Structural studies of Pseudomonas aeruginosa pilY1, a protein central to infections in cystic fibrosis

Pseudomonas aeruginosa is an opportunistic pathogen that is of particular concern to people afflicted with Cystic Fibrosis (CF). Patients with this disease have significantly decreased lung function that leads to airway obstruction and recurrent infections, often by Pseudomonas. This constant infection and its resultant lung damage are eventually fatal in over 90% of CF patients. Pseudomonas aeruginosa is known to adhere to and colonize human host cells using a protein called pilY1. This protein has little available functional data, but its mediation of host cell infection made it an attractive structural target. Here, we present the three dimensional crystal structure of the C-terminal domain (residues 644-1148) of pilY1. This domain is conserved (23% identity, 45% similarity) with the pilC adhesion proteins from both Neisseria gonorrhoeae and Neisseria meningitidis. The structure is a seven-bladed non-canonical beta-propeller and crystallized as a dimer in the asymmetric unit. The protein contains a variety of interesting structural features, including an extensive beta–strand (13 residues) that is integrated within two distinct beta-blades. Structural analysis of the protein led to the hypotheses that certain regions are important in pilY1 function. Through the use of functional assays to test bacterial colonization as well as biophysical methods, we were able to pinpoint two areas of the protein that appear to be relevant to its function. One area is an extended loop region located between two beta-strands on adjacent blades; the other is a calcium binding site found in a short beta-turn within a propeller blade. These regions appear to be necessary to pilY1’s function in twitching motility and pilus formation. Knowledge of the nature of these areas could be critical to the design of small molecules to inhibit pilY1 activity and hence Pseudomonas aeruginosa bacterial infection