Converting staphylococcal pathogenicity islands from malevolence to benevolence

项目概要/摘要 金黄色葡萄球菌，导致各种各样的危及生命的感染，其中许多是无法治疗的 由于抗生素耐药性，因此，迫切需要新的方法来治疗这些疾病。 感染，每年在美国造成约18，000人死亡。我们开发了一种新型的非抗生素 基于高度移动的葡萄球菌致病岛治疗葡萄球菌感染的方法 （SaPI）。SaPI是稳定插入葡萄球菌染色体中的~15 kb遗传元件，但可以通过插入葡萄球菌染色体中的基因片段来表达。 由“辅助”细胞诱导切除和复制。复制的SaPI DNA被包装在感染性噬菌体中- 类似于在噬菌体诱导的裂解时释放的颗粒。SaPI携带并传播编码 超抗原毒素和其他毒力因子。我们没有致力于预防SaPI的传播， 基于利用SaPI传播的想法，通过将这些疾病因子转化为治疗因子- 抗菌无人机（ABD）。为了创建ABD，我们重新设计了SAPI，删除了它们的自然 货物（毒素基因），将其包装容量从15 kb增加到>40 kb，并插入抗菌模块。 我们还修饰了辅助噬菌体，使得ABD颗粒在功能性噬菌体缺失的情况下产生。 噬菌体。将ABD颗粒施用于受感染的动物（或植物），在那里它们附着到感染的细胞（或组织）上。 细菌，插入它们的DNA，表达它们的抗菌货物基因，从而消除感染。就证明了 原则上，我们已经开始通过将CRISPR/cas9或CRISPR/dcas 9模块与 分别靶向染色体基因或全局毒力调节因子启动子区的间隔区。 初步研究表明，含有CRISPR/cas9的ABD可以杀死S.金黄色葡萄球菌体外DNA 裂解，阻断皮下S.金黄色葡萄球菌脓肿，并拯救给予致命剂量的小鼠 色葡萄金黄色葡萄球菌。含有ABD的CRISPR/dcas 9阻断葡萄球菌表达 在体外的毒力，并在体内阻止皮下脓肿的形成。该提案概述了我们的 计划开发ABD系统，并验证我们的基本假设，即SaPI可以转换为 多用途和完全有效的抗葡萄球菌治疗剂。有三个具体目标。在Aim I中，我们将 构建和测试ABD，用于治疗由S.金黄色。在Aim II中，我们将 重点是扩大ABD的宿主范围，以针对不同的S。金黄色葡萄球菌菌株。在Aim III中，我们将 纯化ABD颗粒以提高疗效，检测ABD耐药性和免疫原性， 我们生产和保存高滴度ABD制剂的程序。

Project summary/Abstract Staphylococcus aureus, causes a wide variety of life-threatening infections, many of which cannot be treated effectively, owing to antibiotic resistance. Consequently, there is an urgent need for new ways to treat these infections, which annually cause some 18,000 deaths in the US. We have developed a novel non-antibiotic method, for treating staphylococcal infections based on the highly mobile staphylococcal pathogenicity islands (SaPIs). The SaPIs are ~15 kb genetic elements that are stably inserted in the staph chromosome but can be induced by “helper” phages to excise and replicate. The replicated SaPI DNA is packaged in infectious phage- like particles which are released upon phage-induced lysis. The SaPIs carry and disseminate genes encoding superantigen toxins and other virulence factors. Instead of working on the prevention of SaPI spread, we hit upon the idea of exploiting SaPI spread by converting these agents of disease into agents of therapy – antibacterial drones (ABDs). To create the ABDs, we have re-engineered the SaPIs, deleting their natural cargo (toxin genes), increasing their packaging capacity from 15 to >40 kb, and inserting antibacterial modules. We have also modified the helper phage so that ABD particles are produced in the absence of functional phage. The ABD particles are administered to an infected animal (or plant), where they attach to the infecting bacteria, insert their DNA, express their antibacterial cargo genes and thus abrogate the infection. As proof of principle, we have begun by incorporating into ABDs either CRISPR/cas9 or CRISPR/dcas9 modules with spacers targeting a chromosomal gene or the promoter region of a global virulence regulator, respectively. Preliminary studies have shown that the CRISPR/cas9-containing ABD kills S. aureus in vitro by DNA cleavage, blocks the development of a subcutaneous S. aureus abscess, and rescues mice given a lethal dose of S. aureus intraperitoneally. The CRISPR/dcas9 containing ABD blocks the expression of staphylococcal virulence in vitro and blocks the formation of a subcutaneous abscess in vivo. This proposal outlines our program to develop the ABD system and validate our underlying hypothesis that SaPIs can be converted to versatile and fully effective anti-staphylococcal therapeutic agents. There are 3 specific aims. In Aim I we will construct and test ABDs designed to treat the wide array of infections caused by S. aureus. In Aim II, we will focus on expanding the host range of the ABDs to target diverse S. aureus strains. In Aim III, we will encapsidate the ABD particles for increased efficacy, test for ABD resistance and immunogenicity, and refine our procedures for producing and preserving high titer ABD preparations.