Structural characterization of outer membrane proteins from Yersinia pestis

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Current research Our collaborator at NIAID, Dr. B. Joseph Hinnebusch, has used DNA microarrays to compare expression profiles of Y. pestis recovered from plague infected rats as opposed to Y. pestis that are grown in flask cultures. Using these methods they have identified eight putative outer membrane proteins that are found in higher amounts in infected animals. Almost all of these are proteins found in the outer envelope of Y. pestis and for this reason it is thought that they might make good vaccine targets. This is because portions of these proteins protrude into the surrounding environment of the bacteria where they can be readily detected by the immune system of an infected but vaccinated human being. Additionally almost all are involved in the import of iron into the cell. The ability to acquire iron is essential for the survival of most bacteria including pathogenic bacteria. In Y. pestis the ability to obtain iron from the iron poor environment of the infected host is correlated to virulence and lethality of infection in mice. Our lab has a lot of expertise on the basic and structural biology of iron import pathways E. coli. The experiences gained on the handling of similar proteins in E. coli have already been valuable in the study of Y. pestis proteins. We have proceeded to clone, express, purify and crystallize these 8 proteins with two general aims: first, to generate protein for vaccine development studies and second, to make use of the excess material for structural studies. So far we have expressed and successfully purified 5 out of the 8 proteins. In 2009, we solved two structures of Y. pestis iron transporters, and in one case we have structures of the apo form and a form with bound cognate siderphore. These structures are currently being used to do in silico drug design by docking small molecules into the siderophore binding pocket. Once good candidate molecules are found, they will be synthesized and analyzed for competitive binding. This approach may lead to novel antibiotics specifically targeting Y. pestis. In related work, we also solved the structure of a Y. pestis bacteriocin which we engineered to kill a wide variety of Yersinia strains. The engineered bacteriocin may provide an alternative drug treatment for plague. This work has been submitted for publication and is currently under review. In related work, we recently solved the structure of a Y. pestis outer membrane protein involved in adhesion to host cells and evasion of the host complement response. We carried out functional experiments to identify binding partners for both activities and have determined that this protein produces an immune response in mice. It is currently being investigated as a potential vaccine target by the Hinnebusch lab. 2010 update: We have developed both small molecule and protein drugs targeting a Y. pestis iron transporter one manuscript is under revision and experiments in mice are currently underway. A provisional patent has been filed for the protein drug that we engineered. For the outer membrane adhesin that we solved, we identified adhesion and serum resistance targets and solved a complex structure that maps the binding site for one of the complement components. The manuscript was recently submitted. This outer membrane protein is currently being investigated for vaccine development by our collaborator, and experiments in mice have already demonstrated a robust immune response.