Hospital-acquired infections derived from various pathogens including P. aeruginosa (Pa) require approximately $45 billion in annual medical expenses in the U.S., according to the CDC. Despite intense interest, the function of alveolar macrophages (AM) in Pa infection remains elusive. Macroautophagy (hereafter autophagy) is a conserved homeostasis mechanism by which cellular components are sequestered to autophagosomes for degradation through ubiquitination. The autophagosome machinery may contribute to the innate immunity to enhance bacterial clearance. However, it is unknown whether autophagy impacts Pa infection. Our preliminary data revealed that Pa infection can induce autophagy, and subsequently increasing bacterial degradation. We also showed that the pleiotropic Src kinase, Lyn, interacts with toll like receptors (TLRs) to boost autophagic activity In addition, we found that Atg-7 is critical for phagosome-lysosome fusion. Importantly, starvation increases AM phagocytosis of Pa and blocking autophagy by autophagy inhibitor 3-methyladenine (3MA) decreased phagocytosis and subsequent bacterial clearance. Thus, we hypothesize that Pa-induced autophagy augments host defense by facilitating phagocytosis and bacterial degradation. Our long-term goal is to discover the key factors required for regulating host defense against Pa invasion in order to develop novel therapeutic strategies. The objective of this proposal is to examine the physiological significance of Lyn/Atg-7 modulated phagocytosis with KO mice and to delineate the underlying molecular mechanisms using biochemical and whole animal imaging approaches.
Aim 1 : Define the functional role of Atg-7 in Pseudomonas pulmonary infection. Our working hypothesis is that atg-7 is required for protection against Pa infection.
Aim 2 : Evaluate the impact of autophagy on phagocytosis in pulmonary bacterial infection utilizing a mouse model and primary mouse AM. Based on our finding that Pa infection can induce autophagy and reducing bacterial burdens in AM, our working hypothesis is that Pa-induced autophagy augments macrophage phagocytosis.
Aim 3 : Determine the role of TLR-2, Lyn, and Atg-7 in autophagosome formation and phagolysosome fusion in host response against bacteria. Thus, our working hypothesis is that TLR-2, Lyn, and Atg-7 are key elements for delivery of bacteria to lysosomes for degradation. These proposed studies will reveal a novel mechanism for AM to eradicate bacteria and suggest novel therapeutic targets.
Hospital-acquired infections impose $45 billion in annual medical expenses in the U.S., according to the CDC and Pseudomonas aeruginosa (Pa) is the fourth most commonly-isolated nosocomial bacterium. Using novel mouse models and whole animal imaging, studying novel roles of autophagy (cell self-eating) in bacterial pathogenesis will substantially advance our understanding of the function of alveolar macrophages (AM) in host defense against microbes. This application will ultimately reveal new therapeutic targets for controlling Pa and potentially other Gram-negative bacteria.
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