利用节杆菌B4胞外多糖研究多糖抑制铜绿假单胞菌生物膜形成的关键机制

The mechanism for polysaccharide inhibition of Pseudomonas aeruginosa biofilms is yet to be unclear, which limits the further development and application of these polysaccharides as antibiofilm drugs. In our previous study, an exopolysaccharide with the high antibiofilm activity against P. aeruginosa PAO1 was discovered from Arthrobacter sp.B4,whose inhibition rate was about 81%. Furthermore,it was observed that galactosyl and fucosyl were involved in the constituents of B4 and other antibiofilm polysaccharides, and their inhibitions of biofilms were achieved by non-biocidal modes of action. Thus elucidation of the cause for B4 polysaccharide of inhibition of biofilm will reveal the mechanism for polysaccharide inhibition of biofilm. In this study, B4 polysaccharide and P. aeruginosa PAO1 will be selected as the object of research, according to our previous results and other relative references, it will be hypothesized that polysaccharide inhibition of biofilm is achieved by targeting bacterial lectins. Firstly, Blocking of P. aeruginosa PAO1 lectins will be analyzed by hemagglutination inhibition test and western blotting. Secondly, the difference of biofilms between P. aeruginosa PAO1 wild stain and deletion mutants lacking lectin genes will be investigated in a static model of biofilm formation in vitro and the biofilms were assayed by means of fluorescence labeling and confocal laser scanning microscopy. Lastly, the key mechanism for B4 polysaccharide inhibition of P.aeruginosa PAO1 biofilm formation by targeting and blocking its lectins will be testified and elucidated. It will provide a theoretical reference in the exploration of the mechanisms for the other antibiofilm polysaccharide against other bacteria.

针对多糖抑制铜绿假单胞菌生物膜形成作用机制尚不明确，极大限制了其医药开发与应用的研究现状。前期工作中我们发现节杆菌B4胞外多糖可高效抑制铜绿假单胞菌PAO1生物膜形成，抑制率达81%；它与同类报道多糖均具有半乳糖或甘露糖等组分，且它们都不杀菌而抑制生物膜形成，据此我们认为深入探索B4多糖抑制PAO1生物膜形成原因，将揭示多糖抑制铜绿假单胞菌生物膜形成的关键机制。本课题以B4多糖和PAO1为研究对象， 假设B4多糖抑制PAO1生物膜形成的作用靶位是其凝集素，拟采用红细胞凝集抑制试验、Western blotting研究B4多糖对PAO1凝集素的体外封闭作用，再利用框内缺失突变、荧光标记、共聚焦激光显微镜等技术研究B4多糖抑制PAO1凝集素缺失突变株、野生株生物膜体外形成的差异，阐明B4多糖靶向封闭凝集素是抑制PAO1生物膜形成的关键机制，为研究多糖抑制其它细菌生物膜形成作用机制提供理论参考。

多糖抑制铜绿假单胞菌生物膜形成作用机制尚不明确，极大限制了其医药开发与应用的研究现状。我们研究发现节杆菌B4胞外多糖可高效抑制铜绿假单胞菌PAO1生物膜形成，抑制率达81%，但B4多糖未显示任何杀菌活性。为进一步探索B4多糖的抑膜机制，对B4胞外多糖进行分离纯化和理论性质分析，结果显示B4多糖由半乳糖、甘露糖、葡萄糖、葡萄糖醛酸等组成，含三个主要组分，命名为B4-EPS1、B4-EPS2、B4-EPS3，分子量分别为6.84×103 Da、3.97×104 Da、5.9×106 Da，具有良好的还原、絮凝、红细胞凝集等性质。将B4多糖的三个组分分别作用于铜绿假单胞菌PAO1野生株、凝集素LecA/LecB单双缺失突变株、凝集素过表达株生物膜，结果显示B4-EPS2可通过与PAO1凝集素结合抑制其生物膜形成。以上研究发现为阐明多糖抑制细菌生物膜形成作用机制提供参考、也为进一步开发抗生素辅剂型抗生物膜多糖药物提了充分的理论依据。