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，来自炭疽芽孢杆菌的关键毒力因子水肿因子（EF）和霍乱弧菌的霍乱毒素（Ctx）均抑制蛋白质运输至细胞-细胞连接处，这是一种范式转变。EF是一种高活性的腺苷酸环化酶，Ctx ADP-核糖基化Gsα亚基以组成型激活宿主腺苷酸环化酶。这两种产生cAMP的毒素降低了细胞粘附分子（例如，钙粘蛋白）和信号蛋白（例如，Notch组分）与细胞-细胞连接，导致血管内皮（EF）或肠上皮（Ctx）的破坏。EF和Ctx的这种新作用是在模型系统Drosophila melanogaster（果蝇）中发现和遗传解剖的，并且这些细胞生物学机制在人血管内皮细胞（EF）和肠上皮细胞（Ctx）中以及在小鼠体内（EF和Ctx）中转化为毒素作用。另一个具有重要实际意义的关键发现是Rab 11的过表达可以逆转EF和Ctx在苍蝇和人类细胞中体内的连接破坏作用。在目前修订的补助金，我们提出了三个综合目标，阐明介导的EF和Ctx的屏障破坏性行动的途径，并分析这种新的细胞生物学机制在疾病发病机制的后果。在目的1中，我们将研究EF/Ctx产生的高持续水平cAMP降低Rab 11蛋白水平以破坏连接转运的途径，并探索EF和Ctx与抑制免疫细胞中的外囊功能相关的新的潜在功能。在目标2中，我们将研究如何抑制内吞再循环促进泄漏在人类细胞单层和在血管炭疽感染。由于血管塌陷是炭疽病死亡的常见原因，我们还将确定增加Rab 11水平或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。在目标3中，我们将类似地检查外囊抑制对霍乱特异性的大量液体分泌的贡献，以及通过遗传或药理学手段提高内吞再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相关性，因为当抗生素不再有效时，毒素可以达到临界致命水平，就像患者开始寻求医疗干预一样。因此，基于恢复内吞再循环的治疗可以与现有的抗毒素疗法（例如，抗毒素抗体、小分子抑制剂）来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。这种促进运输的化合物还可以增加流体替代物治疗霍乱和治疗其他屏障破坏性疾病的功效，包括：缺血、哮喘、皮炎、IBD、癌症、睫状体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.