Cloning, Expression, and Sequencing of a Cell Surface Antigen Containing a Leucine-Rich Repeat Motif from Bacteroides forsythus ATCC 43037

Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (bspA) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in Escherichia coli, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response.

福赛拟杆菌（Bacteroides forsythus）是近年来被确认的人类牙周致病菌，与重度及复发性牙周炎密切相关。然而，目前对该菌的致病机制仍知之甚少。本研究旨在鉴定该菌的表面分子，这类分子可能在其黏附口腔组织或触发宿主免疫应答中发挥作用。研究人员通过对以λZAP II载体构建的福赛拟杆菌基因文库进行免疫筛选，成功分离得到编码该菌表观分子量为98 kDa的细胞表面相关蛋白的基因（bspA）。该基因编码的98 kDa蛋白（BspA）包含14个完整的23个氨基酸残基重复序列，该序列与富亮氨酸重复基序具有部分同源性。研究人员在大肠杆菌（Escherichia coli）中表达了包含该重复区域的重组蛋白，对其进行纯化后，将其用于抗体制备及体外结合实验。纯化后的重组蛋白可呈剂量依赖性地强力结合纤连蛋白与纤维蛋白原，且能进一步抑制福赛拟杆菌与这些细胞外基质（extracellular matrix，ECM）成分的结合。此外，福赛拟杆菌相关牙周炎的成年患者体内可检测到针对BspA蛋白的特异性抗体。本研究成功克隆并表达了福赛拟杆菌的免疫原性细胞表面相关蛋白（BspA），并推测该蛋白可介导细菌与细胞外基质成分及凝血因子（分别为纤连蛋白与纤维蛋白原）的结合，这一过程可能对该菌在口腔内的定植至关重要，同时该蛋白也是宿主免疫应答的靶标。