菌膜粘附分子聚N-乙酰葡萄糖胺合成与转运机理研究

Bacterial biofilms are complex surface attached microbial communities that are a significant medical problem because once established they are difficult to eradicate. A key feature of bacterial biofilms is that cellular aggregates grow within a self-produced extracellular matrix that are full of secreted proteins and exopolysaccharides. The extracellular matrix facilitates the adherence between bacteria and the surfaces they colonize while also providing protection against environmental stresses, a diffusion barrier against antibiotics, and isolation from the innate immune system. A wide variety of medically important biofilm-forming bacteria produce N-acetylated poly-β-1,6-N-acetyl-D-glucosamine (PNAG) exopolysaccharides, also referred to as biofilm intercellular adhesin. In E. coli, PNAG production is dependent on the four-gene operon, pgaABCD,and its products PgaC-PgaD complex and PgaA form PNAG-secretion channels across bacterial inner and outer membranes, respectively. To elucidate the molecular mechansism of PNAG biogenesis, we propose structural and functional studies of Pga proteins that are involved in PNAG synthesis and translocation,primarily using X-ray crystallography. These studies will not only improve our undstanding of the secretion mechanisms of exopolysaccharides in general, but also provide important information for developing novel drugs against biofilm formation.

环境中细菌不是游离存在的，而是藏身于自身分泌的蛋白质和多糖等所组成的被称为菌膜(biofilm)的胶状基质中。菌膜为细菌的寄生生活提供了强大的保护屏障，使其对宿主体内的各种免疫环境具有极大的抵抗力，同时赋予了细菌极强的致病性。细菌分泌的胞外多糖聚N-乙酰葡萄糖胺(PNAG)，又称菌膜粘附分子，是介导细菌之间发生初步接触和粘附的关键分子，直接影响菌膜的形成。大肠杆菌膜蛋白PgaC-PgaD和PgaA分别负责PNAG特异性地跨内外膜分泌。我们将以X-射线衍射晶体学为主要手段，开展对参与PNAG 合成和转运的一系列膜蛋白进行结构测定，并结合其他生物物理，生物化学以及细菌遗传学手段，研究PNAG合成与分泌的机理。通过这些研究，可以加深对胞外多糖分泌机制以及菌膜形成的了解，也将为研发抗菌膜生成药物提供重要信息。

环境中细菌不是游离存在的，而是藏身于自身分泌的蛋白质和多糖等所组成的被称为菌膜的胶状基质中，菌膜为细菌的寄生生活提供了强大的保护屏障，使其对宿主体内的各种免疫环境具有极大的抵抗力，同时赋予了细菌极强的致病性。细菌分泌的胞外多糖聚N-乙酰葡萄糖胺（PNAG），又称菌膜黏附分子，是介导细菌之间发生初步接触和粘附的关键分子，直接影响菌膜的形成。大肠杆菌膜蛋白PgaC-PgaD和PgaA,PgaB分别负责PNAG特异性地跨内外膜分泌。我们用X-射线晶体衍射技术解析了关键外膜蛋白PgaA-PgaB复合物3.2 Å的晶体结构，在革兰氏阴性细菌胞外多糖分泌这一研究领域中率先得到了完整的外膜分泌复合体的空间结构；并通过定点突变和体内功能实验确定了该复合物上若干关键的氨基酸位点。并且成功地在体外表达了重组的PgaC-PgaD复合物，获得较为稳定的PgaC-PgaD复合物用于结构学和功能学研究。在上述的基础上，我们提出一种猜想，认为内膜的合酶/转运复合体PgaC-PgaD应该与外膜的分泌复合体PgaA-PgaB存在相互作用，才能够完成将在内膜上新合成的多糖链转移到外膜的分泌复合体中进而分泌到细胞外这一生物学功能。对PgaA-PgaB复合体以及PgaC-PgaD复合体进行的体外生化实验的研究最终为我们提供了这一相互作用的证据。

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