James Bann photoJames Bann
Associate Professor, Biochemistry
Wichita State University

Structure and Mechanism of CS1 Pilus Assembly (2005-2007)

Pili are hair-like surface fibers that mediate attachment and colonization of pathogenic bacteria to host tissues. The long-term goal of my research is to understand the mechanism of assembly of pili as a prelude to the development of therapeutics aimed at preventing pilus assembly. The CS1 pilus system from enterotoxigenic Escherichia coli is encoded on the cooBACD operon, and is composed of multiple CooA subunits that have at the distal tip a single subunit of CooD required for adherence to intestinal cells. Additionally, both CooC, a 95 kDa outer membrane protein, and CooB, a periplasmic chaperone, are required for pilus assembly.

The specific hypothesis is that the assembly of CS1 pili is initiated by a conformational change in the outer membrane protein CooC upon binding of CooB alone or as a complex with CooD or CooA. The hypothesis is based on limited data that indicate that 1) CooC is an integral outer membrane protein, 2) expression of cooC is required for export of pili to the cell surface and 3) CooB copurifies with CooC and stabilizes CooC against degradation. Therefore CooB, either by itself or as a complex with CooA or CooD, initiates a conformational change in CooC that allows transport of subunits to the surface. The specific aims are to:

  1. Characterize the native structure and oligomerization state of CooC. The formation of a defined structure will be determined by (i) the stability to denaturant, (ii) stability to proteolysis, and (iii) dispersion of 19F-NMR chemical shifts. The oligomerization state of CooC will be determined by analytical gel filtration.
  2. Define structural changes in CooC. Purified CooB or CooB-subunit complexes will be added to purified CooC labeled site-specifically with p-fluoro-phenylalanine, and structural changes in CooC determined by 19F-NMR. The 31 phenylalanines are well dispersed throughout the sequence. Specific regions that interact with CooB or with CooB-subunit complexes will be identified, and mutagenesis of residues in regions of CooC will be correlated with in vivo studies of complementation of a cooC- cooBAD operon. The regions identified as important for binding will guide the development of peptide therapeutics aimed at attenuating binding.
  3. Determine the role of CooC in the mechanism of polymerization of CooA. Preliminary data indicate that CooA forms a stable 1:1 complex with a CooB, suggesting that polymerization of CooA requires other components such as CooC. The CooB-A complex will be added to purified CooC in the absence and presence of a CooB-CooD complex, and polymerization of CooA will be determined by (i) gel filtration, (ii) fluorescence polarization and (iii) native PAGE followed by western blotting with a CooA specific antibody.