Cryo-EM Map of the CFA/I Pilus Rod

Abstract

AbstractEnterotoxigenicEscherichia coli(ETEC) are common agents of diarrhea for travelers and a major cause of mortality in children in developing countries. To attach to intestinal cells ETEC express colonization factors, among them CFA/I, which are the most prevalent factors and are the archetypical representative of Class 5 pili. Due to their helical quaternary structure that can be unwound and works as a damper of force, CFA/I pili help ETEC bacteria withstand intestinal fluid motion. We report in this work the CFA/I pilus structure at 4.0 Å resolution and report details of the donor strand complementation. The density map allows us to identify the buried surface area between subunits, and these regions are correlated to quaternary structural stability in class 5 and Chaperone-Usher pili. In addition, from the EM map we also predicted that residue 13 (proline) of the N-terminal β-strand could have a major impact on the filament’s structural stability. Therefore, we used optical tweezers to measure and compare the stability of the quaternary structure of wild type CFA/I and point-mutated CFA/I in which proline 13 was changed to a non-polar residue, phenylalanine. We found that pili with this mutated CFA/I require a lower force to unwind, supporting our hypothesis that Pro 13 is important for structural stability. The high-resolution CFA/I pilus structure presented in this work and the analysis of structural stability will be useful for the development of novel antimicrobial drugs that target the pilus structure to reduce its damping properties, which are needed for initial attachment and sustained adhesion of ETEC.SynopsisThe structure of a common virulence factor expressed on the surface of diarrheacausing bacteria, CFA/I pili, has been determined at 4.0 Å resolution. The role of proline 13 in stabilizing the pilus structure has been confirmed using force-measuring optical tweezers on wild type and point-mutated pili.