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

描述(由申请人提供)：鲍曼不动杆菌(A.baumannii)是一种人类病原体，导致最近军队和平民人群医院感染的增加。病原体对现有抗生素的广泛耐药性的进一步出现，强调了真正新颖的抗菌疗法的必要性。我们此前已经设计了一种自然产生的杀菌蛋白--R-型毒素，它可以杀死超出毒素自然杀伤光谱的细菌病原体，并首先通过其尾部纤维与病原体表面可获得的靶向毒力因子结合来杀死细菌。因此，出现了对工程化腐菌素产生抗药性的病原体，从而降低了毒力。在此，我们建议为鲍曼不动杆菌创造这样的杀菌剂。R型腐菌素是一些铜绿假单胞菌产生的高分子量蛋白质复合体，可以特异性地杀死同一物种的其他菌株。由于R型腐菌素及其工程化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型腐菌素不受细菌抗药性机制的影响。我们的目标是产生具有新的结合表型的尾部纤维，这些表型可以被选择并整合到R型腐殖素中。多样性产生逆转录元件(DGRs)是一种新发现的使DNA序列及其编码的蛋白质多样化的手段。DGR提供了一种方法来生成在精确位置高度可变的蛋白质文库，并可用于选择具有所需物理、化学或生物特性的变体。在与沃尔特里德陆军研究所(WRAIR)的合作中，我们将分离编码针对鲍曼不动杆菌表面可及毒力因子的尾部纤维的基因，并部署这些基因来创造对多种病原体具有特定杀菌作用的R型毒素。在WRAIR的伤口感染动物模型中，将对工程R型腐殖素在体外和体内的有效性进行评估。应在相同的动物模型中评估鲍曼不动杆菌对针对毒力因子的工程腐菌素的抗药性突变株的致病性。这将使我们能够确定耐药鲍曼不动杆菌是否在体内损害了毒力，正如我们之前所显示的那样，对R型腐菌素的抗药性突变体O157：H7发生了这种情况，这种突变体是针对其毒力因子之一而设计的。 与公共卫生相关：鲍曼不动杆菌是一种在土壤和水中很容易发现的细菌，具有神奇的抵抗新旧抗生素的能力。这些细菌通常对健康的人类无害，但在住院患者中发现了一个重要而危险的利基，无论是平民还是军人，它们会导致长期和严重的发病率，表现为广泛耐药的伤口和骨感染、肺炎和尿路感染。我们之前已经设计了自然产生的抗菌蛋白质颗粒，称为R型腐殖素，将它们的细菌杀灭能力重新定向到它们不能自然杀死的致病细菌。我们建议创造一种R型毒素，专门杀死鲍曼不动杆菌。重要的是，R型毒素杀死细菌的机制与任何抗生素使用的机制完全不同；因此，R型毒素不受细菌用来抵抗抗生素的机制的影响。我们以前已经设计了R型腐殖素，通过首先与细菌表面的分子结合来靶向细菌，这些分子是细菌致病所需的(“毒力因子”)。为了对目标毒素产生抗药性，细菌必须失去或修改这种毒力因子，并损害它们引起感染的能力。在与沃尔特里德陆军研究所(WRAIR)的合作中，我们希望改造R型毒素，以靶向从受伤士兵中分离的鲍曼不动杆菌表面普遍存在的毒力因子。此外，我们还将与WRAIR合作，在鲍曼不动杆菌感染伤口的动物模型中评估我们的工程化脓菌素的有效性。

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.