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、水肿因子(EF)和霍乱弧菌、霍乱毒素(CTX)的关键毒力因子都能抑制蛋白质向细胞-细胞连接的运输，这一发现正在转变。EF是一种高活性的腺苷环化酶，CTX-ADP-核糖化Gsα亚基构成激活宿主腺苷环化酶。这两种产生cAMP的毒素降低了细胞黏附分子(如钙粘附素)和信号蛋白(如Notch成分)内吞循环到细胞-细胞连接的最后一步所需的小GTPase(Rab11)的水平和活性，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果蝇(果蝇)中发现并从基因上剖析的，这些细胞生物学机制转化为对人血管内皮细胞(EF)和肠上皮细胞(CTX)以及在小鼠体内(EF和CTX)的毒性作用。另一个具有重要实际意义的关键发现是，Rab11的过度表达可以逆转EF和CTX在体内对果蝇和人类细胞的连接干扰效应。在目前的修订拨款中，我们提出了三个综合目标，以阐明EF和CTX破坏屏障作用的途径，并分析这一新的细胞生物学机制在疾病发病机制中的后果。在目标1中，我们将研究EF/CTX产生的持续高水平的cAMP降低Rab11蛋白水平以破坏连接转运的途径，并探索EF和CTX与抑制免疫细胞外囊功能相关的新的潜在功能。在目标2中，我们将研究在炭疽感染期间，抑制内吞循环如何促进跨越人类细胞单层和血管系统的渗漏。由于血管塌陷是炭疽病死亡的常见原因，我们还将确定提高Rab11水平或使用已知的交通促进剂治疗是否可以逆转EF导致的血管渗漏。在目标3中，我们将类似地检验外囊抑制对霍乱病原体大量液体分泌的贡献，以及通过遗传或药物手段提高内吞循环在体内是否具有保护作用。拟议的研究对炭疽病的治疗具有重要的翻译意义，因为在抗生素不再有效的情况下，当患者开始寻求医疗干预时，毒素可能会达到临界致命水平。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，以中和循环中已经存在的毒素。交通促进剂的一个优势是，当血管完整性崩溃和其他器官系统失效时，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增加液体补充剂治疗霍乱和其他屏障干扰性疾病的疗效，包括：缺血、哮喘、皮炎、IBD、癌症、睫状神经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.