RavD binds PI(3)P and contributes to lysosomal avoidance 1 Legionella effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the Legionella-containing vacuole Colleen M. Pike 1 , Rebecca Boyer-Andersen 1 , Lisa N. Kinch 4 , Jeffrey L. Caplan 1,2,3 , and M. Ramona Neunuebel 1,* From the 1 Delaware Biotechnology Institute, 2 Department of Biological Sciences, 3 Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716; 4 Howard Hughes Medical Institute, Dallas, TX 75390 Running Title: RavD binds PI(3)P and contributes to lysosomal avoidance *To whom the correspondence should be addressed: M. Ramona Neunuebel, Department of Biological Sciences, University of Delaware, 105 The Green, Newark, DE 19716; neunr@udel.edu, Tel. (302) 831- 3450; Fax. (302) 832-2281 Keywords: host-pathogen interaction, bacterial pathogenesis, phosphoinositide, bacterial effectors, Legionella pneumophila, cellular localization, infection, virulence factor, immune evasion, Legionnaires’ disease ABSTRACT Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here, we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism that the pathogen uses to prevent encounters with lysosomes. Protein–lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro. We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD co-localized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD’s interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that upon translocation into macrophages, RavD is retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amoebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways. Legionella pneumophila is a facultative intracellular bacterial pathogen that commonly infects protozoa and in humans it causes a life- threatening pneumonia known as Legionnaires’ disease (1). Following inhalation of contaminated aerosols the bacterium reaches the lung where it infects resident alveolar macrophages as well as neutrophils (2,3). These professional phagocytes ingest microbes by http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.007086 The latest version is at JBC Papers in Press. Published on February 7, 2019 as Manuscript RA118.007086 by guest on April 29, 2019 http://www.jbc.org/ Downloaded from