Cytotoxic mechanisms of Aggregatibacter actinomycetemcomitans leukotoxin

描述（由申请方提供）：伴放线菌聚集杆菌是一种革兰氏阴性病原体，是局部侵袭性牙周炎和其他全身性感染（包括感染性心内膜炎）的病原体。牙周炎影响美国和世界各地医疗服务不足的儿童，其特征是牙周韧带和牙槽骨结构的破坏，这些结构将牙齿固定在适当的位置。如果不进行干预，就会发生牙齿脱落，导致外观畸形和功能缺陷。A.目前尚不清楚伴放线菌引起的细菌性脑膜炎;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。基于实验和临床数据，LtxA被认为是细菌的主要毒力因子。因此，预防或干扰LtxA活性可能是治疗疾病的一种选择。这种毒素杀死人类和灵长类动物的白色血细胞，可能在A.伴随放线菌逃避感染期间的免疫反应。已经证明，在其与宿主细胞的初始反应中， 在膜中，LtxA不形成孔，而是使膜不稳定。此外，LtxA在细胞内内化，在那里它与其受体的细胞内区域结合。在 在本研究的辅导阶段，我们将探讨这两个发现的机制。在我们的第一个目标中，我们将确定负责膜不稳定的LtxA的结构域。第二个目标将使我们能够定义内化机制，并确定负责内化的LtxA结构域。在研究的独立阶段，这些机制和结构域将用于设计治疗器械。在第三个目的中，提供了一种通过干扰LtxA活性来阻断LtxA活性的装置， 与毒素的细胞靶点的关系在第四个目标中，LtxA的内化机制将被用于设计直接携带药物的药物递送装置 到细胞的胞质溶胶中。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，这项工作将导致治疗剂的开发，对LtxA和RTX毒性具有特异性活性，另一种具有更广泛的应用。在这项工作中，主要研究者将接受必要的生物技术培训 在指导阶段的研究，这将是至关重要的，她计划的职业生涯作为一个独立的调查员在微生物发病机制。拟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，将允许首席研究员过渡到一个独立的职业生涯中，她将她的工程背景应用于微生物发病机制的研究。

DESCRIPTION (provided by applicant): Aggregatibacter ctinomycetemcomitans 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, on with specific activity against LtxA and RTX toxicity and another with more general applications. During this work, the principal investigator 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 principal investigator to transition to an independent career in which she applies her engineering background to the study of microbial pathogenesis.