The Inhibition and Resistance Mechanisms of Actinonin, Isolated from Marine Streptomyces sp. NHF165, against Vibrio anguillarum.

Na Yang,Chaomin Sun

doi:10.3389/fmicb.2016.01467

Abstract

Vibrio sp. is the most serious pathogen in marine aquaculture, and the development of anti-Vibrio agents is urgently needed. However, it is extreme lack of high-throughput screening (HTS) model for searching anti-Vibrio compounds. Here, we established a protein-based HTS screening model to identify agents targeting peptide deformylase (PDF) of Vibrio anguillarum. To find potential anti-Vibrio compounds, crude extracts derived from marine actinomycetes were applied for screening with this model. Notably, crude extract of strain Streptomyces sp. NHF165 inhibited dramatically both on V. anguillarum PDF (VaPDF) activity and V. anguillarum cell growth. And actinonin was further identified as the functional component. Anti-VaPDF and anti-V. anguillarum activities of actinonin were dose-dependent, and the IC50 values were 6.94 and 2.85 μM, respectively. To understand the resistance of V. anguillarum against actinonin, spontaneous V. anguillarum mutants with resistance against actinonin were isolated. Surprisingly, for the resistant strains, the region between 774 and 852 base pairs was found to be absent in the gene folD which produces 10-formyl-tetrahydrofolate, a donor of N-formyl to Met-tRNAfmet. When compared to the wild type strain, ΔfolD mutant showed eight times of minimum inhibition concentration on actinonin, however, the folD complementary strain could not grow on the medium supplemented with actinonin, which suggested that folD gene mutation was mainly responsible for the actinonin resistance. To our knowledge, this is the first report showing that marine derived Streptomyces sp. could produce actinonin with anti-VaPDF activity and the resistance against actinonin by V. anguillarum is mediated by mutation in folD gene.

Highlights

Sudden outbreak of diseases is a major setback in aquaculture, and it leads to high mortality and severe economic loss in all producing countries
The sequencing result showed that the length of pdf gene of V. anguillarum YN was 510 bp which encodes a 19.21 kDa “Class I” peptide deformylase (PDF) (Giglione et al, 2000) (Figure 1A), and the GenBank accession number of this gene is KU214433
The purified V. anguillarum PDF (VaPDF) catalyzed the removal of the N-formyl group from formyl-Met-AlaSer (Figure 1B) and the free N-formyl group could reacted with fluorescamine to form highly fluorescent products

Summary

Introduction

Sudden outbreak of diseases is a major setback in aquaculture, and it leads to high mortality and severe economic loss in all producing countries. Marine Vibrio species are associated with large-scale losses of penaeids and cause diseases to fish (Letchumanan et al, 2015b). Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting. Actinonin against Vibrio Anguillarum various marine and fresh/brackish water fish, bivalves and crustaceans. In both aquaculture and larviculture, this disease is responsible for severe economic losses worldwide (Frans et al, 2011). Oxytetracycline, tetracycline, quinolones, sulphonamides and trimethoprim are antimicrobial agents permitted and utilized in the Asian aquaculture industry (Letchumanan et al, 2015a). New resistance mechanisms are constantly being described, such as combined novel gene mph(G) coding macrolide phosphotransferase and gene mef (C) coding efflux pump were found to be responsible for high-level macrolide resistance Vibrio sp. (Nonaka et al, 2015)

Methods

Results

Conclusion