Generating a library of antimicrobial R-type pyocins against enteric bacteria

描述（由申请人提供）：我们的提案是开发一个r型脓毒杆菌作为杀菌剂的平台，该平台可以被设计成具有显著的病原体特异性，同时足够通用，能够快速修改其对各种致病菌的特异性。具体来说，我们将针对产志在大肠杆菌血清群O26、O45、O55、O91、O103、O111、O113、O118、O121、O145、O146，以及肠沙门氏菌血清群D1（包括肠炎和伤寒血清型）和霍乱弧菌血清型O1和O139，开发一系列高度特异性的治疗和预防药物。提出的工作是基于我们的发现，r型脓毒蛋白是高度特异性和高效的杀菌蛋白复合物，已被证明对动物全身细菌感染有效。虽然对目标生物具有高度特异性，但我们已经证明，这些杀菌剂可以通过用噬菌体的相关尾部蛋白修饰其尾部纤维来重新靶向其他细菌。因此，我们假设r型pyocins可以作为一个通用平台来靶向广泛的人类致病菌。噬菌体的普遍性和多样性对几乎所有类型的细菌都具有特异性，这为该平台提供了广泛的应用前景。为了实现我们的目标，我们将首先分离出能够识别和杀死目标病原体并识别其尾部纤维的足病毒科噬菌体（Specific Aim #1）。然后，该尾纤维将融合到r2型脓毒蛋白的截短的天然尾纤维中，并与剩余的脓毒蛋白基因组一起表达，从而产生具有针对感兴趣病原体特异性改变的活脓毒蛋白（Specific Aim #2）。这种有效的靶向杀菌剂组合将在这些肠道病原体广泛感染的公共卫生管理和临床方面具有广泛的应用。这些特定的治疗药物可用于管理意外或故意，大规模或小规模的感染。由于其非凡的特异性，它们有望消除目标病原体，同时保持正常，健康，完整的微生物群。重要的是，鉴于噬菌体对多种细菌的多样性，这种多功能平台可以开发出远远超出本文所提出的特异性，对几乎所有已知细菌具有更广泛的适用性。

DESCRIPTION (provided by applicant): Our proposal is to develop a platform of R-type pyocins as bactericidal agents that can be engineered to be remarkably pathogen-specific, while versatile enough to enable rapid modification of their specificity towards a diverse range of pathogenic bacteria. Specifically, we will generate a battery of highly specific therapeutic and prophylactic agents targeting STEC serogroups O26, O45, O55, O91, O103, O111, O113, O118, O121, O145, O146, as well as Salmonella enterica serogroup D1, including serotypes enteriditis and typhi, and Vibrio cholera serotypes O1 and O139. The work proposed is based on our findings that R-type pyocins are highly specific and highly potent bactericidal protein complexes that have been shown to be efficacious for systemic bacterial infections in animals. While highly specific to their target organism, we have shown that these bactericidal agents can be retargeted to other bacteria by modifying their tail fibers with the related tail proteins of bacteriophages. We therefore hypothesize that R-type pyocins can be used as a versatile platform to target a broad range of human pathogenic bacteria. The ubiquity and diversity of phages specific against virtually every type of bacteria, promise broad utility for this platform. In order to achieve our goals, we will first isolate Podoviridae bacteriophages that recognize and kill the pathogen of interest and identify its tail fiber (Specific Aim #1). That tail fiber will then be fused to a truncated native tail fiber of the R2-type pyocin and expressed together with the remaining pyocin genome, creating a viable pyocin with altered specificity against the pathogen of interest (Specific Aim #2) This portfolio of potent, targeted bactericidal agents will have broad applications in the management of the public health and clinical aspects of widespread infection by these enteric pathogens. These specific therapeutic agents could be used to manage accidental or intentional, large- or small scale infections. Due to their extraordinary specificity, they are expected to eliminate the target pathogens while leaving normal, healthy, microflora intact. Importantly, given the diversity of bacteriophages available against a wide array of bacteria, this versatile platform can be developed for specificities far beyond what is proposed herein, have far wider applicability against nearly all known bacteria. PUBLIC HEALTH RELEVANCE: We propose to develop a versatile, pure protein-based platform for defense against a diverse array of dangerous bacteria that cause severe intestinal infections and potentially death. Our platform, based on altering the binding and killing specificity of the basic R-type pyocin structure by attaching only the specific tail proteins of bacterial viruses, offers a potent response to a wide range of pathogens which pose a threat to public health in the form of accidental or intentional food- or waterborne outbreak.