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

TFEB最近被确定为调节自噬过程的关键蛋白，在自噬过程中，细胞消化和回收部分细胞内内容物。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一过程是否受到TFEB的控制，这一问题仍未得到充分研究。为了研究这个问题，我们将使用一个感染细菌病原体福氏志贺氏菌的上皮细胞模型，并将结合细胞微生物学技术、生物信息学（RNAseq和ChIP-seq）和合成生物学（生成工程细菌来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：我们的主要假设是TFEB在细菌感染期间被激活，并且该蛋白在控制细胞内细菌的自噬反应中起重要作用。特别是，我们提出TFEB激活对于感染细胞的转录重编程至关重要，从而允许自噬和溶酶体生物发生途径的上调。科学方法：为了解决上述假设，我们的具体目标如下：目标1：确定感染过程中控制TFEB激活的宿主途径和细菌决定因素。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“细菌效应物”）是否可以操纵TFEB调节。目的2：利用合成生物学方法描述志贺氏菌劫持丙酮酸对TFEB激活的影响。我们观察到，突变的志贺氏菌不能使用宿主的细胞内丙酮酸池，不会引起TFEB激活。为了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的必要条件和充分条件，我们建议使用合成生物学方法进一步改造志贺氏菌。目的3：研究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中的作用。TFEB对(i)自噬、（ii）细菌生长和（iii）感染细胞中基因表达的影响将通过用志贺氏菌感染野生型和TFEB缺陷细胞来评估。新颖性和预期意义：总体而言，该项目将结合前沿技术，阐明TFEB在宿主防御中的作用。这将为几名学生的培训提供一个独特的环境，并使他们接触到综合研究主题和轴，包括细胞微生物学，生物信息学和合成生物学。

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.