Single Particle Localization and a Novel Topology of the Enteropathogenic Escherichia coli Type IV Pilus Outer Membrane Secretin
AuthorLieberman, Joshua Abraham
AdvisorDonnenberg, Michael S.
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AbstractType IV Pili (T4P) are filamentous surface appendages required for 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 are a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4Ps, type II secretion, type III secretion, and phage assembly. We determined the role of the lipoprotein transport pathway in targeting the BfpB secretin protein of the EPEC T4P to the OM. We demonstrated that concurrent mutation of genes encoding both the secretin and retraction ATPase can result in viable cells and find that these cells display paradoxically low levels of cell envelope stress response activity. To study the sub-cellular distribution of BfpB, we used Photo-activated Localization Microscopy to localize single BfpB molecules fused to photo-activated mCherry. Contrary to findings in other T4P systems, we find that BFP components predominantly have an uneven distribution throughout the cell envelope and are only found at one or both poles in a minority of cells. Furthermore, we have used an array of biophysical and biochemical techniques to construct a model topology of BfpB. We provide evidence that the N- and C-termini of this protein have a trans configuration with respect to the OM. We propose that BfpB and all secretins constitute a novel structural class of OM proteins.
DescriptionUniversity of Maryland in Baltimore. Molecular Microbiology and Immunology. Ph.D. 2011
Keywordouter membrane protein
type IV pilus
Bacterial Outer Membrane Proteins
Enteropathogenic Escherichia coli