title: Outer membrane targeting, ultrastructure, and single molecule localization of the enteropathogenic Escherichia coli type IV pilus secretin BfpB.
abstract: Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Lieberman
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Frost
authors.forename: Michael
authors.initials: M
authors.lastname: Hoppert
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Fernandes
authors.forename: Stefanie L
authors.initials: SL
authors.lastname: Vogt
authors.forename: Tracy L
authors.initials: TL
authors.lastname: Raivio
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Blanpied
authors.forename: Michael S
authors.initials: MS
authors.lastname: Donnenberg
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D054308:Enteropathogenic Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D001425:Bacterial Outer Membrane Proteins; C433794:BfpB protein, E coli; D029968:Escherichia coli Proteins; D008074:Lipoproteins