Cytotoxic mechanisms of Aggregatibacter actinomycetemcomitans leukotoxin

描述(申请人提供)：伴生革兰氏杆菌是一种革兰氏阴性病原体，是局限性侵袭性牙周炎(LAP)和其他全身感染的病原体，包括感染性心内膜炎。LAP在美国和世界各地都会影响医疗服务不足的儿童，其特征是支持牙齿就位的牙周膜和牙槽骨结构崩溃。如果不进行干预，就会出现牙齿脱落，导致整容畸形和功能缺陷。伴生放线菌引起LAP的机制尚不清楚，但已知它可产生几种可能的毒力因子，包括重复毒素(RTX)家族的一种白毒素(LtxA)。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，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白细胞，并可能在伴随放线菌逃避感染期间的免疫反应中发挥作用。已经证明，在它与宿主细胞的最初反应中 膜，LtxA不形成孔，而是破坏膜的稳定。此外，LtxA被内化在细胞内，在那里它与其受体的细胞内区域结合。在 在本研究的指导阶段，我们将探讨这两个发现的机制。在我们的第一个目标中，我们将确定导致膜不稳定的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Ltd.A的结构域。在研究的独立阶段，将在治疗设备的设计中利用这些机制和结构域。在第三个目标中，一种通过干扰来阻止LtxA活动的设备 随着毒素的细胞靶标将被开发出来。在第四个目标中，将利用LtxA的内化机制来设计一种直接携带药物的药物输送装置 到细胞的胞浆中。这项研究将回答有关LtxA杀死宿主细胞的机制的关键问题。此外，这项工作还将导致治疗药物的开发，这些药物具有对抗LtxA和RTX毒性的特定活性，以及另一种具有更广泛应用的药物。在这项工作中，首席调查员将接受必要的生物技术培训。 在研究的指导阶段，这将是她作为微生物发病机制独立研究员计划的职业生涯的关键。拟议的培训计划的其他要素，如课程作业、撰写补助金和寻找有指导的教员职位，将允许首席研究员过渡到独立的职业生涯，在这一职业中，她将把自己的工程学背景应用到微生物发病机制的研究中。 与公共卫生相关：牙周病是由定植在牙齿周围区域的细菌引起的感染。这是一个恶劣的环境，为了生存，细菌产生的因子将保护它免受宿主的各种防御机制的影响。我们正在研究这些因素之一--蛋白质毒素--如何通过杀死宿主白细胞来帮助细菌生存的机制。

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. PUBLIC HEALTH RELEVANCE: Periodontal diseases are infections caused by bacteria that colonize the area around the teeth. This is a harsh environment, and to survive, a bacterium produces factors that will protect it from the various defense mechanisms of the host. We are studying the mechanism of how one of these factors a protein toxin, helps the bacteria to survive by killing the host white blood cells.