Analyzing a novel mechanism of action of bacterial cAMP producing toxins

描述（由申请人提供）：我们最近发现炭疽芽孢杆菌的关键毒力因子，水肿因子（EF）和霍乱弧菌，霍乱毒素（Ctx），都抑制蛋白质运输到细胞-细胞连接处，这是范式转变。EF是一种高活性的腺苷酸环化酶，Ctx adp核糖化Gsα亚基，组成性地激活宿主腺苷酸环化酶。这两种产生cAMP的毒素降低了细胞黏附分子（如钙粘蛋白）和信号蛋白（如Notch成分）内吞再循环的最后一步所需的小GTPase （Rab11）的水平和活性，从而导致血管内皮（EF）或肠上皮（Ctx）的破坏。EF和Ctx的这种新作用在果蝇模型系统中被发现并进行了遗传解剖，这些细胞生物学机制转化为毒素作用于人血管内皮细胞（EF）和肠上皮细胞（Ctx）以及小鼠体内（EF和Ctx）。另一个具有重要实际意义的关键发现是，Rab11的过表达可以逆转EF和Ctx在果蝇体内和人类细胞中的连接破坏作用。在当前修订的拨款中，我们提出了三个综合目标，以阐明介导EF和Ctx的屏障破坏作用的途径，并分析这种新的细胞生物学机制在疾病发病机制中的影响。在Aim 1中，我们将研究EF/Ctx产生的高持续水平cAMP降低Rab11蛋白水平以破坏连接运输的途径，并探索EF和Ctx在免疫细胞中抑制囊泡功能的新潜在功能。在目标2中，我们将研究抑制内吞循环如何促进炭疽感染期间人类细胞单层和脉管系统的渗漏。由于血管塌陷是炭疽热的常见死亡原因，我们还将确定增加Rab11水平或使用已知的交通促进剂治疗是否可以逆转EF引起的血管渗漏。在Aim 3中，我们将类似地研究胞囊抑制对大量液体分泌的贡献，这是霍乱的病理特征，以及通过遗传或药物手段提高内吞循环是否在体内具有保护作用。拟议的研究对治疗炭疽具有重要的转化意义，因为当患者开始寻求医疗干预时，当抗生素不再有效时，毒素可能达到临界致死水平。因此，基于恢复内吞循环的治疗可以与现有的抗毒素治疗（例如，抗毒素抗体，小分子抑制剂）结合使用，以中和已经存在于循环中的毒素。交通促进剂的一个优点是，当血管完整性崩溃和其他器官系统衰竭时，它们会在最后一步进行干预。这种促进交通的化合物还可能提高液体替代品治疗霍乱和其他破坏屏障的疾病的功效，包括：缺血、哮喘、皮炎、IBD、癌症、纤毛疾病和神经退行性疾病。

DESCRIPTION (provided by applicant): Our recent discovery that key virulence factors from Bacillus anthracis, edema factor (EF), and Vibrio cholerae, cholera toxin (Ctx), both inhibit protein trafficking to cell-cell junctions is paradigm shifting. EF is a highly active adenylate cyclase and Ctx ADP-ribosylates Gsα subunits to constitutively activate host adenylate cyclase. These two cAMP producing toxins reduce the levels and activity of a small GTPase (Rab11) required in the final step of endocytic recycling of cell adhesion molecules (e.g., cadherins) and signaling proteins (e.g., Notch components) to cell-cell junctions, resulting in disruption of the vascular endothelium (EF) or intestinal epithelium (Ctx). This novel effect of EF and Ctx was discovered and genetically dissected in the model system Drosophila melanogaster (fruit fly), and these cell biological mechanisms translate to toxin action in human vascular endothelial cells (EF) and intestinal epithelial cells (Ctx) as well as in vivo in mice (both EF and Ctx). Another key finding, with important practical implications, was that over-expression of Rab11 can reverse the junction disrupting effects of EF and Ctx in vivo in flies and in human cells. In the current revised grant, we propose three integrated aims to elucidate the pathways mediating the barrier disruptive actions of EF and Ctx and to analyze the consequences of this novel cell biological mechanism in disease pathogenesis. In Aim 1 we will examine the pathways by which high sustained levels of cAMP produced by EF/Ctx reduce Rab11 protein levels to derail junctional transport and explore new potential functions of EF and Ctx related to inhibition of exocyst function in immune cells. In Aim 2, we will investigate how inhibition of endocytic recycling promotes leakage across human cell monolayers and in the vasculature during anthrax infection. Since vascular collapse is a frequent cause of death in anthrax, we will also determine whether increasing Rab11 levels or treating with known traffic-promoting agents can reverse the vascular leakage caused by EF. In Aim 3, we will similarly examine the contribution of exocyst inhibition to the massive fluid secretion that is pathognomonic of cholera and whether elevating endocytic recycling via genetic or pharmacological means is protective in vivo. The proposed studies have important translational relevance to treating anthrax since toxins can reach critical lethal levels just as patients begin to seek medical intervention, when antibiotics are no longer effective. Thus, treatments based on restoring endocytic recycling could be used in conjunction with existing anti-toxin therapies (e.g., anti-toxin antibodies, small molecule inhibitors) to neutralize toxins already present in the circulation. An advantage of traffic-promoting agents is that they would intervene at the very last step when vascular integrity collapses and other organ systems fail. Such traffic-promoting compounds might also increase the efficacy of fluid replacements to treat cholera and to treat other barrier disruptive diseases including: ischemia, asthma, dermatitis, IBD, cancer, ciliary diseases, and neurodegenerative disorders.