Inhibition of human neutrophil function by Francisella tularensis

描述（由申请人提供）：土拉菌病是一种潜在致命的人类人畜共患疾病，由兼性细胞内细菌土拉弗朗西斯菌（Ft）引起。事实上，吸入少至10个微生物就足以引起严重的肺炎、组织坏死和死亡。由于其通过气溶胶途径的极端毒性，Ft是一种潜在的生物武器，已被列为A类选择剂。最近的一项研究结果表明，基质金属蛋白酶-9上调Ft和细胞外基质的后续裂解产生的趋化因子，驱动中性粒细胞迁移到肺。尽管在急性感染期间（第2 - 14天），中性粒细胞是肺泡和细支气管中的主要白色血细胞，并且在该部位吞噬大量细菌，但Ft未被杀死，并且细菌负荷显著增加。这些数据表明，Ft对中性粒细胞功能的破坏可能是毒力的一个重要方面。事实上，如果中性粒细胞聚集迁移到肺中受到损害，小鼠在致死剂量的氟中毒中存活。土拉热。虽然相对较少的病原体抵抗PMN的消除，但在分子水平上对嗜中性粒细胞-Ft相互作用几乎一无所知。我们的初步数据表明，Ft对人类中性粒细胞功能有深远的影响，包括快速和完全抑制氧化爆发。在这方面，值得注意的是，异源刺激的细胞活化也受损，因此我们的数据表明，Ft的影响超出了其自身吞噬体的范围。弗朗西斯菌还阻断中性粒细胞的非氧化性杀伤武器库，并且脱粒被抑制或严重延迟。在感染后期，Ft突破吞噬体膜并驻留在营养丰富的细胞质中。因此，我们假设Ft破坏NADPH氧化酶活性和吞噬体-溶酶体融合作为逃避细胞内杀伤的手段。我们的长期目标是在分子水平上确定Ft破坏中性粒细胞功能的机制。为了验证这个假设，我们将：1。阐明Ft对NADPH氧化酶组装和活性的影响。2.定义吞噬体的组成并量化颗粒动员;以及3.通过Ft毒力决定因子对中性粒细胞基因表达和关键细胞内信号传导途径的影响，辨别PMN功能受损的程度。为此，我们将利用化学发光荧光和其他生化测定来定量活性氧;共聚焦显微镜定位NADPH氧化酶组分;免疫荧光显微镜和免疫电子显微镜评估吞噬体组成;透射电子显微镜定量吞噬体逃逸和PMN活力;体外激酶测定和免疫印迹测量细胞内信号传导; DNA微阵列，以确定在中性粒细胞基因表达的变化;和两个有针对性的等位基因替换策略和转座子突变库筛选，开始，以确定在这个系统中的毒力所需的Ft基因。 项目叙述：吸入称为土拉弗朗西斯菌的细菌会导致严重的，有时是致命的肺炎，并且没有疫苗可用。在这项研究中，我们将开始确定弗朗西斯菌如何避免被一种称为中性粒细胞的白色血细胞（在这种感染过程中积聚在肺部）杀死。这项研究的结果可能会导致新的治疗方法来对抗这种严重且往往致命的肺部感染。

DESCRIPTION (provided by applicant): Tularemia is a potentially fatal zoonosis of humans caused by the facultative intracellular bacterium, Francisella tularensis (Ft). Indeed, inhalation of as few as ten organisms is sufficient to cause severe pneumonic disease, tissue necrosis and death. Because of its extreme virulence via the aerosol route, Ft is a potential bioweapon and has been classified as a Category A Select Agent. The results of a recent study demonstrate that matrix metalloprotease-9 is upregulated by Ft and that subsequent cleavage of extracellular matrix generates a chemoattractant that drives neutrophil migration into the lung. Although neutrophils are the dominant white blood cell in alveoli and bronchioles during acute infection (day 2-14), and engulf large numbers of bacteria in this locale, Ft are not killed and bacterial load increases markedly. These data suggest that disruption of neutrophil function by Ft may be an important aspect of virulence. Indeed, if PMN accumulation migration into the lung is compromised, mice survive an otherwise lethal dose of F. tularensis. Although relatively few pathogens resist elimination by PMN, almost nothing is known about neutrophil-Ft interactions at the molecular level. Our preliminary data now demonstrate that Ft has profound effects on human neutrophil function that include rapid and complete inhibition of the oxidative burst. In this regard it is noteworthy that cell activation by heterologous stimuli is also impaired, and as such our data suggest that effects of Ft extend beyond the confines of its own phagosome. Francisella also blocks non-oxidative killing arsenal of the neutrophil, and degranulation is inhibited or severely delayed. Later in infection, Ft breaches the phagosome membrane and resides in the nutrient-rich cytosol. Thus, we hypothesize that Ft disrupts both NADPH oxidase activity and phagosome-lysosome fusion as a means to evade intracellular killing. Our long term goal is to define at the molecular level the mechanisms by which Ft disrupts neutrophil function. To test this hypothesis we will: 1. Elucidate the effect of Ft on NADPH oxidase assembly and activity. 2. Define the composition of the phagosome and quantify granule mobilization; and 3. Discern the extent to which PMN function is impaired via effects of Ft virulence determinants on neutrophil gene expression and key intracellular signaling pathways. Toward this end we will utilize chemiluminescence fluorescence and other biochemical assays to quantify reactive oxygen species; confocal microscopy to localize NADPH oxidase components; immunofluorescence microscopy and immuno-electron microscopy to assess phagosome composition; transmission electron microscopy to quantify phagosome escape and PMN viability; in vitro kinase assays and immunoblotting to measure intracellular signaling; DNA microarrays to define changes in neutrophil gene expression; and both targeted allelic replacement strategies and transposon mutant library screening to begin to define Ft genes required for virulence in this system. Project Narrative: Inhalation of the bacterium called Francisella tularensis causes severe, sometime fatal pneumonia and no vaccine is available. In this study we will begin to determine how Francisella avoid being killed by a type of white blood cell called neutrophils (which accumulate in the lung during this infection). The results of this study may lead to new treatments to combat this severe and often fatal lung infection.