The role of TFEB in innate immune responses to a bacterial pathogen

TFEB 在针对细菌病原体的先天免疫反应中的作用

• 批准号: RGPIN-2019-04443
• 负责人: Girardin, Stephen
• 金额: $ 2.62万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=757968
• 关键词: role; TFEB; innate; immune; responses

TFEB has been recently identified as a key protein regulating a process known as autophagy, in which cells digest and recycle parts of their intracellular content. Because of the known importance of autophagy in host defense against bacterial pathogens, a crucial question is whether this process is under the control of TFEB, which remains understudied. To study this question, we will use an epithelial cell model of infection with the bacterial pathogen Shigella flexneri, and will combine cellular microbiology techniques, bioinformatics (RNAseq and ChIP-seq) and synthetic biology (generation of engineered bacteria to monitor and analyze intracellular pyruvate extraction) to uncover the role of TFEB in host-bacterial interaction. Hypothesis: Our over-arching hypothesis is that TFEB is activated during bacterial infection and that the protein plays essential roles in controlling autophagic responses to intracellular bacteria. In particular, we propose that TFEB activation is critical for the transcriptional reprogramming of infected cells, to allow up-regulation of autophagic and lysosomal biogenesis pathways. Scientific approach: To address the above hypothesis, our specific objectives are the following: Objective 1: To identify the host pathways and bacterial determinants controlling TFEB activation during infection. We will first focus on host signaling pathways controlling TFEB activation during infection. Next, we will investigate if proteins injected in the host cell by bacteria (known as "bacterial effectors") can manipulate TFEB regulation. Objective 2: To use a synthetic biology approach to delineate the impact of pyruvate highjacking by Shigella on TFEB activation. We observed that a mutated Shigella, unable to use the host's intracellular pools of pyruvate, did not cause TFEB activation. We propose here to use a synthetic biology approach to engineer Shigella further in order to demonstrate that dynamic extraction of pyruvate from host's intracellular pools is a condition necessary and sufficient for activating TFEB in infected cells. Objective 3: To characterize the role of TFEB in the control of bacterial autophagy, lysosomal biogenesis and transcriptional reprogramming in Shigella-infected cells. The impact of TFEB on (i) autophagy, (ii) bacterial growth and (iii) gene expression in the infected cells will be assessed by infecting wild type and TFEB-deficient cells with Shigella. Novelty and expected significance: Overall, the project will combine cutting-edge techniques to shed light on the function of TFEB in host defense. This will provide a unique environment for the training of several students and their exposure to integrative research themes and axes, including cellular microbiology, bioinformatics and synthetic biology.

TFEB最近已被确定为调节称为自噬的过程的关键蛋白质，在该过程中细胞消化并回收其细胞内内容物的一部分。由于已知自噬在宿主防御细菌病原体中的重要性，一个关键的问题是这个过程是否在TFEB的控制下，这仍然是研究不足的。为了研究这个问题，我们将使用细菌病原体福氏志贺菌感染的上皮细胞模型，并将结合联合收割机细胞微生物学技术，生物信息学（RNAseq和ChIP-seq）和合成生物学（生成工程细菌以监测和分析细胞内丙酮酸提取）来揭示TFEB在宿主-细菌相互作用中的作用。 假设：我们的过度假设是TFEB在细菌感染期间被激活，并且该蛋白在控制对细胞内细菌的自噬反应中起重要作用。特别地，我们提出TFEB激活对于受感染细胞的转录重编程是至关重要的，以允许上调自噬和溶酶体生物合成途径。 科学方法：为了解决上述假设，我们的具体目标如下：目标1：确定感染期间控制TFEB激活的宿主途径和细菌决定簇。我们将首先关注感染过程中控制TFEB激活的宿主信号通路。接下来，我们将研究细菌注射到宿主细胞中的蛋白质（称为“细菌效应子”）是否可以操纵TFEB调节。 目的2：利用合成生物学方法研究志贺菌丙酮酸劫持对TFEB激活的影响。我们观察到，不能利用宿主细胞内丙酮酸池的突变志贺氏菌不会引起TFEB激活。我们在这里建议使用合成生物学方法进一步工程化志贺氏菌，以证明从宿主细胞内池中动态提取丙酮酸是激活感染细胞中TFEB的必要和充分条件。目标3：表征TFEB在志贺氏菌感染细胞中控制细菌自噬、溶酶体生物发生和转录重编程的作用。通过用志贺氏菌感染野生型和TFEB缺陷型细胞来评估TFEB对感染细胞中（i）自噬、（ii）细菌生长和（iii）基因表达的影响。 新奇和预期意义：总体而言，该项目将联合收割机结合尖端技术，阐明TFEB在宿主防御中的功能。这将为几名学生的培训和他们接触综合研究主题和轴，包括细胞微生物学，生物信息学和合成生物学提供一个独特的环境。