The Anti-Autophagy Arsenal of Legionella pneumophila

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PROJECT SUMMARY Autophagy is a highly conserved, primarily degradative pathway defined by the growth of a cup-shaped membrane that envelopes and delivers cytosolic cargo (such as bacteria) to the lysosome for degradation. Legionella pneumophila is a species of facultative intracellular bacteria that secretes over 300 effector proteins that subvert host pathways such as the degradative endosomal and autophagy pathways to promote intracellular survival. Legionella lacking currently known autophagy-inhibiting effectors are still capable of evading this pathway, indicating that there are additional, undiscovered autophagy-inhibiting effectors. Taking advantage of the highly conserved nature of this pathway, a recent screen identified several Legionella effectors that blocked autophagy in yeast. One of those effectors, Lem26, was confirmed to inhibit autophagy in mammalian cells. With the central hypothesis that Lem26 inhibits autophagy to prevent the capture and lysosomal degradation of Legionella, the proposed research is designed to identify the mechanism by which Lem26 inhibits autophagy (Aim 1) and determine its physiological relevance in the context of infection (Aim 2). To discover the mechanism by which Lem26 inhibits autophagy, Aim 1 utilizes both an unbiased mass spectrometry-based approach to identify the host targets of Lem26 as well as targeted experimentation on putative targets based on epistasis data identifying the autophagic step inhibited by Lem26. To determine the physiological relevance of Lem26 in the context of infection, Aim 2 utilizes multiple approaches to identify the localization and interactome of bacterially translocated Lem26 as well as assess the impact of Lem26 on the progression of autophagy and the intracellular growth of Legionella. The research and training plans laid out in this proposal will be executed in a highly collaborative environment that is suited for the development of the critical thinking skills and technical expertise required for a future research group leader. Given the broad implication of autophagy in various diseases and the common virulence strategies employed by intracellular pathogens, this proposed work will have broad implications in human disease.