Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY Bacterial pathogens modulate the physiology of human cells by secreting effector proteins that assist pathogen immune evasion and proliferation. The diverse repertoire of secreted pathogenic effectors re-wire an array of cellular signaling pathways that are at the core of host cell function. One class of these effector proteins enter the human cell nucleus and directly modulate transcriptional programs of the host by altering our epigenetic code. These so-called epigenetic “nucleomodulins” represent an important aspect of host-pathogen interaction, however very little is understood about how these effectors might interact with and sense the epigenetic landscape of the host cell during infection. One of the most well-studied epigenetic nucleomodulins is RomA, a histone lysine methyltransferase from Legionella pneumophila, the causative agent of Legionnaires Disease. RomA mono-, di- and tri-methylates histone H3K14, silences the expression of host cell immune response genes and enables efficient replication of L. pneumophila inside the host cytoplasm. RomA contains several domains that are conserved in eukaryotic chromatin-binding proteins, suggesting that its H3K14 methylation activity may be regulated by interacting with histone modifications of the host. In eukaryotes, the phenomenon where an existing histone modification controls the recognition or deposition of another is called histone modification crosstalk. These histone crosstalk mechanisms underlie the complex regulation of transcription and genome architecture in eukaryotes. However, there are no known examples of a bacterial pathogen that can interpret human histone modifications through histone crosstalk and alter the human epigenome. Such an observation would greatly expand our understanding of host-pathogen interactions during infection. In this R21 proposal, we will use the L. pneumophila effector RomA as a model to establish if pathogenic bacteria can interact with and interpret existing histone modifications through crosstalk mechanisms. In Aim 1, we will subject RomA and individual RomA domains to a comprehensive three-part screen to establish if any human histone modifications can bind to RomA or regulate its catalytic activity. Then, in Aim 2 we will establish a cryo- EM screening workflow to identify optimal substrates and freezing conditions that will be necessary to determine a structure of RomA bound to its preferred substrate nucleosome.
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