Pili are critical in host recognition, colonization and biofilm formation during bacterial infection. Here, we report the crystal structures of SafD-dsc and SafD-SafA-SafA (SafDAA-dsc) in Saf pili. Cell adherence assays show that SafD and SafA are both required for host recognition, suggesting a poly-adhesive mechanism for Saf pili. Moreover, the SafDAA-dsc structure, as well as SAXS characterization, reveals an unexpected inter-molecular oligomerization, prompting the investigation of Saf-driven self-association in biofilm formation. The bead/cell aggregation and biofilm formation assays are used to demonstrate the novel function of Saf pili. Structure-based mutants targeting the inter-molecular hydrogen bonds and complementary architecture/surfaces in SafDAA-dsc dimers significantly impaired the Saf self-association activity and biofilm formation. In summary, our results identify two novel functions of Saf pili: the poly-adhesive and self-associating activities. More importantly, Saf-Saf structures and functional characterizations help to define a pili-mediated inter-cellular oligomerizaiton mechanism for bacterial aggregation, colonization and ultimate biofilm formation.

菌毛在细菌感染过程中对于宿主识别，定植和生物膜形成至关重要。在这里，我们报告了Saf pili中SafD- dsc和SafD-SafA-SafA（SafDAA- dsc）的晶体结构。细胞粘附试验表明，SafD和SafA都是宿主识别所必需的，这表明Saf菌毛具有多粘附机制。此外，SafDAA- dsc的结构，以及SAXS表征，揭示了意料之外的分子间低聚反应，促使人们对生物膜形成中Saf驱动的自缔合的研究。珠/细胞聚集和生物膜形成测定法用于证明Saf pili的新功能。针对SafDAA- dsc中分子间氢键和互补结构/表面的基于结构的突变体二聚体显着损害Saf的自缔合活性和生物膜形成。总而言之，我们的结果确定了Saf pili的两个新功能：多胶粘和自缔合活动。更重要的是，Saf-Saf的结构和功能表征有助于定义菌毛介导的细胞间寡聚机制，用于细菌聚集，定居和最终生物膜形成。