Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species.
Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species.
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DOI：
10.1042/bj20131415
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发表时间：
2014-03-15
期刊：
影响因子：
--
通讯作者：
Stafford GP
中科院分区：
文献类型：
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作者：
Phansopa C;Roy S;Rafferty JB;Douglas CW;Pandhal J;Wright PC;Kelly DJ;Stafford GP
许多人类居住的细菌为了生长或表面展示而获取唾液酸。我们以前在连翘中发现了一个唾液酸利用操纵子，它包括一个新的外膜唾液酸转运系统(NANOU)，其中NANO(神经氨酸外膜渗透酶)是一个假定的TonB依赖的受体，而NANU(细胞外神经氨酸摄取蛋白)是一个预测的SusD家族蛋白。通过将Nanou基因异源互补到一株没有外膜唾液酸渗透的大肠杆菌中，我们证明了肠道细菌脆弱类杆菌的Nanou系统是有功能的，并证明了它的功能依赖于TonB。我们还表明，Nanu是运输系统发挥最大功能所必需的，并且以唾液酸响应的方式表达。我们还通过分离和免疫荧光实验证明了它在外膜上的细胞定位。配体结合研究表明，唾液酸与两种拟杆菌的NaNU(Kd~400nM)高亲和力结合，并与一系列唾液酸类似物结合。对Nanu晶体结构的测定揭示了一个单体的SusD样结构，其中包含一个新的基序，其特征是一个延长的扭结螺旋，这可能决定了糖结合的特异性。本研究的结果鉴定了第一个细菌胞外唾液酸结合蛋白，并确定了唾液酸特异的PUL(多糖利用位点)。我们从生化和结构上表征了人类类杆菌中存在的一种新的可诱导的表面相关唾液酸结合蛋白，它代表了一种新型的唾液酸特异的多糖利用位点。
Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), where NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus). We biochemically and structurally characterize a novel inducible surface-associated sialic acid-binding protein present in human-dwelling Bacteroidetes species that represents a novel type of sialic acid-specific polysaccharide utilization locus.