Antiparallel stacking of Csu pili drives Acinetobacter baumannii 3D biofilm assembly

Some of the most troublesome Gram-negative nosocomial pathogens rely on archaic chaperone-usher pili to establish stress- and drug-resistant, multi-layered biofilms. Here, we unveil the mechanism of three-dimensional (3D) biofilm assembly. In situ analyses of Acinetobacter baumannii biofilms using electron microscopy (EM) reveal an extensive network of ultrathin, flat stacks of archaic Csu pili interconnecting bacterial cells in 3D space. Cryo-EM structures of a single native pilus, pilus pairs, and two types of multi-pilus stacks show that the pili pack into antiparallel sheets, with their rods connected laterally by junctions at their zigzag corners. This antiparallel arrangement ensures that contacts form primarily between pili from interacting cells rather than pili from the same cell. With a remarkably short helical repeat, archaic chaperone-usher pili spontaneously establish a high density of junctions that determines the biofilm’s 3D architecture. These findings suggest that pilus-pilus interactions represent a critical aspect of combating multidrug-resistant bacterial infections.