Sunday, May 5, 2024
5/5/2024
Project Summary/Abstract While phagocytosis is an effective immune strategy against many infectious microbes, some, such as Mycobacterium tuberculosis, have evolved the ability to subvert the phagosome maturation that normally follows engulfment, and establish replicative niches inside macrophages. Intriguingly, triggering the autophagy pathway through nutrient withdrawal or chemical treatment can stimulate macrophages to overcome the block, and deliver the phagosomal contents to the lysosome for degradation. It was also found that recruitment of canonical autophagy effectors to the phagosome is required for this process, which suggested that the autophagy pathway promotes phagosome maturation. We have discovered that an evolutionarily conserved protein, Psidin, plays opposite roles in these processes: it inhibits autophagy but promotes phagosome maturation. The existence of a protein with such opposing roles indicates that, rather than co-operating, autophagy and phagocytosis may be divergently regulated, and also offers an entry point to understanding how. Using the model system Drosophila, our goal is to understand how Psidin exerts these differential effects, with the expectation that these investigations will help us understand how cells regulate these two critical processes. In Aim 1, we will test whether known biochemical activities of Psidin, such as actin regulation or protein acetylation, contribute to its regulation of autophagy vs. phagocytosis, or whether novel activities are responsible, by analysing psidin mutants that selectively impair known activities of Psidin. Immunoblot and mass spectrometric analysis will determine whether Psidin affects the levels of the Acetyl CoA metabolite, or of Lysine acetylation of total cellular proteins or candidate autophagy effectors. Finally, we will identify post-translational modifications that could direct Psidin's regulation of autophagy vs phagocytosis. In Aim 2, we will determine where in phagosome maturation and the autophagy regulatory cascade Psidin exerts its effects, and test for roles for Psidin in mediating a possible tradeoff between autophagy and phagocytosis. Phenotypic analysis of the autophagy and phagosome defects in psidin mutants, along with epistasis analysis of double mutants will allow us to determine whether Psidin regulates phagocytosis and autophagy at similar stages of the two processes but in opposite ways, addressing the exciting possibility that Psidin acts as a nutrient-dependent switch between them. Finally, we will test whether Psidin activity can boost phagosomal clearance of Mycobacteria. Overall, by applying the power of Drosophila genetics towards important and unresolved questions about the interactions between autophagy and phagocytosis, this work will clarify how these pathways engage to combat serious infections, and thus offer new avenues for therapeutic approaches to treat tuberculosis.
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