Abstract
The infectious diseases caused by Vibrio species have posed a major challenge in the shrimp aquaculture industry. Owing to the increasing prevalence of multi-drug resistance in Vibrios, newer targets and antibiotics are urgently needed to combat these pathogens. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the first committed enzyme of the methyl erythritolphosphate (MEP) isoprenoid biosynthetic pathway, which is essential for bacteria growth but is absent in hosts. DXR inhibitor fosmidomycin could inhibit the growth of Vibrio vulnificus E1758, and this effect could be reversed by supplementation with isoprenoid precursor isopentenyl diphosphate and DXR product MEP. This finding suggests that DXR is critical for the growth of V. vulnificus and could serve as a potential target for the screening of anti-Vibrio agents. Gene encoding DXR was cloned from V. vulnificus E1758 and overexpressed in Escherichia coli. The recombinant enzyme (rVvDXR) was purified as a soluble protein, and the enzymatic properties and fosmidomycin sensitivity were characterized. Overexpressed rVvDxr can effectively overcome the growth inhibitory effects of fosmidomycin on E. coli, indicating its similar functions to E. coli DXR. A protein-based high throughput screening model was established to identify agents targeting rVvDXR. Methanolic fractions of ethyl acetate extracts from 53 fungal fermentations were subjected for the screening, and hits of interest were confirmed by subsequent enzyme assays. On the basis of the antibacterial activity analysis against three pathogenic Vibrio species and the toxic activity evaluation in brine shrimp Artemia spp., fraction 251-18 obtained from Fusarium oxysporum MCCC 3A00251 was selected for in vivo challenge experiment. A remarkable decrease in mortality was found in the Litopenaeus vannamei post-larvae given with 100 and 200 μg/g fraction-supplemented feed compared with that in the control group fed with un-supplemented feed. These results revealed that DXR can serve a good target for anti-Vibrio agent screening. The application of feed additives with inhibitory effects against DXR may constitute a practical approach to control pathogenic Vibrios for sustainable shrimp cultivation.
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