Cryo-EM structure of the CFA/I pilus rod.

Weili Zheng,Edward Egelman,Esther Bullitt,Narges Mortezaei,Magnus Andersson

doi:10.1107/s2052252519007966

Weili Zheng, Edward Egelman + Show 3 more

Open Access

https://doi.org/10.1107/s2052252519007966

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Journal: IUCrJ	Publication Date: Jul 9, 2019
Citations: 10	License type: CC BY 4.0

Affiliation: University of Virginia, Boston University, Umeå University

Abstract

Enterotoxigenic Escherichia 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. The helical quaternary structure of CFA/I can be unwound under tensile force and it has been shown that this mechanical property helps bacteria to withstand shear forces from fluid motion. We report in this work the CFA/I pilus structure at 4.3 Å resolution from electron cryomicroscopy (cryo-EM) data, and report details of the donor strand complementation. The CfaB pilins modeled into the cryo-EM map allow 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 model built using the EM structure 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 a point-mutated CFA/I with a propensity for unwinding. We found that pili with this mutated CFA/I require a lower force to unwind, supporting our hypothesis that Pro13 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 adhesion pili needed for initial attachment and sustained adhesion of ETEC.

Highlights

Enterotoxigenic Escherichia coli (ETEC) bacteria are common agents of infectious diarrheal diseases that cause disease in over 200 million people each year, with over 18 000 deaths of children younger than five years of age and over 50 000 total deaths (Khalil et al, 2018)
Adhesion pili subunits are assembled into helical polymers after being chaperoned across the periplasm, transported through an usher protein that resides in the outer membrane, and pilins are sequentially added to the growing pilus filament
Our electron cryomicroscopy three-dimensional helical reconstruction of CFA/I pili [Fig. 1(a), Supplemetary Fig. S1] shows pilin subunits assembled into 7.8 nm diameter helical filaments with an 8.6 Arise per CfaB subunit, and 3.18 CfaB subunits per turn of the helix

Summary

Introduction

Enterotoxigenic Escherichia coli (ETEC) bacteria are common agents of infectious diarrheal diseases that cause disease in over 200 million people each year, with over 18 000 deaths of children younger than five years of age and over 50 000 total deaths (Khalil et al, 2018). The bioassembly of CFA/I pili is initiated by the attachment of the chaperone/minor pilin (CfaA/CfaE) heterodimer to the usher protein CfaC at the outer membrane assembly site followed by cyclic incorporation of the major pilin, CfaB (Poole et al, 2007; Li et al, 2009). Both pilin subunits, CfaE and CfaB, require the chaperone CfaA for folding and stability and the CfaC usher protein to facilitate donor-strand exchange. This growing chain of subunits forms a helical structure 1–3 mm in length and with an outer diameter of approximately 8 nm (Mu et al, 2005, 2008)

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