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.