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

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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.