Cytotoxic mechanisms of Aggregatibacter actinomycetemcomitans leukotoxin

4.4.7项目总结 伴放线聚集杆菌是一种革兰氏阴性病原体，是局部性放线菌病的病原体。 侵袭性牙周炎（aggressive periodontitis，牙周炎）和其他全身性感染，包括感染性心内膜炎。，其中 影响美国和世界各地医疗服务不足的儿童，其特点是 牙周韧带和牙槽骨结构的破坏，使牙齿保持在适当的位置。没有 如果进行干预，会发生牙齿脱落，导致外观畸形和功能缺陷。的方式 其中A。伴放线菌引起放线菌病的原因尚不清楚;然而，已知它产生几种 推定的毒力因子，包括白细胞毒素（LtxA），毒素重复序列（RTX）家族的成员。基于 根据实验和临床数据，LtxA被认为是该细菌的主要毒力因子。因此，在本发明中， 预防或干扰LtxA活性可能是治疗疾病的一种选择。这种毒素会杀死人类 和灵长类动物的白色血细胞，并可能在A.伴放线菌逃避免疫 感染时的反应。 已经证明，在其与宿主细胞膜的初始反应中，LtxA不形成孔， 而是使膜不稳定。此外，LtxA在细胞内内化，在细胞内它结合到细胞内的蛋白质。 其受体的胞内区域。 在本研究的指导阶段，我们将研究这两个发现的机制。我们的第一个目标， 我们将鉴定LtxA的结构域，其负责膜去稳定化。第二 目的将使我们能够定义内化机制，并确定LtxA的结构域， 负责内化。在研究的独立阶段，这些机制和结构 将在治疗设备的设计中利用这些领域。在第三个目标中，阻断LtxA活性的装置 通过干扰毒素的细胞靶点。第四个目标，内部化 LtxA的机制将被用于设计药物递送装置以将药物直接携带到胞质溶胶 一个细胞。 这项研究将回答有关LtxA杀死宿主细胞的机制的重要问题。此外该 这项工作将导致治疗剂的开发，一种对LtxA和RTX毒性具有特异性活性的治疗剂 而另一个具有更一般的应用。 在这项工作中，PI将在指导阶段接受必要的生物技术培训。 这项研究对她作为微生物发病机理独立研究者的计划职业生涯至关重要。 建议的培训计划的其他要素，如课程，赠款写作和指导教师 职位搜索，将允许PI过渡到一个独立的职业生涯中，她应用她的工程 微生物致病机理研究的背景。

4.4.7 Project Summary Aggregatibacter actinomycetemcomitans is a Gram-negative pathogen that is the etiologic agent of localized aggressive periodontitis (LAP) and other systemic infections, including infective endocarditis. LAP, which affects medically-underserved children in both the United States and throughout the world, is characterized by a breakdown of the periodontal ligament and alveolar bone structure that holds the teeth in place. Without intervention, loss of teeth occurs, causing both a cosmetic deformity and a functional defect. The manner in which A. actinomycetemcomitans causes LAP is not known; however, it is known that it produces several putative virulence factors, including a leukotoxin (LtxA), a member of the repeats-in-toxin (RTX) family. Based on experimental and clinical data, LtxA is believed to be a primary virulence factor for the bacterium. Thus, preventing or interfering with LtxA activity may be one option for treatment of disease. The toxin kills human and primate white blood cells and likely plays a role in A. actinomycetemcomitans evasion of the immune response during infection. It has been demonstrated that in its initial response with the host cell membrane, LtxA does not form a pore, but rather destabilizes the membrane. Additionally, LtxA is internalized within the cell, where it binds to the intracellular region of its receptor. In the mentored phase of this study, we will investigate the mechanisms of these two findings. In our first aim, we will identify the structural domains of LtxA that are responsible for membrane destabilization. The second aim will allow us to define the mechanism of internalization and determine the structural domains of LtxA that are responsible for internalization. In the independent phase of the study, these mechanisms and structural domains will be exploited in the design of therapeutic devices. In the third aim, a device to block LtxA activity by interfering with the cellular targets of the toxin will be developed. In the fourth aim, the internalization mechanism of LtxA will be utilized in the design of a drug-delivery device to carry a drug directly to the cytosol of a cell. The research will answer vital questions about the mechanisms by which LtxA kills host cells. In addition, the work will lead to the development of therapeutic agents, one with specific activity against LtxA and RTX toxicity and another with more general applications. During this work, the PI will be trained in the necessary biological techniques during the mentored phase of the research that will be vital to her planned career as an independent investigator in microbial pathogenesis. Other elements of the proposed training plan, such as coursework, grant-writing, and a mentored faculty position search, will allow the PI to transition to an independent career in which she applies her engineering background to the study of microbial pathogenesis.