Role of DeoR-family transcription regulators in metabolic adaptation of Listeria monocytogenes to stressful food-related environmental conditions and intracellular host cells

项目摘要 单核细胞增生李斯特菌（Listeria monocytogenes，LM）是一种非常危险的食源性致病菌，致死率高达20 - 30%。一 LM之所以危险，主要是因为它可以在各种环境压力下生存 在其感染周期中遇到的包括低pH，高盐，低温和细胞内 环境. LM在不同环境胁迫下的生存能力与其在不同环境胁迫下的存活能力直接相关。 根据压力的类型和营养的可用性来调整其新陈代谢。在我们的知识中存在着一个鸿沟。 了解控制LM代谢适应压力环境的调节机制 条件我们有证据表明，DeoR家族调节因子通过调节LM来发挥这一关键功能 新陈代谢. DeoR家族调节因子的成员通常作为转录抑制因子或激活因子， 糖代谢通过对LM株F2365的基因组分析，我们发现了七个DeoR家族成员 监管部门我们通过靶向编码FruR的DeoR家族调节子构建了LM缺失菌株 （LMOf2365_2307）。我们发现F2365ΔfruR在巨噬细胞中被减弱并具有降低的生长 相对于强毒亲本菌株F2365。此外，fruR的缺失导致了蛋白质表达的强烈上调。 磷酸果糖激酶（fruK）和果糖特异性PTS系统转运蛋白亚基IIABC（fruA）。我们的中央 一个假设是，DeoR家族转录调节因子有助于LM适应环境的能力。 环境变化和复制在宿主细胞中作为全球调节许多代谢基因， 包括磷酸转移酶转运系统（PTS）和ABC转运蛋白。这项提案的具体目标是 确定DeoR系列调节剂对LM适应恶劣环境条件的贡献。 这一目标将通过以下两个具体目标来实现：目标1阐明FruR在L. 单核细胞增多症细胞内复制。在这一目标中，我们将分析葡萄糖掺入和代谢 F2365ΔfruR在巨噬细胞中的复制。我们将比较 亲本和F2365ΔfruR菌株在巨噬细胞系中生长期间的生长。我们将决定是否 F2365ΔfruR的细胞内生长缺陷是由于延迟的空泡逃逸或由于F2365Δ fruR的细胞内生长缺陷。 溶血素的产生。目的2确定其他DeoR家族调节因子在李斯特菌对应激反应中的作用 条件这一目标将确定DeoR家族基因在毒力和生长缺陷中的作用， 巨噬细胞系。我们还将确定DeoR家族调节剂在LM适应食物相关疾病中的作用。 应力条件，包括暴露于酸和高盐。该项目的基本原理是确定 使LM能够耐受与食物相关的环境条件并在其中生长的机制， 在细胞内宿主细胞中存活，这将有助于开发控制LM的干预策略 并将导致对抗食源性感染的治疗方法的进步。

Project Summary Listeria monocytogenes (LM) is a very dangerous foodborne pathogen with a high mortality rate (20 - 30%). A major reason why LM is dangerous is because it can survive in a variety of environmental stresses encountered during its infection cycle, including low pH, high salt, low temperature, and the intracellular environments. The ability of LM to survive in different environmental stresses is directly related to its ability to adjust its metabolism depending on the type of stress and nutrient availability. There is a knowledge gap in our understanding of the regulatory mechanisms controlling LM metabolic adaptations to stressful environmental conditions. We have evidence that DeoR-family regulators serve this critical function by regulating LM metabolism. Members of the DeoR-family regulators often serve as transcriptional repressors or activators in sugar metabolism. By analyzing the genome of LM strain F2365, we found seven members of the DeoR-family regulators. We constructed a LM deletion strain by targeting FruR-encoding DeoR-family regulator (LMOf2365_2307). We found that F2365ΔfruR is attenuated and has decreased growth in macrophages relative to the virulent parent strain F2365. Furthermore, deletion of fruR causes strong upregulation of phosphofructokinase (fruK) and fructose-specific PTS system transporter subunit IIABC (fruA). Our central hypothesis is that the DeoR-family transcriptional regulators contribute to the ability of LM to adapt to environmental changes and replicate in host cells by acting as global regulators for many metabolic genes, including phosphotransferase transport system (PTS) and ABC transporters. The specific goal of this proposal is to determine the contribution of DeoR-family regulators to LM adaptation to harsh environmental conditions. This goal will be achieved by performing the following two specific aims: Aim 1 elucidate the role of FruR in L. monocytogenes intracellular replication. In this aim, we will analyze the glucose incorporation and metabolism in F2365ΔfruR replicating in macrophage by 13C-perturbation techniques. We will compare the transcriptome of parent and F2365ΔfruR strain during growth in macrophage cell line. We will determine whether the intracellular growth defect of F2365ΔfruR is due to delayed vacuolar escape or due to a defect in the hemolysin production.; Aim 2 define the role of other DeoR-family regulators in Listeria's response to stressful conditions. This aim will determine the effect of DeoR-family genes in virulence and in growth defect in macrophages cell lines. We will also identify the role of DeoR-family regulators in LM adaptation to food related stress conditions, including exposure to acid and high salt. The rationale for this project is to determine mechanisms that allow LM to tolerate and grow within stressful food-related environmental conditions and survive in intracellular host cells, which will assist in the development of intervention strategies to control LM and will lead to advancements in therapies to combat foodborne infection.