PROJECT SUMMARY Environmental sensing is at the basis of an organism's ability to adapt its physiology to respond to changes in its niche. Over the past years, it has emerged that bacteria not only perceive and respond to chemical stimuli, but are capable of sensing and processing physical forces as an environmental cue. In the case of bacterial pathogens, we and others have shown that mechanical stimuli can act as a hallmark of host colonization and lead to activation of virulence genes. Despite this realisation, our mechanistic understanding of pathways and systems involved in mechanosensing, transduction and processing of physical forces is currently limited, as is our knowledge regarding the role of mechanoregulation within the host environment. Over the course of this grant, we have made important discoveries regarding the mechanistic basis of mechanoregulation in the human pathogen enterohemorrhagic E. coli (EHEC). In the human host, ingestion of EHEC can lead to enterocolitis and severe complications such as haemolytic uremic syndrome. These hallmarks of infection are caused by the concerted action of virulence factors, including colonization factors and Shiga toxins. We discovered that the transcriptional regulator GrlA regulates the LEE pathogenicity island encoded type 3 secretion system, an important colonization factor, in response to mechanical cues. We showed that GrlA is regulated by subcellular compartmentalisation, and is sequestered on the inner membrane in resting cells. When activated by shear force GrlA dissociates from the membrane and is released into the cytoplasm, where it binds and induces the LEE masterregulator Ler. We also showed the biochemical basis for this mechanism, by identifying both constitutively active GrlA variants, and variants that are `blind' to mechanical cues. Regarding the interplay between intestinal motility and EHEC infection, we have identified Shiga toxin 2 as an effector that leads to enteric nervous system stimulation and increased gut transit. We discovered that the Shiga toxin 2-converting phage, but not Shiga toxin 1 is also subject to mechanical induction via GrlA. In this competing renewal application, we will (1) determine the mechanistic basis for the observed mechanoregulation of Shiga toxin and the toxin encoding phage, (2) characterize Shiga toxin induction in vivo and its impact on gastrointestinal motility, and (3) define the role of Shiga toxin during competition of EHEC with the endogenous microbiota. This work will reveal an alternative regulatory mechanism for a phage-encoded toxin, and define the role of Shiga toxin during bacterial colonization and inter-species competition in the host niche. .