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（鲍曼不动杆菌）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。该病原体对现有抗生素的广泛耐药性的进一步出现，强调了对真正新型抗菌疗法的需求。我们之前设计了一种自然产生的杀菌蛋白，r型脓毒素，它可以杀死脓毒素自然杀伤范围之外的细菌病原体，并通过其尾部纤维首先与病原体表面可到达的目标毒力因子结合。结果，病原体对工程化脓毒素产生抗药性，毒性受损。在此，我们建议研制鲍曼不动杆菌杀菌剂。r型pyocins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，可特异性杀死同一物种的其他菌株。由于r型pyocins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pyocins不受细菌耐药机制的影响。我们的目标是产生具有新型结合表型的尾纤维，这些尾纤维可以被选择并整合到r型脓毒蛋白中。多样性生成逆转录因子（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）的合作下，我们将分离出从受伤的战士身上分离出的鲍曼不动杆菌表面可达毒力因子的尾部纤维编码基因，并利用这些基因制造出对多种病原体具有特异性杀菌作用的r型脓毒杆菌。工程R型脓毒蛋白将在体外和体内进行有效性评估，后者在WRAIR伤口感染动物模型中进行评估。选择鲍曼不动杆菌对靶向毒力因子的工程化脓毒杆菌产生抗性的突变体，应在同一动物模型中评估其致病性。这将使我们能够确定耐药鲍曼不动杆菌生物体内的毒力是否受损，正如我们之前所显示的那样，大肠杆菌O157:H7突变体对一种r型脓毒素具有抗性，该r型脓毒素被设计为针对其毒力因子之一。

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.