Department of Biotechnology, Alagappa University, Science Campus, Karaikudi, 630 003, India.
Centre for Nanoscience and Nanotechnology, Sathyabama University, Chennai, 600 119, India.
Mol Cell Probes. 2018 Jun;39:14-24. doi: 10.1016/j.mcp.2018.03.003. Epub 2018 Mar 21.
This study aimed to determine the antibiofilm activity of seawater microbes against Vibrio cholerae (VCO1) through functional metagenomics approach. A metagenomic library was constructed from Palk Bay seawater and the library was screened to identify the biofilm inhibitory metaclone. Metaclone SWMC166 (harbouring ∼30 kb metagenomic insert) was found to exhibit antibiofilm activity against VCO1. The biofilm inhibitory potential of partially purified ethyl acetate extract of SWMC166 (EA166) was further evaluated through microscopic studies and biochemical assays. Further, EA166 treated VCO1 divulged up-regulation of genes involved in high cell density-mediated quorum sensing (QS) pathway which was analysed by real-time PCR. In order to identify the genes of interest (within ∼30 kb insert), subcloning was performed through shotgun approach. Small molecules from positive subclones SC5 and SC8 were identified through HRLC-MS analysis. Resulted small molecules were docked against QS receptors of V. cholerae to identify the bioactive metabolites. Docking studies revealed that totally seven metabolites were able to interact with QS receptors that can possibly trigger the QS cascade and sequentially inhibit the biofilm formation and virulence factors of VCO1.
本研究旨在通过功能宏基因组学方法确定海水微生物对霍乱弧菌（VCO1）的抗生物膜活性。从帕尔克湾海水中构建了宏基因组文库，并对文库进行筛选，以鉴定具有生物膜抑制活性的宏克隆。发现宏克隆 SWMC166（含有约 30kb 宏基因组插入片段）对 VCO1 具有抗生物膜活性。通过显微镜研究和生化分析进一步评估了 SWMC166 的部分纯化乙酸乙酯提取物（EA166）的生物膜抑制潜力。此外，EA166 处理后的 VCO1 上调了与高细胞密度介导的群体感应（QS）途径相关的基因表达，这通过实时 PCR 进行了分析。为了鉴定感兴趣的基因（在约 30kb 插入片段内），通过鸟枪法进行了亚克隆。通过 HRLC-MS 分析鉴定了阳性亚克隆 SC5 和 SC8 中的小分子。对接研究表明，总共七种代谢物能够与霍乱弧菌的 QS 受体相互作用，可能触发 QS 级联反应，从而依次抑制 VCO1 的生物膜形成和毒力因子。
