Mechanisms of histone crosstalk with bacterial pathogens

项目总结 细菌病原体通过分泌辅助病原体的效应蛋白来调节人类细胞的生理。 免疫逃避和增殖。各种不同的分泌型致病效应器重新连接了一系列 处于宿主细胞功能核心的细胞信号通路。其中一类效应器蛋白进入 人类细胞核并通过改变我们的表观遗传学直接调节宿主的转录程序 密码。这些所谓的表观遗传“核调节蛋白”代表了宿主-病原体相互作用的一个重要方面， 然而，关于这些效应器如何与表观遗传相互作用并感知表观遗传，人们知之甚少。 感染期间宿主细胞的景观。最受研究的表观遗传核调蛋白之一是Roma，一种 军团菌组蛋白赖氨酸甲基转移酶，嗜肺军团菌组蛋白赖氨酸甲基转移酶。 罗马单甲基化组蛋白H3K14、二甲基化组蛋白H3K14和三甲基化组蛋白H3K14抑制宿主细胞免疫反应的表达 基因，使嗜肺性乳杆菌能够在宿主细胞质内高效复制。罗马包含几个 真核细胞染色质结合蛋白中保守的结构域，提示其H3K14甲基化 活性可能通过与宿主的组蛋白修饰相互作用来调节。在真核生物中，这种现象 其中现有的组蛋白修饰控制另一组蛋白的识别或沉积，称为组蛋白 修改串扰。这些组蛋白串扰机制构成了复杂的转录调控机制。 以及真核生物的基因组结构。然而，目前还没有已知的细菌病原体的例子 可以通过组蛋白串扰来解释人类的组蛋白修饰，并改变人类的表观基因组。这样的一种 观察将极大地扩大我们对感染过程中宿主与病原体相互作用的理解。在此R21中 建议，我们将使用嗜肺乳杆菌效应器Roma作为模型来确定致病菌是否可以 通过串扰机制与现有的组蛋白修饰相互作用并进行解释。在目标1中，我们将 对吉普赛人和个人吉普赛人进行全面的三部分筛选，以确定是否有人 组蛋白修饰可以与ROMA结合或调节其催化活性。然后，在目标2中，我们将建立一个低温- EM筛选工作流程，以确定满足以下要求的最佳基质和冷冻条件 确定与其首选底物核小体结合的ROMA的结构。

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.