Survival of pathogenic bacteria in their host is correlated with their capacity to maintain attachment to host tissue. Often this adherence is facilitated by the presence of filamentous adhesion pili on the bacterial surface, as in ETEC (enterotoxigenic Escherichia coli). ETEC cause severe diarrhea, presenting a significant worldwide health risk, particularly for infants and small children. In addition, the ease with which ETEC-caused diarrhea spreads via tainted food or water, places international travelers at high risk when entering regions where infection is endemic. In order to prevent or limit outbreaks, it is vital that we understand the mechanism of sustained bacterial attachment that can lead to colonization and illness. ? Structural information about adhesion pili will provide a basis for rational design of new therapies for prevention of bacterial binding or for removal of pathogenic bacteria already bound to the human host. The long-term goal of this project is to elucidate how the structure of pili supports their role as a virulence factor for pathogenic bacteria. In the proposed project period, studies on the structure and function of ETEC pili, in-depth computer modeling of P-pili expressed on the surface of pyelonephritic Escherichia coli (which cause urinary tract infections involving the kidneys), pilus damage/recovery experiments, and localization of type 1 adhesins will be used to examine the relationship between the structure and the function of adhesion pili. A combined approach will be employed to 1) elucidate the structural features of virulent ETEC pili that enable them to withstand peristaltic motility and other intestinal cleansing systems. Studies will include electron microscopy and image processing of negatively stained and frozen-hydrated ETEC pili. 2) examine the mechanism by which the P-pilus helical filament can unwind to a thin fibrillar structure five times its original length. Energy minimization and spatial constraints will be used with genetic algorithms to model, from individual monomeric subunits, this prototypic pilus filament into both intact and damaged pili. 3) investigate a means for reducing bacterial binding through damage to pili, with the aim of reducing the bacterial load and thus permitting the body's natural defenses to eradicate the remainder. Studies will use optical tweezers to measure the forces necessary to damage P-pili expressed on uropathogenic bacteria, and to investigate whether recovery occurs. 4) localize the adhesins on type 1 pili using immunoelectron microscopy. ? ? ?
