Snodgrass J L, Mohamed N, Ross J M, Sau S, Lee C Y, Smeltzer M S
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Infect Immun. 1999 Aug;67(8):3952-9. doi: 10.1128/IAI.67.8.3952-3959.1999.
The Staphylococcus aureus collagen adhesin (CNA) occurs in at least four forms that differ in the number (one, two, three, or four) of B domains. The B domains contain 187 amino acids and are located between the domains that anchor CNA to the cell envelope and the ligand-binding A domain. To determine whether a B domain is required for functional expression of CNA, we cloned the 2B cna gene from S. aureus strain Phillips and then eliminated both B domains by overlapping PCR. The absence of a B domain did not affect processing of the collagen adhesin to the cell surface or the ability to bind collagen. Based on our recent demonstration that the capsule can mask CNA on the surface of S. aureus cells (A. F. Gillaspy et al., Infect. Immun. 66:3170-3178, 1998), we also investigated the possibility that multiple B domains can extend the ligand-binding A domain outward from the cell surface and thereby overcome the inhibitory effect of the capsule. Specifically, we cloned the naturally occurring 4B CNA variant from S. aureus UAMS-639 and, by successive elimination of B domains, generated 1, 2, and 3B variants that are isogenic with respect to the 4B clone. After introducing each variant into microencapsulated and heavily encapsulated strains of S. aureus and growing cells under conditions known to affect capsule production (e.g., growth on Columbia agar), we correlated capsule production with exposure of CNA on the cell surface and the ability to bind collagen. Under no circumstance was the masking effect of the capsule reduced by the presence of multiple B domains. These results indicate that the B domains do not extend the ligand-binding A domain outward in a fashion that can overcome the inhibition of collagen binding associated with capsule production.
金黄色葡萄球菌胶原黏附素（CNA）至少以四种形式存在，这些形式在B结构域的数量（一个、两个、三个或四个）上有所不同。B结构域包含187个氨基酸，位于将CNA锚定到细胞膜的结构域与配体结合A结构域之间。为了确定B结构域对于CNA的功能性表达是否必需，我们从金黄色葡萄球菌菲利普斯菌株中克隆了2B cna基因，然后通过重叠PCR消除了两个B结构域。B结构域的缺失并不影响胶原黏附素向细胞表面的加工过程或结合胶原的能力。基于我们最近的证明，即荚膜可以掩盖金黄色葡萄球菌细胞表面的CNA（A.F.吉拉斯皮等人，《感染与免疫》66:3170 - 3178，1998），我们还研究了多个B结构域是否能够将配体结合A结构域从细胞表面向外延伸，从而克服荚膜的抑制作用。具体而言，我们从金黄色葡萄球菌UAMS - 639中克隆了天然存在的4B CNA变体，并通过连续消除B结构域，产生了与4B克隆同基因的1B、2B和3B变体。在将每个变体引入微囊化和高度囊化的金黄色葡萄球菌菌株，并在已知影响荚膜产生的条件下（例如在哥伦比亚琼脂上生长）培养细胞后，我们将荚膜产生与细胞表面CNA的暴露以及结合胶原的能力进行了关联。在任何情况下，多个B结构域的存在都不会降低荚膜的掩盖作用。这些结果表明，B结构域不会以能够克服与荚膜产生相关的胶原结合抑制作用的方式将配体结合A结构域向外延伸。
