Large Scale Chemical Screen Against Pathogenic Bacteria

描述（由申请人提供）：革兰氏阴性菌被两层膜包围，使其与外界隔绝。细菌的生存能力取决于这种膜屏障的完整性。许多革兰氏阴性细菌如耶尔森氏菌、沙门氏菌、志贺氏菌、欧文氏菌和致病性大肠杆菌的致病性取决于它们将其毒素输出穿过这些膜而不损害膜屏障的完整性的能力。在用于丝状噬菌体输出的输出途径、用于将毒素或降解酶输出到细胞外环境中的II型分泌和III型分泌之间存在相当大的同源性，在III型分泌中，蛋白毒素被分泌并直接注入真核宿主细胞的胞质溶胶中，引起细胞毒性。 丝状噬菌体f1的输出途径形成跨膜水通道，噬菌体在生物发生期间穿过该跨膜水通道。这一出口渠道的开放受到严格管制。该通道通常是封闭的，并且仅打开以允许丝状噬菌体的挤出（或在II型或III型分泌的情况下，毒素的输出）。如果这个通道不适当地打开，宿主细菌的生存能力就会受到严重损害。该通道的渗透性可以用比色测定来研究，该比色测定与可以打开这些通道的试剂的高通量筛选相容。 这些输出通道只存在于致病菌中。它们在致病岛上编码，不是宿主细菌基因组的一部分。这是一个大规模化学筛选的应用程序，用于影响这些通道的门控并打开它们。这可以产生一种试剂，它可以选择性地靶向特定的病原菌，而使其余的细菌植物群保持完整。从革兰氏阴性病原体中的每种输出蛋白将被克隆并在E. coli系统，该系统已被优化用于开放通道的比色检测。这些E.然后用化学文库筛选大肠杆菌，以检测打开这些通道的物质。

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.