Abstract:

The intestinal epithelium plays a central role of protection against pathogens by acting as a physical barrier and contributing to the initiation of an innate immune response. Indeed, although intestinal epithelial cells are not considered as professional immune cells, they are equipped with a set of pathogen recognition receptors allowing them to detect a large spectrum of microbes and respond by the production of pro-inflammatory cytokines and chemokines, which recruit and activate immune cells at the sites of infection.

Shigella flexneri is a gram-negative entero-invasive bacterium responsible for shigellosis in humans. During infection of epithelial cells by Shigella flexneri, cytosolic sensing of ADP-heptose (ADPH), a small metabolite of the LPS biosynthesis pathway, is recognized by the atypical kinase ALPK1 which triggers an innate immune response dependent on the adaptor protein TIFA and its constitutive binding partner TRAF6. Upon recognition of ADPH, ALPK1 phosphorylates TIFA leading to the formation of large protein oligomers, named TIFAsomes. In turn, this induces the activation of NF-kB and the expression of pro-inflammatory genes. The ADPH/ALPK1/TIFA axis is also activated during infection with Yersinia pseudotuberculosis, Helicobacter pylori and Campylobacter jejuni and several actors of this signalling pathway are also involved in several non-infectious diseases, including cancers. Yet, the mode of regulation of this pathway and the fate of TIFAsomes remain unknown.

In my thesis, I show that TIFAsomes form within minutes of ADPH detection and last for several hours in cells. This leads to the rapid and transient activation of the NF-κB and the JNK and p38 MAPK signaling pathways, and the recruitment of the kinases TAK1 and TBK1 on TIFAsomes, suggesting that they act like signalling platforms to activate downstream pathways. Single TIFAsome tracking by live cell imaging reveals that TIFAsomes are dynamic structures with limited movement within the cells. It also shows that TIFAsomes fuse to each before being degraded. This last process depends on the proteasome and the E3 ubiquitin ligase activity of TRAF6. Altogether, our results provide the first insights into the spatio-temporal regulation of TIFAsomes and contribute to better characterize the ADPH/ALPK1/TIFA signalling pathway, which constitutes a new pathway of innate immunity against Gram-negative bacteria.