Evaluating Mycobacterium avium glycopeptidolipids as key factors in the transition from biofilm to macrophages

项目总结 像禽类分枝杆菌这样的非结核分枝杆菌是广泛存在的环境生物， 存在于许多栖息地，既有天然的，也有人工培育的()。禽类分枝杆菌的定植能力 家用管道、热水浴缸和花园土壤，通常将它们放置在靠近 人类。大多数由禽类分枝杆菌引起的疾病是肺部和患者群体。 包括老年人、免疫功能受损或既有肺损伤的人 由于潜在的呼吸系统疾病()。禽类分枝杆菌病的病例持续上升， 美国的病例估计每年增长5%-10%()。禽类分枝杆菌肺 疾病既难诊断又难治疗。一旦确诊，使用多个 抗生素可以持续18-24个月，临床指南显示持续治疗12个月 几个月后，患者的痰中不再检测到感染细菌。即使有了这个 疗程延长，感染复发仍较常见。 禽类支原体是一种以巨噬细胞为主要宿主细胞的胞内病原体。因此 为了成功感染，分枝杆菌必须从生活在它的自然水库中进行调整。 在生物膜中入侵并在巨噬细胞中复制。我们对此知之甚少 分枝杆菌完成了这一关键转变。我们假设修改了 糖肽类脂类是禽类支原体的主要细胞表面分子，在这一过程中起着关键作用。 过渡及其在生物膜形成和巨噬细胞中的重要性 激活支持这一论点。在特定的目标1中，我们将通过分离 从生物膜和巨噬细胞中分离出不同的禽类支原体GPL，并对其能力进行了表征 来调节巨噬细胞的功能。我们还将产生基因敲除来产生GPL 绵羊分枝杆菌生物膜形成和巨噬细胞突变体的筛选及评价 侵袭/存活，以确定观察到的表型需要哪些GPL物种。在……里面 目的2我们将对在肉汤培养、生物膜和 巨噬细胞定义不同基因的表达水平 各种GPL变种。转录分析将具有额外的好处，即定义更多 在全球范围内，当禽类分枝杆菌从生长过渡到 生物膜在巨噬细胞中的复制。

Project summary Non-tuberculosis mycobacteria like M. avium are widespread environmental organisms, occurring in many habitats, both natural and engineered (). The ability of M. avium to colonize household plumbing, hot tubs, and garden soils, often places them in close proximity with humans. Most cases of disease caused by M. avium are pulmonary and the patient population includes individuals who are elderly, immune-compromised, or have preexisting lung damage due to an underlying respiratory disease (). Cases of M. avium disease have continued to rise, with cases increasing an estimated 5-10% annually in the United States (). M. avium pulmonary disease is both difficult to diagnose and treat. Once diagnosed, treatment with multiple antibiotics can last 18-24 months, with clinical guidelines indicating continuous treatment for 12 months after the infecting bacteria are no longer detected in patient sputum. Even with this extended treatment course, recurrence of infection is still common. M. avium is an intracellular pathogen with macrophages being the primary host cell. Therefore for the infection to be successful, the mycobacteria must adjust from living in its natural reservoir within a biofilm to invading and replicating in a macrophage. We know very little about how the mycobacteria makes this critical transition. We hypothesize that modifications of glycopeptidolipids, which are major cell surface molecules of M. avium, play a critical role in this transition and evidence showing their importance in biofilm formation and macrophage activation supports this argument. In Specific aim 1 we will test this hypothesis by isolating, from biofilm and macrophages, the different M. avium GPL species and characterize their ability to modulate macrophage function. We will also generate gene knockouts to produce GPL mutants and evaluate the M. avium mutants for biofilm formation and macrophage invasion/survival to define what GPL species are necessary for the observed phenotypes. In aim 2 we will perform a transcriptional analysis of M. avium grown in broth culture, biofilms and macrophages to define expression levels of the different genes involved in producing the various GPLs variants. The transcriptional analysis will have the added benefit of defining more globally, changes in gene expression which occur as the M. avium transitions from growth in biofilm to replication in macrophages.