Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via type 4 secretion systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a second copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggests that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

革兰氏阳性细菌之间的水平基因转移导致毒力因子和抗生素抗性的快速传播。通常通过通常在结合质粒上编码的4型分泌系统（T4SS）促进这种转移。然而，已经含有特定结合质粒的供体细胞抵抗了所述质粒的第二拷贝的获得。他们利用不同的机制，包括为此目的进行表面排斥。粪肠球菌由结合质粒pCF10编码的PrgA是一种表面蛋白，已被暗示在毒力和表面排斥中均起着作用，但尚未完全解释其实现机理。在这里，我们报告了全长PrgA的结构，该结构表明PrgA远离细胞壁（大约40 nm）突出，并在那里呈现蛋白酶结构域。体内实验表明，PrgA为细胞粘附提供了物理屏障，从而减少了细胞聚集。PrgA的这种功能有助于表面排斥，将其同源质粒的摄取降低约一个数量级。使用在催化位点具有突变的PrgA的变体，我们表明表面排斥作用取决于PrgA的蛋白酶结构域的活性。电脑分析表明，PrgA可以与另一种肠球菌粘附素PrgB相互作用，并且这两种蛋白已经共同进化。PrgB是强毒力因子，PrgA参与PrgB的翻译后加工。最后，竞争交配实验表明PrgA为携带质粒的细胞提供了显着的适应性优势。