2011 update: Our structures of Y. pestis FyuA and pesticin allowed us to engineer a novel phage therapy drug against a Gram-negative pathogen: We solved the structure of FyuA, a TonB-dependent iron transporter required for virulence in bubonic plague, with and without its cognate siderophore, ferric yersiniabactin. At the same time, we determined the structure of a bacteriocin called pesticin that uses FyuA to cross the outer membrane. Once inside the periplasm, pesticin kills the cell by degrading the peptidoglycan layer. From our structure we discovered that the killing domain of pesticin resembles phage T4 lysozyme, so we engineered a hybrid bacterial-phage toxin that contains a bacterial targeting domain (to FyuA) and a phage killing domain. We showed that the hybrid lysine evades the natural protection mechanism of toxin-producing strains and kills all Yersinia strains tested, both in vitro and in vivo (mouse model of bubonic plague). This is the first demonstration of phage therapy for Gram-negative pathogens because until now, no one knew how to transport the toxin across the outer membrane. This work is under revision at Nature, awaiting final statistics for the animal experiments. Structures of Y. pestis Ail reveal how a bacterial outer membrane protein binds to host cells: In our efforts to contribute to vaccine and drug design against Gram-negative pathogens, we have worked for the past nine years on Y. pestis outer membrane proteins that appear important for disease progression. One of the most highly expressed Y. pestis outer membrane proteins is Ail, a small outer membrane protein that functions to bind host cells and also to evade the host immune response by interfering with the complement cascade. In unpublished work, we found that Ail is immunogenic and confers protection in a mouse model of bubonic plague, and we are continuing efforts to formulate a protein component vaccine based on this work. Because Ail is so important, we solved to structures of it and characterized adhesion to target cells. We determined that Ail selectively binds the extracellular matrix proteins laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yersinia outer proteins (Yops) to Hep-2 cells. We solved crystal structures of Ail alone and in complex with a heparin analogue, providing a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells. This manuscript is in press at Structure.
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