Create, evaluate and develop pre-clinically an engineered R-type pyocin to specif

性状（由申请方提供）：鲍曼不动杆菌（A.鲍曼不动杆菌）是一种人类病原体，是近来军队和平民人群医院感染增加的原因。病原体对可用抗生素的广泛耐药性的进一步出现强调了对真正新颖的抗菌治疗剂的需要。我们之前已经设计了一种天然存在的杀菌蛋白，R型绿脓菌素，以杀死绿脓菌素的天然杀伤谱之外的细菌病原体，并通过首先经由其尾纤维结合到病原体表面上可接近的靶向毒力因子来杀死。因此，对工程化绿脓菌素产生抗性的病原体具有受损的毒力。在此，我们建议创建这样的杀菌剂，为A。鲍曼不动杆菌。 R型绿脓菌素是由一些铜绿假单胞菌菌株产生的高分子量蛋白质复合物，其特异性地杀死相同物种的其他菌株。由于R型绿脓菌素及其工程衍生物的杀菌机制与传统抗生素的作用机制完全不同，因此R型绿脓菌素不受细菌抵抗抗生素的机制的影响。我们的目标是产生具有新的结合表型的尾纤维，其可以被选择并整合到R型绿脓菌素中。 Diversity Generating Retroelements（DGRs）是一种新发现的使DNA序列及其编码的蛋白质多样化的方法。DGR提供了一种产生蛋白质文库的方法，所述蛋白质文库在精确位置高度可变，并且可用于选择具有所需物理、化学或生物学特性的变体。 在与沃尔特里德陆军研究所（WRAIR）的合作中，我们将分离出编码尾纤维的基因，这些尾纤维靶向A.鲍曼不动杆菌从受伤的战士中分离出来，并利用这些基因产生R型绿脓菌素，对多种病原体菌株具有特异性杀菌作用。工程化的R型绿脓菌素应在体外和体内评估其功效，后者在WRAIR的伤口感染动物模型中进行。 突变体A.选择对靶向毒力因子的工程化绿脓菌素具有抗性的鲍曼不动杆菌应在相同的动物模型中评估其致病性。这将使我们能够确定是否耐药A。鲍曼不动杆菌在体内的毒力降低，正如我们以前所证明的，大肠杆菌O 157：H7突变体对R型绿脓菌素具有抗性，该R型绿脓菌素被工程化以靶向其毒力因子之一。 公共卫生相关性：鲍曼不动杆菌是一种容易在土壤和水中发现的细菌，具有抵抗新旧抗生素的不可思议的能力。这些细菌通常对健康人无害，但在住院患者中发现了一个重要而危险的利基，无论是平民还是军事，它们以广泛耐药的伤口和骨骼感染，肺炎和尿路感染的形式引起长期和严重的发病率。 我们之前已经设计了天然存在的抗菌蛋白颗粒，称为R型绿脓菌素，将其细菌杀伤能力重新定向到它们不会自然杀死的致病细菌。我们建议创造R型绿脓菌素，杀死A。尤其是鲍曼不动杆菌。重要的是，R型绿脓菌素通过与任何抗生素所使用的机制完全不同的机制杀死细菌;因此，R型绿脓菌素不受细菌用于抵抗抗生素的机制的影响。我们之前已经通过首先结合细菌表面上的分子（细菌引起疾病所需的分子）来工程化R型绿脓菌素以靶向细菌（“毒力因子”）。为了对靶向绿脓菌素产生抗性，细菌必须失去或修改该毒力因子并损害其引起感染的能力。 在与沃尔特里德陆军研究所（WRAIR）的合作中，我们希望设计R型绿脓菌素，以靶向A.从伤兵身上分离出的鲍曼不动杆菌。我们还将与WRAIR合作，评估我们的工程化绿脓菌素在A.鲍曼不动杆菌。

DESCRIPTION (provided by applicant): Acinetobacter baumannii (A. baumannii) is a human pathogen responsible for recent increases in nosocomial infections of both military and civilian populations. Further emergence of the pathogen's broad resistance to available antibiotics has emphasized the need for truly novel antibacterial therapeutics. We have previously engineered a naturally occurring bactericidal protein, R-type pyocin, to kill bacterial pathogens beyond the natural killing spectrum of the pyocin and to kill by first binding via its tail fibers to a targeted virulence factor accessible on the pathogen's surface. As a result, pathogens emerging resistant to the engineered pyocin have compromised virulence. Herein we propose to create such bactericidal agents for A. baumannii. R-type pyocins are high molecular weight protein complexes produced by some Pseudomonas aeruginosa strains and which specifically kill other strains of the same species. Because the bactericidal mechanism of R-type pyocins and their engineered derivatives is completely different from the mechanisms of action of traditional antibiotics, R-type pyocins are unaffected by the mechanisms deployed by bacteria to resist antibiotics. Our goal is to generate tail fibers with novel binding phenotypes that can be selected and integrated into R-type pyocins. Diversity Generating Retroelements (DGRs) are a newly discovered means to diversify DNA sequences and the proteins they encode. The DGR provides a means to generate libraries of proteins that are highly variable at precise locations and can be used to select variants with desired physical, chemical or biological properties. In collaboration with Walter Reed Army Institute of Research (WRAIR) we shall isolate genes encoding tail fibers that target surface accessible virulence factors of A. baumannii isolates from wounded warfighters and deploy those genes to create R-type pyocins specifically bactericidal for multiple strains of the pathogen. The engineered R- type pyocins shall be evaluated for their efficacy in vitro and in vivo, the latter in an animal model of wound infection at WRAIR. Mutants of A. baumannii selected for resistance to engineered pyocins that target virulence factors shall be assessed for their pathogenicity in the same animal model. This will allow us to determine whether the resistant A. baumannii organisms have compromised virulence in vivo, as we have previously shown occurs for E. coli O157:H7 mutants resistant to an R-type pyocin that was engineered to target one of its virulence factors. PUBLIC HEALTH RELEVANCE: Acinetobacter baumannii is a species of bacteria readily found in soil and water and has an uncanny ability to resist old and new antibiotics. These bacteria are usually harmless to healthy humans but have found a significant and dangerous niche in hospitalized patients, both civilian and military, where they cause prolonged and serious morbidity in the form of broadly antibiotic-resistant wound and bone infections, pneumonia, and urinary tract infections. We have previously engineered naturally occurring antibacterial protein particles, called R-type pyocins, to redirect their bacterial killing ability towards disease-causing bacteria that they do not kill naturally. We propose to create R-type pyocins that kill A. baumannii specifically. Importantly, R-type pyocins kill bacteria by a mechanism completely different than those mechanisms used by any antibiotic; and therefore, R- type pyocins are not subject to the mechanisms that bacteria use to resist antibiotics. We have previously engineered R-type pyocins to target bacteria by first binding to molecules on the bacterial surface that are required for the bacteria to cause disease ("virulence factors"). To become resistant to the targeted pyocin, bacteria must lose or modify that virulence factor and compromise their ability to cause infections. In collaboration with Walter Reed Army Institute of Research (WRAIR) we expect to engineer R-type pyocins to target virulence factors prevalent on the surfaces of A. baumannii bacteria isolated from wounded soldiers. Also in collaboration with WRAIR we shall evaluate the effectiveness of our engineered pyocins in an animal model of wounds infected by A. baumannii.