Periodic Reporting for period 4 - PyraSig (Pyrazine Signalling in Commensal and Pathogenic Bacteria)
Période du rapport: 2021-04-01 au 2023-06-30
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 QS in the major human pathogen, Vibrio cholerae. We found that the recently discovered AI molecule DPO (3,5-dimethylpyrazin-2-ol) controls virulence gene expression in V. cholerae by inhibiting the expression of the major virulence regulator, AphA, via the small regulatory RNA (sRNA) VqmR. Indeed, we further discovered that many more sRNAs affect QS regulation and virulence gene production in vibrios and thus could become suitable drug targets. Moreover, these sRNAs also affect how pathogens respond to antibiotic treatment. For example, the VadR sRNA is produced in response to cell wall-damaging antibiotics and improves the tolerance of V. cholerae towards these types of antibiotics. Understanding how pathogens counteract antibiotic treatment is key for the development of innovative antimicrobial treatments targeting multi-resistant microbial pathogens and we believe that our work will add to this process. In addition, we have discovered a new QS regulator in V. cholerae (called QrrX) that modulates virulence and other collective functions and thus could be exploited for future intervention strategies.
In this project, we have been investigating the physiological, regulatory, and mechanistic underpinnings of quorum sensing (QS) and virulence gene expression in Vibrio cholerae. Using a combination of genetic and biochemical methods, we have explored the regulatory principles of QS-mediated signaling in vibrio species and discovered several new regulators involved in this process. Specifically, we have discovered that the recently discovered AI molecule DPO (3,5-dimethylpyrazin-2-ol) affects biofilm formation and virulence expression in V. cholerae and that this function critically depends on the regulatory roles of the VqmR small RNA (sRNA), which base-pairs with and inhibits the production of the major virulence determinant AphA. We further showed that DPO works in concert with other signaling molecules and that full QS behavior requires the combined activity of at least three signaling molecules.
These findings prompted us to search for additional sRNAs regulators controlling QS and/or virulence. Indeed, we discovered three additional sRNAs (FarS, VcdRP, and VadR) involved in these processes, however, their exact regulatory functions differed. For example, FarS controls fatty acid metabolism, which is key for host colonization, whereas VcdRP modulates cholera toxin production in response to changes in carbohydrate metabolism. In contrast, VadR modulates biofilm formation and antibiotic tolerance in V. cholerae. Taken together, these results suggest that post-transcriptional regulation is fundamental for QS signaling (incl. DPO-mediated signaling) and virulence in V. cholerae, and thus could be exploited as a potential drug target. Of note, all of these sRNAs are conserved in other pathogenic vibrio species suggesting that our findings could be relevant beyond the realm of a single organism.
Recently, we further expanded our research into QS regulation in V. cholerae and discovered a novel regulatory module consisting of the transcription factor, QrrT, and the non-coding regulator, QrrX. We showed that both, QrrT and QrrX, are required for rapid conversion from one QS state to another and that this function relies on inactivation of key QS regulators by the QrrX transcript. Interestingly, QrrX does not function as a typical sRNA controlling the expression of mRNAs, but rather base-pairs with other non-coding regulators to control their function. This type of regulation was previously unknown in vibrio species and studying their functions could help to improve our general understanding of QS pathway architectures in bacteria.
These findings prompted us to search for additional sRNAs regulators controlling QS and/or virulence. Indeed, we discovered three additional sRNAs (FarS, VcdRP, and VadR) involved in these processes, however, their exact regulatory functions differed. For example, FarS controls fatty acid metabolism, which is key for host colonization, whereas VcdRP modulates cholera toxin production in response to changes in carbohydrate metabolism. In contrast, VadR modulates biofilm formation and antibiotic tolerance in V. cholerae. Taken together, these results suggest that post-transcriptional regulation is fundamental for QS signaling (incl. DPO-mediated signaling) and virulence in V. cholerae, and thus could be exploited as a potential drug target. Of note, all of these sRNAs are conserved in other pathogenic vibrio species suggesting that our findings could be relevant beyond the realm of a single organism.
Recently, we further expanded our research into QS regulation in V. cholerae and discovered a novel regulatory module consisting of the transcription factor, QrrT, and the non-coding regulator, QrrX. We showed that both, QrrT and QrrX, are required for rapid conversion from one QS state to another and that this function relies on inactivation of key QS regulators by the QrrX transcript. Interestingly, QrrX does not function as a typical sRNA controlling the expression of mRNAs, but rather base-pairs with other non-coding regulators to control their function. This type of regulation was previously unknown in vibrio species and studying their functions could help to improve our general understanding of QS pathway architectures in bacteria.
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.