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Abstract：
The exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm-dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de-N-acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site-directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the -1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.
摘要：
胞外多糖聚-β-（1→6）-N-乙酰葡糖胺（PNAG）是导致持续和医院感染的细菌生物膜的主要结构决定因素。通过PNAG水解糖苷酶酶对生物膜的酶分散，例如分散剂B(DspB)，是治疗生物膜依赖性细菌感染的可能方法。由天然PNAG的部分去N-乙酰化产生的阳离子电荷对于PNAG依赖性生物膜形成是关键的。我们最近证明，DspB对去N-乙酰化PNAG寡糖的催化活性增加，但这种活性增加的分子基础尚不清楚。这里,我们分析了周围的阴离子氨基酸DspB的催化口袋中的PNAG底物识别和水解使用定点突变的组合的作用，使用合成的PNAG寡糖类似物进行活性测量，和体外生物膜分散试验。这些研究的结果支持一种模型，其中结合的PNAG与DspB蛋白表面上的浅阴离子凹槽弱相关，识别由与催化袋中的-1GlcNAc残基的相互作用驱动。驱动了阳离子PNAG水解速率的增加，在某种程度上,通过与阴离子表面上的D147相互作用。此外，我们发现,DspB突变体对完全乙酰化的PNAG寡糖的水解改善与PNAG依赖性表皮葡萄球菌生物膜的体外分散改善相关.这些结果为DspB识别底物的机制提供了见解，并提出了一种通过阴离子结合表面内氨基酸的突变来改善DspB生物膜分散活性的方法。
