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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PATHSENSE (Training Network to Understand and Exploit Mechanisms of Sensory Perception in Bacteria)

Teaser

Summary

The PATHSENSE (Pathogen Sensing) ETN brings together an interdisciplinary team of world-leading researchers from Europe to tackle a highly ambitious scientific project, focusing on the molecular mechanisms of sensory perception in bacterial pathogens. PATHSENSE has established an innovative doctoral training programme that will deliver 13 PhD graduates and high-impact scientific outputs. The relationship between molecular structures and biological function is central to understanding any living system; however the research methodologies required to unravel these relationships are often complex and fast-changing.

The team participating in this Network has the infrastructure and track-record to train ESRs in these state-of-the art methodologies, including structural biology, proteomics & protein biochemistry, molecular biology, bacterial genetics, food microbiology, mathematical modelling, cell biology, microscopy and comparative genomics. PATHSENSE will investigate the poorly understood structure function relationships that exist within a large multi-protein complex called a â€œstressosomeâ€, which acts as a sensory organelle in bacteria. The goal is to understand the molecular mechanisms of sensory perception in pathogenic bacteria so that better preventative strategies can be developed to control them.

The project will involve has extensive inter-sectoral mobility of the ESRs across 7 EU countries to make full use of the complementary skills available at each of the hosting institutions. The inter-sectoral Network comprises 8 leading Universities, 1 public research institution, 4 companies (from spin-off to large multi-national) and 1 governmental agency.

A major objective of this Network will is be to exploit the fundamental research to develop novel antimicrobial treatments that have applications in the food and public health sectors. This project will deliver high-impact science, 13 highly-trained innovative researchers and will produce a long-lasting inter-sectoral network of collaborators who will continue to work together to exploit fundamental research for the socio-economic benefit of Europe.

Work performed

13 ESRâ€™s were successfully recruited to the PATHSENSE Network and are based in 7 different European countries. In addition to the in-house training that each ESR receives while being immersed in their Individual Research Projects, all partners in the consortium are taking a coordinated and active role in network-wide training activities, scheduled at various times over the implementation period. So far, the Network-wide training that was organised has been a success and has consisted of four workshops (Workshop 1, Entrepreneurship; Workshop 2, Outreach; Workshop 3, Scientific Integrity & Ethics; Workshop 4, The Power of Microscopy: Seeing is Believing) and one Summer School (Translating Postgenomic Microbiology to Advancing Food Science & Safety).

As summarised below, significant progress has been made towards fulfilling the projectâ€™s technical objectives.

Stressosome Structure: Progress has been made towards elucidating the structure of the stressosome in both Listeria and Vibrio. Cryo-electron microscopy, purification and crystallisation of the N-terminal domains and X-ray diffraction studies are providing very useful structural information and allowing the stressosome structure of these two genera to be compared. Thus far three of N-terminal domains of the RsbR paralogues from L. monocytogenes have been crystallised and the structure of two of these have been solved at a high resolution. A potential ligand binding pocket has been identified in one of these structures.

Stressosome Function: There has been a focus on constructing mutants in Listeria monocytogenes and Vibrio vulnificus to examine the function of the stressosome in these pathogens. The PATHSENSE consortium has established a fruitful collaboration with Prof Mathew Cabeen (Oklahoma State University) to further our understanding of stress sensing in B. subtilis and L. monocytogenes. The phosphatase RsbX from L. monocytogenes is shown to be required for motility. The SigB regulon in B. cereus has been compared to that of L. monocytogenes and found to be much smaller and somewhat distinct.

Stressosome & Virulence: Mutants have been constructed in Listeria, Bacillus and Vibrio that are allowing the role of the stressosome components in virulence to be tested. In the chick embryo model there is evidence that L. monocytogenes SigB may play a negative role in virulence, probably through effects on the virulence gene regulator PrfA.

Sensory Mechanisms: The genetic tools for dissecting the sensory mechanisms underpinning the stressosome are being developed for Bacillus, Listeria and Vibrio. An assay to measure the phosphorylation of RsbR during stress sensing has been developed. 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.

Sensory Applications: A collection of plant extracts with potential antimicrobial activity has been generated, and are being tested for anti-Listeria activity. Extensive testing is underway to determine if these are effective in food matrices, with the goal of using them improve food preservation efficiency. The role SigB plays in L. monocytogenes during growth in a real food matrix is being mathematically modelled.

Final results

This project has already uncovered novel findings pertaining to the biological structure and function of the stressosome in bacteria. In addition we are continually learning more about the sensory mechanisms of this large multi-protein complex. As this structure is highly conserved in several important bacterial phyla this knowledge is giving us new insights into how these bacteria sense and respond to their environment. PATHSENSE has new high-resolution structural data on the stressosome that will shed light on the molecular mechanisms that underpin stress sensing. Ultimately understanding how bacteria â€œseeâ€ their environment will facilitate the development of new and improved control measures, with positive impacts on food spoilage and safety and on the treatment of infections.

The project is seeking to identify novel natural antimicrobials using a combination of chemical extractions from plant material and microbial activity assays. Compounds that inhibit pathogen growth have been identified and these are under investigation to examine the efficacy of these natural inhibitors in food preservation. The involvement of highly experienced industrial beneficiaries in the project is expected to ensure that these discoveries will be translated into new technologies that can give a competitive advantage to EU industry and improve the lives of its citizens.