Large Scale Chemical Screen Against Pathogenic Bacteria

描述(申请人提供)：革兰氏阴性细菌被两层膜包围，使它们与外部世界隔绝。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依赖于它们通过这些膜输出毒素的能力，而不损害膜屏障的完整性。在用于出口丝状噬菌体的出口途径之间存在相当大的同源性，II型分泌用于将毒素或降解酶出口到细胞外环境，而III型分泌则是蛋白质类毒素被分泌并直接注入真核宿主细胞的胞浆，引起细胞毒性。 丝状噬菌体F1的输出途径形成了一个跨膜水溶液通道，在生物发生过程中该噬菌体穿过该通道。这一出口渠道的开放受到严格监管。该通道通常是关闭的，并且仅打开以允许丝状噬菌体的挤出(或者在II型或III型分泌物的情况下，允许毒素输出)。如果这个通道打开不当，宿主细菌的生存能力就会受到严重影响。该通道的通透性可以用与高通量筛选可以打开这些通道的试剂相兼容的比色分析来研究。 这些出口渠道只存在于病原菌中。它们是在致病岛上编码的，不是宿主细菌基因组的一部分。这是一个大规模化学筛查的应用程序，用于筛选影响这些通道的门控和打开它们的试剂。这可能会产生一种试剂，选择性地针对特定的病原菌，而保持其余的细菌菌群不变。来自革兰氏阴性病原体的每一种输出蛋白都将被克隆并在大肠杆菌系统中表达，该系统已经过优化，用于对开放的通道进行比色检测。然后用化学文库对这些大肠杆菌进行筛选，以检测打开这些通道的试剂。

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.