Abstract 1095 Structural Divergence of Type IV Pilus Extension ATPases in Pathogenic Bacteria: Cryo-EM Analysis of Pseudomonas aeruginosa PilB

Bacterial pathogenicity often requires adhesive surface structures, including Type IV pili (T4P). These filamentous appendages, common to many bacterial and archaeal species, serve as versatile molecular machines crucial for surface motility, adhesion, biofilm formation, and pathogenesis. T4P are a remarkable example of divergent evolution, likely present in the last universal common ancestor. T4P extension is a coordinated and energy-dependent process performed by a complex machine of more than ten proteins spanning the bacterial membrane. An extension ATPase(s) often called PilB supplies the required energy. How the chemical energy from ATP hydrolysis is converted to mechanical energy is a central question in T4P biology. In a recent study, we presented cryo-EM structures of the type IVb pilus (T4bP) ATPase BfpD from the lethal human pathogen enteropathogenic Escherichia coli (EPEC). T4bP diverged from type IVa pili (T4aP) very early in evolution. All previously reported T4P ATPases were from non-pathogenic thermophiles that make T4aP were solved by X-ray crystallography and displayed two-fold symmetry. In contrast, BfpD displayed six-fold symmetry. Intriguingly, BfpD demonstrated simple enzyme kinetics, while an ATPase from a thermophilic T4aP system was reported to exhibit complex enzyme kinetics. The striking differences between BfpD and T4aP extension ATPase despite a similar role in pilus biogenesis pose several questions. Do these differences represent divergence between T4aP and T4bP systems? Do they reflect differences between non-pathogenic thermophiles and mesophilic pathogens? Is the apparent difference in quaternary structure an artifact of crystallography? To investigate these questions, we synthesized a codon-optimized extension ATPase pilB gene and purified the T4aP extension ATPase PilB from Pseudomonas aeruginosa, a highly-antimicrobial-resistant opportunistic human pathogen. We obtained high-quality cryo-EM images of PilB in the presence of ADP. A total of 463,000 particles from 6,685 movies were analyzed using CryoSPARC, resulting in consensus maps with a resolution of 3.4 Å. The presence of two-fold rotational symmetry was confirmed through examination of 2D class averages and a 3D map. Further, we employed AlphaFold structure prediction to generate a single subunit PilB model which we used for refinement through chain tracing in Coot. The monomeric model was further expanded into a hexameric model and refined using Chimera-ISOLDE and Coot, alternating with real space refinement in PHENIX to fit into the density map. Our work represents the first example of a T4aP extension ATPase structure from a pathogenic bacterium. We found that PilB retains the quaternary structure previously reported for several T4aP extension ATPases from thermophiles but diverges from the symmetry observed in the EPEC T4bP BfpD ATPase. Ongoing studies, including investigations into enzyme kinetics and interactions with transmembrane partner proteins, will shed further light on the functioning of the T4P machine. This work was supported by an Accelerate Award from the Virginia Commonwealth University Office of the Vice President for Research and Innovation.