Peters Britta, Stein Johanna, Klingl Stefan, Sander Nathalie, Sandmann Achim, Taccardi Nicola, Sticht Heinrich, Gerlach Roman G, Muller Yves A, Hensel Michael
Abt. Mikrobiologie, Universität Osnabrück, Osnabrück, Germany.
Lehrstuhl für Biotechnik, FAU Erlangen-Nürnberg, Erlangen, Germany.
PLoS Pathog. 2017 May 30;13(5):e1006418. doi: 10.1371/journal.ppat.1006418. eCollection 2017 May.
The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion.
肠炎沙门氏菌的巨型非菌毛黏附素SiiE介导了与上皮细胞顶端部位的首次接触，并促成随后的侵袭。SiiE是一种595 kDa的蛋白质，由53个重复的细菌免疫球蛋白（BIg）结构域组成，是SPI4编码的1型分泌系统（T1SS）唯一已知的底物。SiiE的BIg50 - 52的晶体结构显示每个BIg结构域有两个由保守的天冬氨酸或谷氨酸残基形成的不同的Ca2+结合位点。在一项突变分析中，通过在SiiE的BIg结构域的不同位置将天冬氨酸替换为丝氨酸来破坏Ca2+结合位点。随着完整Ca2+结合位点数量的减少，分泌的SiiE量也减少。SiiE的BIg结构域包含不同的Ca2+结合位点，I型位点与其他T1SS分泌的蛋白质相似，II型位点是在SiiE中新发现的。我们从功能和结构上剖析了SiiE中I型和II型Ca2+结合位点的作用，以及SiiE不同位置的Ca2+结合位点的重要性。I型Ca2+结合位点对SiiE的有效分泌至关重要，I型位点数量的减少与分泌减少相关。II型位点对SiiE的分泌、稳定性和表面表达不太重要，然而，C末端部分II型位点的完整性是SiiE介导黏附和侵袭功能所必需的。
