Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.
Infect Immun. 2012 Aug;80(8):2868-77. doi: 10.1128/IAI.00372-12. Epub 2012 Jun 11.
Aggregatibacter actinomycetemcomitans is hypothesized to colonize through the interaction with collagen and establish a reservoir for further dissemination. The trimeric adhesin EmaA of A. actinomycetemcomitans binds to collagen and is modified with sugars mediated by an O-antigen polysaccharide ligase (WaaL) that is associated with lipopolysaccharide (LPS) biosynthesis (G. Tang and K. Mintz, J. Bacteriol. 192:1395-1404, 2010). This investigation characterized the function and cellular localization of EmaA glycosylation. The interruption of LPS biogenesis by using genetic and pharmacological methods changed the amount and biophysical properties of EmaA molecules in the outer membrane. In rmlC and waaL mutant strains, the membrane-associated EmaA was reduced by 50% compared with the wild-type strain, without changes in mRNA levels. The membrane-associated EmaA protein levels were recovered by complementation with the corresponding O-polysaccharide (O-PS) biosynthetic genes. In contrast, another trimeric autotransporter, epithelial adhesin ApiA, was not affected in the same mutant background. The inhibition of undecaprenyl pyrophosphate recycling by bacitracin resulted in a similar decrease in the membrane-associated EmaA protein. This effect was reversed by removal of the compound. A significant decrease in collagen binding activity was observed in strains expressing the nonglycosylated form of EmaA. Furthermore, the electrophoretic mobility shifts of the EmaA monomers found in the O-PS mutant strains were associated only with the membrane-associated protein and not with the cytoplasmic pre-EmaA protein, suggesting that this modification does not occur in the cytoplasm. The glycan modification of EmaA appears to be required for collagen binding activity and protection of the protein against degradation by proteolytic enzymes.
聚集放线杆菌（Aggregatibacter actinomycetemcomitans）被假设通过与胶原蛋白的相互作用而定植，并建立进一步传播的储库。聚集放线杆菌的三聚体黏附素 EmaA 与胶原蛋白结合，并通过与脂多糖（LPS）生物合成相关的 O-抗原多糖连接酶（WaaL）介导的糖基化修饰。本研究对 EmaA 糖基化的功能和细胞定位进行了表征。通过使用遗传和药理学方法中断 LPS 生物合成，改变了外膜中 EmaA 分子的数量和生物物理特性。在 rmlC 和 waaL 突变株中，与野生型菌株相比，膜相关的 EmaA 减少了 50%，而 mRNA 水平没有变化。用相应的 O-多糖（O-PS）生物合成基因进行互补，恢复了膜相关的 EmaA 蛋白水平。相比之下，另一种三聚体自转运蛋白上皮黏附素 ApiA 在相同的突变背景中不受影响。十一异戊烯基焦磷酸循环的抑制通过杆菌肽导致膜相关的 EmaA 蛋白相似减少。去除该化合物可逆转该效应。在表达非糖基化形式的 EmaA 的菌株中，观察到胶原结合活性显著降低。此外，在 O-PS 突变株中发现的 EmaA 单体的电泳迁移率变化仅与膜相关蛋白有关，而与细胞质前 EmaA 蛋白无关，这表明这种修饰不会发生在细胞质中。EmaA 的糖基化修饰似乎是胶原结合活性所必需的，并且可以保护该蛋白免受蛋白水解酶的降解。
