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

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.

Construction and Use of Transposon MycoTetOP2 for Isolation of Conditional Mycobacteria Mutants

“…There is renewed interest in PG biosynthesis as a target for anti-TB therapies as multidrug resistance becomes a serious threat (Catalao et al, 2019). In addition, GcpE/IspG (locus II) constitutes part of the methylerythritol phosphate (MEP) pathway which is considered promising targets for the development of antimycobacterial and antiparasitic agents (He et al, 2018;Wang and Dowd, 2018). The findings of these critical genes in this study illustrate the usefulness of our newly constructed transposon in the studies of mycobacteria.…”

N-Glycosylation in Piroplasmids: Diversity within Simplicity

“…The MEP pathway is a seven-step process that starts with pyruvate and ends with the synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), and, in apicomplexans, takes place in the apicoplast [ 25 ]. All MEP pathway enzymes were shown to be encoded in the genomes of B. bovis , B. microti , and B. orientalis , and expression of two of them (BoDXS and BoDXR) was experimentally demonstrated in merozoites (mz) of the latter [ 26 , 27 ]. The MEP pathway is absent in mammals, and inhibitors of this route hamper the in vitro growth of Babesia spp.…”

“…The MEP pathway is absent in mammals, and inhibitors of this route hamper the in vitro growth of Babesia spp. mz, making it an attractive therapeutic target against piroplasmids [ 26 , 27 , 28 ].…”

Erythrocyte Adhesion of Merozoite Surface Antigen 2c1 Expressed During Extracellular Stages of Babesia orientalis

“…Duplicate blots were incubated with B. orientalis-positive serum (1:500 in PBS, 0.1% Tween 20) or 1:500-diluted normal water buffalo serum (negative control) overnight at 4°C. Both sera were collected during previous animal experiment and stored in −20°C in our lab (23). The membranes were then subjected to probing using 1:2000-diluted secondary antibodies (BovIgG/HRP, Bioss Inc., Woburn, MA, USA) at 37°C for 30 min.…”