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

描述（由申请人提供）：我们的建议是开发一种R型绿脓菌素作为杀菌剂的平台，其可以被工程化为具有显著的病原体特异性，同时具有足够的通用性以能够快速修饰其对多种病原菌的特异性。具体而言，我们将产生一系列高度特异性的治疗和预防剂，靶向STEC血清群O26、O45、O55、O91、O103、O111、O113、O118、O121、O145、O146以及肠道沙门氏菌血清群D1，包括血清型痢疾和伤寒，以及霍乱弧菌血清型O1和O139。提出的工作是基于我们的发现，R型绿脓菌素是高度特异性和高效的杀菌蛋白复合物，已被证明是有效的全身性细菌感染的动物。虽然对它们的靶生物具有高度特异性，但我们已经表明，这些杀菌剂可以通过用噬菌体的相关尾蛋白修饰它们的尾纤维而重新靶向其他细菌。因此，我们假设R型绿脓菌素可以用作靶向广泛的人类病原菌的通用平台。针对几乎所有类型细菌的特异性抗菌素的普遍性和多样性，为该平台提供了广泛的实用性。为了实现我们的目标，我们将首先分离出能够识别和杀死感兴趣的病原体并鉴定其尾纤维的短尾病毒科噬菌体（特异性目标#1）。然后将该尾纤维融合到R2型绿脓菌素的截短的天然尾纤维，并与剩余的绿脓菌素基因组一起表达，产生具有针对感兴趣的病原体的改变的特异性的可行的绿脓菌素（特异性目标#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和临床方面的管理中具有广泛的应用。这些特定的治疗剂可用于管理意外或有意的、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，健康的微生物菌群完整。重要的是，考虑到可用于对抗多种细菌的噬菌体的多样性，可以开发出远超出本文提出的特异性的通用平台，对几乎所有已知细菌具有更广泛的适用性。 公共卫生相关性：我们建议开发一种多功能的纯蛋白质平台，用于防御各种导致严重肠道感染和潜在死亡的危险细菌。我们的平台基于通过仅连接细菌病毒的特定尾蛋白来改变基本R型绿脓菌素结构的结合和杀伤特异性，对以意外或故意的食物或水传播爆发的形式对公共卫生构成威胁的各种病原体提供了有力的响应。

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.