The secretin, a homomultimeric outer membrane channel, is the only protein common to type IV pilus (T4P), type 2 secretion and type 3 secretion systems. Therefore, secretins enjoy a unique position of prominence among virulence factors. Although the structures of several periplasmic secretin domains have been solved by X-ray crystallography, and the overall shape of secretins has been described by electron microscopy, sufficient detail to understand the mechanism that allows secretins to interact with other components of their respective machines and to open selectively to allow passage of their substrates has not be achieved. In this proposal we describe how we will advance our understanding of secretin function. Using a variety of techniques, including cysteine mutagenesis and Frster resonance energy transfer, we described a topology model for BfpB, the secretin from the T4P of enteropathogenic Escherichia coli.
The first aim of this proposal will build on that knowledge by using in vivo disulfide cross-linking to trap the pilin in the secretin and thereby map the walls of the secretin channel. Recent advances in cryo-electron microscopy have permitted the structural analysis of large complexes at resolutions approaching the atomic range.
In aim two we will capitalize on these advances to visualize the secretin and proteins with which it associates in unprecedented detail. The experiments described in this proposal will provide critical structural information that is essential for our lng-term goal of a complete understanding of T4P biogenesis. Furthermore, this information will also have implications for secretion systems and may ultimately have practical implications for new therapeutics.

Public Health Relevance

The secretin is a protein channel in the outer membrane of bacteria, through which disease-causing toxins and surface fibers called Type 4 pili that attach to human cells must pass. The structure of this channel has been described only in vague outline, and therefore how it performs its function remains obscure. We will use sophisticated biochemical techniques to trap proteins in the channel and map its walls and new high-resolution microscopic techniques to visualize its architecture in unprecedented detail.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI123964-03
Application #
9245656
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Baqar, Shahida
Project Start
2016-04-01
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2019-03-31
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298