Inhibition of human neutrophil function by Francisella tularensis

描述(申请人提供)：图拉热症是一种潜在的致命的人畜共患病，由兼性细胞内细菌，弗朗西斯图拉氏菌(Ft)引起。事实上，吸入10种微生物就足以导致严重的肺炎疾病、组织坏死和死亡。由于Ft通过气溶胶途径具有极强的毒力，因此是一种潜在的生物武器，并已被归类为A类选择剂。最近的一项研究结果表明，基质金属蛋白酶-9被Ft上调，随后细胞外基质的裂解产生一种趋化物质，驱动中性粒细胞向肺内迁移。虽然在急性感染期间(2-14天)，肺泡和细支气管内的白细胞以中性粒细胞为主，并吞噬了大量的细菌，但Ft没有被杀死，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PMN聚集到肺中的迁移受到损害，小鼠就可以存活下来，否则就会受到致命剂量的图拉氏丝虫感染。虽然相对较少的病原体抵抗PMN的清除，但在分子水平上对中性粒细胞与Ft的相互作用几乎一无所知。我们的初步数据显示，Ft对人的中性粒细胞功能有深远的影响，包括快速和完全地抑制氧化爆发。在这一点上，值得注意的是，异源刺激对细胞的激活也受到了损害，因此我们的数据表明，Ft的影响超出了自身吞噬小体的范围。弗朗西塞拉还阻止中性粒细胞的非氧化性杀伤库，脱颗粒被抑制或严重延迟。在随后的感染中，Ft打破了吞噬体膜，并停留在营养丰富的细胞质中。因此，我们假设Ft破坏NADPH氧化酶活性和吞噬小体-溶酶体融合，作为逃避细胞内杀伤的一种手段。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。为了验证这一假说，我们将：1.阐明Ft对NADPH氧化酶组装和活性的影响。2.确定吞噬小体的组成并量化颗粒动员；3.通过Ft毒力决定因素对中性粒细胞基因表达和关键细胞内信号通路的影响，识别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分析来量化活性氧物种；共聚焦显微镜来定位NADPH氧化酶成分；免疫荧光显微镜和免疫电子显微镜来评估吞噬小体组成；透射电子显微镜来量化吞噬小体逃逸和PMN活性；体外激酶分析和免疫印迹来测量细胞内信号；DNA微阵列来定义中性粒细胞基因表达的变化；以及靶向等位基因替换策略和转座子突变文库筛选，以开始确定在这个系统中毒力所需的Ft基因。 项目简介：吸入一种名为图拉方济氏菌的细菌会导致严重的、有时是致命的肺炎，而且没有疫苗可用。在这项研究中，我们将开始确定弗朗西塞拉如何避免被一种名为中性粒细胞的白细胞杀死(中性粒细胞在感染期间聚集在肺部)。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的、往往是致命的肺部感染。

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.