Peng Z, Wu H, Ruiz T, Chen Q, Zhou M, Sun B, Fives-Taylor P
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, USA.
Oral Microbiol Immunol. 2008 Feb;23(1):70-8. doi: 10.1111/j.1399-302X.2007.00401.x.
BACKGROUND/AIMS: Streptococcus parasanguinis is a primary colonizer of the tooth surface. Its adhesion is mediated by the long fimbriae, which are composed of multiple subunits of a serine-rich glycoprotein, Fap1. Previous studies revealed that a chromosomal region located downstream of fap1 is involved in the secretion and glycosylation of Fap1. In this study, we investigated the role of a glycosylation-associated gene, gap3, in Fap1 biogenesis.
A gap3 non-polar mutant was constructed by insertional inactivation. The phenotype of the mutant and the subcellular distribution of its products were investigated. The binding ability of the mutant was tested with saliva-coated hydroxyapatite (SHA). Electron microscopy was used to observe the morphological changes on the mutant cell surface. Confocal microscopy was utilized to determine biofilm formation ability.
The gap3 mutant produced a partially glycosylated Fap1 precursor, that was less stable than mature Fap1. The Fap1 precursor was distributed in all subcellular fractions including the cell surface and culture medium although in decreased amounts. These data suggest a role for Gap3 in Fap1 glycosylation as well as a link between glycosylation and secretion of Fap1. The gap3 mutant had reduced binding to saliva-coated hydroxyapatite. Electron microscopy revealed that the gap3 mutant had lost its long fimbriae. Biofilm formation was also inhibited by the gap3 mutation. Fewer gap3 mutant cells adhered to the biofilm surface and microcolony formation was decreased.
Gap3 is required for the complete glycosylation and secretion of Fap1, which is important for fimbrial assembly, bacterial adhesion, and in vitro biofilm formation.
背景/目的：血链球菌是牙面的主要定植菌。其黏附由长菌毛介导，长菌毛由富含丝氨酸的糖蛋白Fap1的多个亚基组成。先前的研究表明，位于fap1下游的一个染色体区域参与Fap1的分泌和糖基化。在本研究中，我们调查了一个糖基化相关基因gap3在Fap1生物合成中的作用。
通过插入失活构建gap3非极性突变体。研究了突变体的表型及其产物的亚细胞分布。用唾液包被的羟基磷灰石（SHA）测试突变体的结合能力。用电子显微镜观察突变体细胞表面的形态变化。利用共聚焦显微镜确定生物膜形成能力。
gap3突变体产生了部分糖基化的Fap1前体，其稳定性低于成熟Fap1。Fap1前体分布在包括细胞表面和培养基在内的所有亚细胞组分中，但其含量有所减少。这些数据表明Gap3在Fap1糖基化中发挥作用，以及Fap1糖基化与分泌之间的联系。gap3突变体与唾液包被的羟基磷灰石的结合减少。电子显微镜显示gap3突变体失去了其长菌毛。gap3突变也抑制了生物膜形成。黏附到生物膜表面的gap3突变体细胞减少，微菌落形成也减少。
Gap3是Fap1完全糖基化和分泌所必需的，这对菌毛组装、细菌黏附和体外生物膜形成很重要。
