Structure and Function of Legionella pneumophila Lysine Methyltransferases

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PROJECT ABSTRACT Legionella pneumophila is a intracellular bacterial pathogen that is the primary etiological agent of Legionnaires’ Disease. This disease is categorized as an atypical pneumonia that afflicts the elderly and individuals who are immunocompromised or suffer from chronic lung disease. L. pneumophila infects host cells, such as environmental amoeba and lung macrophages, through the secretion of an array of effector proteins that subvert signaling pathways, membrane trafficking, and gene expression in host cells to promote bacterial replication. Among these effectors are the protein lysine methyltransferases (KMTs) RomA and its highly conserved strain ortholog LegAS4. RomA has been shown to localize to the nucleus where it trimethylates Lys14 in histone H3 (H3K14me3). This novel chromatin modification silences the macrophage gene expression, including loci responsible for innate immunity. In agreement with these findings, deletion of the RomA gene in L. pneumophila hinders bacterial replication within infected host cells, illustrating the importance of this KMT in pathogenesis. Despite the role of RomA in L. pneumophila virulence, outstanding questions regarding the biological functions of RomA and LegAS4 remain unresolved. These questions include the molecular basis of their unique substrate specificity for H3K14, how they interact with chromatin, and the biological roles of LegAS4 in host cell infection. The Trievel, Swanson, Cho, and Ohi laboratories at the University of Michigan have established an interdisciplinary collaboration to address these questions by employing an integrated approach combining biochemistry, microbiology, and structural biology. Our overall objective is to elucidate the mechanism by which RomA and LegAS4 recognize and methylate H3K14 in nucleosomes in vitro and in host cells infected by L. pneumophila. We propose the following specific aims to accomplish this objective: 1) Define the molecular mechanism of H3K14 methylation by RomA and LegAS4. 2) Elucidate the structural basis of nucleosome recognition. 3) Determine the functions of LegAS4 in macrophage infection. We envision that these studies will yield a comprehensive framework for understanding the mechanisms underlying nucleosome recognition and H3K14 methylation by RomA and LegAS4 that promote L. pneumophila replication during host cell infection. These results will provide avenues for developing selective inhibitors of these KMTs that can be applied as novel therapeutics or biocides to combat Legionnaires Disease.