Molecular mechanisms and novel biological-based therapies for anthrax lethal toxin-induced mortality

摘要 炭疽杆菌是炭疽病的病原体，自2000年以来一直是生物恐怖主义的首要问题。 2001年炭疽袭击B。炭疽病通过细菌感染和毒血症的组合引起炭疽病。作为 炭疽致死毒素（LT）是炭疽病的主要毒力因子，在炭疽病的多个阶段起着重要作用。 由于炭疽病病程迅速，特别是吸入性肺炎的非特异性流感样症状， 炭疽病患者通常在疾病已处于中/晚期时寻求医疗援助， 炭疽病患者的临床治疗是一项极具挑战性的任务。目前的治疗方法包括抗生素 以及分别消灭病原体和中和毒素的抗毒素抗体。但没有 可用于处理由已经达到其分子水平的LT引起的细胞/组织损伤的疗法 细胞内的目标。当宿主不能修复这种损伤时，死亡通常随之而来，即所谓的“无点”， 返回”当前治疗。因此，即使有重症医学护理，系统性炭疽的死亡率也很高， 达到> 50%。因此，迫切需要开发更好的靶向治疗以避免肿瘤复发。 炭疽病引起的死亡。我们在这项应用中的目标是发现LT的分子机制， 诱导致死性，并开发潜在的靶向治疗，以治疗患者超越“不归点”。 在这里，我们着手确定破坏炭疽中ERK、p38和JNK通路的具体作用。 诱导致死性，发现潜在的分子机制，并发展再激活的概念 /动员这些途径作为炭疽诱导死亡率的靶向治疗。在目标1中，我们将确定 特异性阻断ERK通路在炭疽致死中的作用及探讨ERK通路 作为靶向治疗的重新激活。在LT靶向的三种核心MAPK途径中，ERK途径是 对许多生物过程至关重要，包括细胞增殖和存活。因此，我们假设， 破坏ERK途径是炭疽诱导致死的主要原因。我们将创造和使用新的 含有MEK 1和MEK 2等位基因的小鼠模型对LT切割具有抗性，以了解精确的 ERK途径失活在炭疽发病机制中的作用。我们将进一步验证这一假设，并探讨ERK 通路再激活作为炭疽诱导的组织损伤的靶向治疗。重要的是，我们的初步数据 证明LT破坏的ERK途径可以通过加入有效的有丝分裂原重新激活，如 表皮生长因子在目标2中，我们将进一步确定破坏p38和JNK通路在细胞凋亡中的作用。 炭疽发病机制，并探讨在炭疽靶向治疗调动这些途径的可行性。 在完成这些研究后，我们期望我们将在以下方面提供重大的概念性进展： 我们对炭疽LT的潜在分子机制的理解，并提供了一个基于证据的 制定炭疽靶向疗法的框架，这将补充目前的疗法， 抗生素和抗毒素抗体，以防止炭疽死亡，即使在炭疽感染的晚期。

Abstract Bacillus anthracis, the causative agent of anthrax disease, has remained as a top bioterrorism concern since the 2001 anthrax attack. B. anthracis causes anthrax through a combination of bacterial infection and toxemia. As a major virulence factor, the anthrax lethal toxin (LT) plays an essential role during multiple steps of the disease. Due to the rapid course of anthrax disease, in particular, the non-specific, flu-like symptoms of inhalational anthrax, patients usually seek medical assistance when the disease is already in the middle/late stages, making the clinical management of anthrax patients an extremely challenging task. Current treatments include antibiotics and anti-toxin antibodies that respectively eliminate the pathogen and neutralize the toxin. However, there is no therapy available to deal with the cellular/tissue damage caused by LT already having reached its molecular targets inside cells. Mortality usually follows when the host fails to repair this damage, the so called “point-of-no- return” for current therapy. Thus, even with intensive medical care, the mortality rate of systemic anthrax is high, reaching > 50%. Therefore, there is an urgent unmet clinical need to develop better targeted therapies to avert anthrax -induced mortality. Our goal in this application is to discover the molecular mechanisms underlying LT- induced lethality and to develop potential targeted therapeutics to treat patients beyond the “point-of-no-return”. Here, we set out to determine the specific roles of disrupting each of the ERK, p38, and JNK pathways in anthrax- induced lethality, discover the underlying molecular mechanisms, and develop the concept of reactivation /mobilization of these pathways as a targeted therapy for anthrax-induced mortality. In Aim 1, we will determine the role of specifically disrupting the ERK pathway in anthrax-induced lethality and explore ERK pathway reactivation as a targeted therapy. Among the three core MAPK pathways targeted by LT, the ERK pathway is fundamental to many biological processes, including cell proliferation and survival. Thus, we hypothesize that disrupting the ERK pathway is the major cause of anthrax-induced lethality. We will generate and use novel mouse models containing MEK1 and MEK2 alleles that are resistant to LT-cleavage to understand the precise role of ERK pathway inactivation in anthrax pathogenesis. We will further test this hypothesis and explore ERK pathway reactivation as a targeted therapy for anthrax-induced tissue damage. Importantly, our preliminary data demonstrate that the LT-disrupted ERK pathway can be reactivated by the addition of potent mitogens, such as epidermal growth factor. In Aim 2, we will further determine the roles of disrupting the p38 and JNK pathways in anthrax pathogenesis and explore the feasibility of mobilizing these pathways in anthrax-targeted therapy. Upon completion of these studies, it is our expectation that we will provide significant conceptual advances in our understanding of the underlying molecular mechanisms of anthrax LT and offer an evidence-based framework for developing anthrax-targeted therapies, which will complement the current therapies with antibiotics and anti-toxin antibodies, to prevent anthrax mortality, even at advanced stages of anthrax infection.