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

项目摘要 非结核分枝杆菌如M.鸟类是广泛分布的环境生物， 存在于许多自然和人工的栖息地中（）。M的能力。鸟类定居 家庭管道，热水浴缸和花园土壤，往往把他们靠近 人类大多数病例由M.禽流感是肺部疾病， 包括老年人、免疫功能低下或已有肺损伤的个体 由于潜在的呼吸系统疾病（）。M的情况。禽流感继续上升， 在美国，病例估计每年增加5-10%（）。M.肺结核 这种疾病既难以诊断，也难以治疗。一旦确诊， 抗生素可以持续18-24个月，临床指南指示连续治疗12个月。 几个月后，在患者痰中不再检测到感染细菌。即使有这种 疗程延长，感染复发仍常见。 M.禽流感是一种细胞内病原体，巨噬细胞是主要宿主细胞。因此 为了使感染成功，分枝杆菌必须适应其自然宿主的生活， 在生物膜内入侵并在巨噬细胞中复制。我们对这些人是如何 分枝杆菌使这一关键转变。我们假设， 糖肽是M. avium，在这方面发挥关键作用， 过渡和证据表明其在生物膜形成和巨噬细胞 激活支持这一论点。在具体目标1中，我们将通过分离， 从生物膜和巨噬细胞中，不同的M.鸟类GPL物种和表征他们的能力 调节巨噬细胞功能。我们还将产生基因敲除以产生GPL 突变体，并对M.用于生物膜形成和巨噬细胞的鸟突变体 入侵/生存来定义观察到的表型所需的GPL物种。在 目的2我们将进行M.在肉汤培养物中生长的禽类，生物膜和 巨噬细胞，以确定参与产生的不同基因的表达水平， 各种GPL变体。转录分析将有额外的好处， 在全球范围内，基因表达的变化发生在M. avium从增长过渡到 生物膜在巨噬细胞中复制。

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.