Periodic Reporting for period 3 - PyraSig (Pyrazine Signalling in Commensal and Pathogenic Bacteria) Reporting period: 2019-10-01 to 2021-03-31 Summary of the context and overall objectives of the project Quorum sensing (QS) is a cell–cell communication process that allows bacteria to modify their behaviour in response to changes in the cell number and species composition of the vicinal community. Many, if not all, bacteria use QS to orchestrate the gene expression programs underlying collective behaviors. QS depends on the production, detection, and response to extracellular signaling molecules called autoinducers (AIs). In the past few years, new QS-dependent functions have been discovered in various bacterial organisms frequently involving new regulatory traits and previously unknown signaling components. However, the molecular and physiological underpinnings underlying these systems are typically unknown. In this project, we study the regulatory and functional roles of a recently discovered QS pathway. The AI of this system is a novel pyrazine molecule, called DPO (3,5-dimethylpyrazin-2-ol), which is recognized by the transcriptional regulator, VqmA. DPO and VqmA were identified in the major human pathogen, Vibrio cholerae, however, DPO production is wide-spread and occurs in virulent bacteria, but also in commensal species of the intestinal microbiota. How pathogens communicate with other species to orchestrate virulence and other collective functions is largely unknown. Studying DPO signaling will provide key insights into this fascinating process, which could also become relevant for the development of innovative treatment strategies targeting multi-resistant microbial pathogens. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In the first half of the PyraSig project, we made several important discoveries regarding the DPO-mediated gene expression control in Vibrio cholerae and – more general - collective behaviors of microbial pathogens. Specifically, we discovered that the DPO pathway of V. cholerae inhibits virulence gene expression by reducing the production of the AphA transcription factor at the post-transcriptional level. We pinpointed the VqmR small non-coding RNA (sRNA) as the key regulator of this signaling pathway and studied the molecular underpinnings of gene regulation by this sRNA. We were able to identify a new principle of RNA-based gene regulation, which prompted us to identify and additional sRNAs from V. cholerae. Here, we were able to identify the sRNAs VrrA, MicV, FarS, and VadR as important regulators of quorum sensing and virulence gene expression in this major pathogen. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In addition, to the results outlined above, our work on regulatory RNAs in Vibrio cholerae inspired us to develop a new genetic tool allowing us to the screen and characterize complex microbial phenotypes using a library of >250.000 synthetic small RNAs (see Peschek et al, EMBO J. 2019 Aug 15;38(16):e101650). This new approach provides several advantages over currently existing methods and we expect that is suitable for a variety of commensal and pathogenic bacteria and thus will have broad applications in microbiology. erc-b2-figure-4-v01.png
