In addition to the in-house training that each ESR received while being immersed in their Individual Research Projects, all partners contributed to the network-wide training activities that were scheduled throughout the programme. This consisted of 8 workshops (Entrepreneurship; Outreach; Ethics; Microscopy; Structural Biology; Proteomics; Career Planning; Grant Writing Skills) and 2 Summer Schools (SS1:Translating Postgenomic Microbiology to Advancing Food Science & Safety and SS2: Virulence & Stress Responses: Two Sides of the Same Coin?).
The project’s technical objectives were fulfilled and significant novel findings have been published in high impact journals and have been disseminated at international conferences and symposia.
Stressosome Structure: Cryo-electron microscopy, protein purification and crystallisation of the N-terminal domains and X-ray diffraction studies have each provided detailed structural information and have allowed the stressosome structures of Vibrio and Listeria to be modelled. A model of the Listeria stressosome is now available at 3.45 Å and the Vibrio vulnificus stressosome at 8.3 Å. High resolution structures of three of the sensory turrets from the RsbR1, RsbR2 and RsbR3 proteins from Listeria monocytogenes were determined by X-ray crystallography and ligand binding pockets were identified in RsbR3. Although the ligand was not established definitively, candidate molecules were identified. Significant new insights into the allosteric regulation of the stressosome by phosphorylation have been gained through this collaborative project.
Stressosome Function: We have provided the first insight into the function of the stressosome in a Gram-negative bacterium, V. vulnificus. Proteomic analyses demonstrated the effects of the stressosome on global protein expression, in particular at entry into stationary phase. In this pathogen we found that the stressosome plays a critical role in in acid survival and in regulation of iron metabolism, suggesting a role in virulence since high blood iron is a risk factor for infections. We have established a direct role of the stressosome in acid sensing in L. monocytogenes. The sensory protein RsbR1 and stressosome phosphorylation are essential for signal transduction of acid stress signals. Since acid stress is important in food preservation and in the gastrointestinal tract this suggests that the stressosome might play a role during food-borne infections. In L. monocytogenes stressosome phosphorylation was also found to be crucial for sensing salt stress.
Stressosome & Virulence: A direct link between stressosome-mediated acid sensing in L. monocytogenes and host invasion gene expression was demonstrated, suggesting that the stressosome likely primes bacteria in the gastrointestinal tract for invasion. Furthermore, we demonstrated that L. monocytogenes stressosome proteins are expressed during infection of eukaryotic host cells and SigB is activated during the intracellular stage of the infection, providing further experimental evidence for the importance of the stressosome in virulence. Evidence generated in this project suggests that the configuration of stressosome proteins might differ between ex vivo culture conditions and within host cells, and that signalling can be modulated by the spatio temporal localization of stressosome proteins. For L. monocytogenes a link between the cell´s surface structure and SigB activity was uncovered, potentially influencing interactions within the host. In V. vulnificus a direct role for the stressosome in regulating motility has been established and this could contribute to virulence in the host. Additionally, the stressosome regulates iron metabolism, which hints at a role in vivo as individuals with elevated blood iron are particularly susceptible to infection.
Sensory Mechanisms: The genetic tools for dissecting the sensory mechanisms underpinning the stressosome have been developed for Bacillus, Listeria and Vibrio. The kinase activity of RsbT in L. monocytogenes plays a crucial role in sensing as shown by the acid sensitive phenotype of an RsbT N49A kinase-defective mutant. Residues in RsbR1 and RsbS were identified as playing key roles in signal transduction. A new mathematical model has been developed that successfully predicts the dynamic phenotypes that have been observed experimentally for stressosome mediated sigma factor regulation. In addition, we have advanced the field by developing a model encompassing the stressosome explicitly in a model of the SigB regulation circuit in B. subtilis. This will allow specific experimental predictions thereby stimulating further development in this fast-moving field of research. Our data suggest that the V. vulnificus stressosome has a unique regulatory output: a two-component system which metabolises the secondary metabolite c-di-GMP.
Sensory Applications: The possibility of using stress responsive promoters to detect and quantify preservation stress in food matrices was explored using known SigB promoters from L. monocytogenes and B. subtilis. While the reporters were effective in vitro the background signal noise made the systems difficult to apply in food matrices. Mathematical modelling indicated that SigB plays a role in surviving food preservation stresses in the minutes immediately after the stress was applied but had only a minimal long-term role. Finally, a collection of plant extracts with antimicrobial activity has been generated, and will be commercially exploited for use in foods.
