Structure and host-receptor recognition studies of Gram-negative bacterial fimbriae assembled via the chaperone/Usher Pathway

Abstract: Enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and Yersinia enterocolitica (YE) are the principle etiological agents of acute and persistent diarrhea worldwide including resource-poor and industrialized regions. Adhesion of these pathogens to host receptor is the first important step to deliver toxins and virulent factors to initiate pathogenicity. Coli surface antigen (CS6) of ETEC, aggregative adherence factor-1 (AAF-1) of EAEC, and mucoid Yersinia fimbriae (Myf) of YE are the widely expressed colonization factors (fimbriae or pili) that mediate bacterial adhesion to the small intestinal epithelium. CS6 consists of CssA and CssB, AAF-1 consists of AggA and AggB, and Myf is made of MyfA, which are secreted and assembled on the cell surface via the chaperone/usher pathway. Using X-ray crystallography, we present atomic resolution insight into the fibre forming subunits and show that subunits of these organelles are assembled into a linear polymer by donor strand complementation. Myf is a homo-polymer, AAF-1 is a polymer of AggA subunits decorated by a single copy of the AggB subunit at the tip of the fiber, and CS6 is hetero-polymer of CssA and CssB subunits that are assembled alternately into a single fiber. Using spectroscopic and biochemical studies, we demonstrate that all subunits of these three fimbriae bind to receptors on intestinal epithelial cells, establishing multipoint attachment. The crystal structure of a MyfA-galactose complex reveals the receptor binding site and responsible residues. Structure guided mutagenesis of AggA shows that it binds to fibronectin via a novel electrostatic binding mechanism. Based on our structural and biochemical studies we conclude that CS6, AAF-1, and Myf are adhesive fimbriae of a novel type: they can act as poly-adhesins with rich binding properties. These findings steer the research field forward by revealing insight into receptor-fimbria interactions. This opens a window to search for receptor inhibitor molecules to decrease the infantile death rate and associated malnutrition.