| Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cenocepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In this dissertation, we aimed to elucidate a strategy for controlling B. cenocepacia infection in CFTR DeltaF508 (DeltaF508) mouse model. We found that, in DeltaF508 macrophages, B. cenocepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of the inflammatory cytokine (IL-1beta). It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Autophagy is a basic fundamental survival mechanism. An autophagosome is a compartment which engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Here we showed for the first time that in wild-type macrophages (WT), many B. cenocepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cenocepacia are higher in DeltaF508 macrophages than in WT macrophages. Interestingly, B. cenocepacia down-regulates autophagy genes in WT and DeltaF508 macrophages. Here, we demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cenocepacia infection in vitro by enhancing the clearance of B. cenocepacia via induced autophagy. In vivo, rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. As a second strategy to stimulate autophagy in DeltaF508 macrophages, was to target SQSTM1/p62. p62 is a ubiquitin binding adaptor protein which is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 downregulation and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, downregulation of p62 in DeltaF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cenocepacia intracellular growth. Interestingly, the depletion of p62 from DeltaF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1), from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cenocepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cenocepacia infection in WT and DeltaF508 macrophages. Therefore, targeting p62 inside the lung of CF patients could be a novel approach to control B. cenocepacia infection. Together, these studies reveal that if efficiently activated, autophagy is capable of controlling B. cenocepacia infection and the associated inflammation. |
