Large Scale Chemical Screen Against Pathogenic Bacteria

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DESCRIPTION (provided by applicant): Gram negative bacteria are surrounded by two membranes that shield them from the outside world. The viability of the bacteria depends upon the integrity of this membrane barrier. The pathogenicity of many gram-negative bacteria such as Yersinia, Salmonella, Shigella, Erwinia and pathogenic Escherichia coli depend upon their ability to export their toxins across these membranes without compromising the integrity of the membrane barrier. Considerable homology exists between the export pathways used for export of filamentous phage, type II secretion for the export of toxins or degradative enzymes into the extracellular milieu and type III secretion, in which proteacous toxins are secreted and injected directly into the cytosol of eukaryotic host cells, causing cytotoxicity. The export pathway for filamentous phage f1 forms a transmembrane aqueous channel ) through which the phage traverse during biogenesis. The opening of this export channel is tightly regulated. The channel is normally closed and only opens to allow the extrusion of filamentous phage (or in the case of type II or type III secretion, the export of toxin). If this channel opens inappropriately, the viability of the host bacteria is significantly compromised . The permeability of this channel can be studied with a colormetric assay that is compatible with a high-throughput screen for agents that could open these channels. These export channels only exist in pathogenic bacteria. They are encoded on the pathogenicity islands and are not part of the host bacterial genome. This is an application for a large-scale chemical screen for agents that affect the gating of these channels and open them. This could generate an agent that would selectively target a particular pathogenic bacteria and leave the rest of bacterial flora intact. Each of the export proteins from the gram-negative pathogens will be cloned and expressed in an E. coli system that has been optimized for the colorimetric detection of channels that are open. These E. coli will then be screened with a chemical library to detect agents that open these channels.