bovis and Plasmodium falciparum were reported to possess an apicoplast which contains the methylerythritol phosphate (MEP) pathway inhibitable by fosmidomycin, suggesting that the pathway could serve as a drug target for screening new drugs. However, it remains unknown in B. orientalis.MethodsPrimers were designed according to the seven MEP pathway genes of Babesia microti and Babesia bovis. The genes were cloned, sequenced and analyzed. The open reading frames (ORFs) of the first two enzyme genes, 1-deoxy-D-xylulose 5-phosphate synthase (BoDXS) and 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (BoDXR), were cloned into the pET-32a expression vector and expressed as a Trx-tag fusion protein. Rabbit anti-rBoDXS and rabbit anti-rBoDXR antibodies were generated. Western blot was performed to identify the native proteins of BoDXS and BoDXR in B. orientalis. Fosmidomycin and geranylgeraniol were used for inhibition assay and rescue assay, respectively, in the in vitro cultivation of B. orientalis.ResultsThe seven enzyme genes of the B. orientalis MEP pathway (DXS, DXR, IspD, IspE, IspF, IspG and IspH) were cloned and sequenced, with a full length of 2094, 1554, 1344, 1521, 654, 1932 and 1056 bp, respectively. BoDXS and BoDXR were expressed as Trx-tag fusion proteins, with a size of 95 and 67 kDa, respectively. Western blot identified a 77 kDa band for the native BoDXS and a 49 kDa band for the native BoDXR. The drug assay results showed that fosmidomycin could inhibit the growth of B. orientalis, and geranylgeraniol could reverse the effect of fosmidomycin.ConclusionsBabesia orientalis has the isoprenoid biosynthesis pathway, which could be a potential drug target for controlling and curing babesiosis. Considering the high price and instability of fosmidomycin, further studies should focus on the screening of stable and cheap drugs.

Highlights

The apicomplexan parasite Babesia orientalis, the causative agent of water buffalo babesiosis in China, is widespread in central and south China, resulting in a huge economic loss annually
The full lengths of D-xylulose 5-phosphate synthase (DXS), Dxylulose 5-phosphate reductoisomerase (DXR), 4diphosphocytidyl-2C-methyl-D-erythritol cytidylyltransferase (IspD), 4-diphosphocytidyl2C-methyl-D-erythritol kinase (IspE), IspF, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG) and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH) were 2094, 1554, 1344, 1521, 654, 1932 and 1056 bp, respectively (Fig. 1). These results indicated that all seven genes of the methylerythritol phosphate (MEP) pathway exist in the genome of B. orientalis
The nucleotide sequences of the DXS, DXR, IspD, IspE, IspF, IspG and IspH were submitted to the GenBank database under the accession numbers MH429606, MH429607, MH429608, MH448076, MH429609, MH429610 and MH429611, respectively

Summary

Introduction

The apicomplexan parasite Babesia orientalis, the causative agent of water buffalo babesiosis in China, is widespread in central and south China, resulting in a huge economic loss annually. Babesia bovis and Plasmodium falciparum were reported to possess an apicoplast which contains the methylerythritol phosphate (MEP) pathway inhibitable by fosmidomycin, suggesting that the pathway could serve as a drug target for screening new drugs. There are three pathways in the apicoplast of Plasmodium and Toxoplasma, including the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis, FAS pathway and heme operate. In B. bovis and Babesia microti, the isoprenoid biosynthesis MEP pathway is the only metabolic pathway in the apicoplast [2, 3]. Isoprenoids comprise a large group of diverse intracellular metabolites with multiple cellular functions, including roles in membrane structure, cellular respiration and cell signaling [4]

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Conclusion