Defining the Role of Yersinia pestis Ail in Host Cell Interactions and Plague Virulence Functions.

Abstract

Adhesion to host cells and delivery of cytotoxic Yops are essential steps in the infection process of the plague pathogen Yersinia pestis. The Y. pestis adhesin Ail was recently demonstrated to mediate host cell binding and be critical for Yop delivery and virulence. Thus, the goal of my thesis was to identify a host cell substrate with which Ail interacts and elucidate the mechanism by which Ail engages host cells. To identify Ail ligands, the Ail protein was purified following overexpression in E. coli and reconstituted in detergent micelles. Purified Ail bound specifically to fibronectin (Fn), an extracellular matrix protein that may act as a bridge between Ail and the host cell. Fibronectin is a large, complex molecule with many known binding domains. We demonstrated Ail binds to host Fn specifically and this Ail-Fn interaction is important for efficient delivery of Yops. Thus, I sought to understand this interaction in more detail. I wanted to identify the Ail binding site along fibronectin and also to identify the amino acids of Ail that are important for host cell binding and Yop delivery. Ail binds the 120kDa fragment, but less effectively than full-length Fn. Antibody studies provided additional evidence that Ail binds the 8-9FNIII modules within the 120kDa fragment. However, inhibitor studies suggest that the Ail-binding site may include a neighboring 45kDa fragment. Together, these data suggest there may be two sites within fibronectin that are required for optimal Ail binding, the collagen/gelatin binding domain in the N-terminal half of fibronectin and the 8-9FNIII modules within the 120kDa cell binding domain more C-terminally located. To study residues that contribute to the various functions of Ail, we used a mutagenesis scheme termed SWIM (selection without isolation of mutants). We found a serine and a phenylalanine in the third exposed loop of Ail that are defective for cell binding and Yop delivery when substituted with alanine. The double S128A/F130A mutant is modestly defective for Fn binding and Yop delivery. Together, my thesis work has contributed to the understanding of how Y. pestis Ail uses fibronectin to engage host cells and lead to virulence functions.