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个R型腐菌素作为杀菌剂的平台，该平台可以被设计成具有显著的病原体特异性，同时具有足够的通用性，能够快速修改其对不同范围的病原菌的特异性。具体地说，我们将针对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(包括肠炎和伤寒杆菌血清型，以及霍乱弧菌血清O1和O139)生产一系列高度特异的治疗和预防药物。提出的工作是基于我们的发现，R型腐菌素是一种高度特异和高效的杀菌蛋白复合体，已被证明对动物的全身细菌感染有效。虽然这些杀菌剂对它们的目标生物体具有高度特异性，但我们已经证明，这些杀菌剂可以通过用噬菌体相关尾部蛋白修饰它们的尾部纤维来重定向到其他细菌。因此，我们假设R型毒素可以作为一个多用途的平台来靶向广泛的人类病原菌。针对几乎每一种细菌的噬菌体的无处不在和多样性，预示着这个平台的广泛用途。为了实现我们的目标，我们将首先分离识别和杀死目标病原体的Podoviridae噬菌体，并鉴定其尾部纤维(特定目标#1)。然后，该尾部纤维将与截断的R2-型脓毒素的天然尾部纤维融合，并与剩余的脓菌素基因组一起表达，产生对感兴趣的病原体(特定目标2)具有改变的特异性的有活性的脓菌素。这一有效、有针对性的杀菌剂组合将在公共卫生管理和由这些肠道病原体广泛感染的临床方面有广泛的应用。这些特定的治疗剂可用于管理意外或故意的、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、健康的微生物群不变。重要的是，鉴于针对各种细菌的噬菌体的多样性，这种多功能平台可以开发出远远超出本文建议的特异性，对几乎所有已知的细菌具有更广泛的适用性。 公共卫生相关性：我们建议开发一个通用的、纯蛋白质为基础的平台，以防御导致严重肠道感染和潜在死亡的各种危险细菌。我们的平台基于改变基本R型腐殖素结构的结合和杀伤特异性，只附着细菌病毒的特定尾蛋白，对意外或故意通过食物或水传播的形式威胁公众健康的广泛病原体提供有效的反应。

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.