Periodic Reporting for period 2 - CBASS (CBASS: Life, Death and cyclic nucleotides)
Periodo di rendicontazione: 2023-02-01 al 2024-07-31
All living things are infected by viruses. In order to survive this attack, organisms have evolved a wide range of defence systems that recognise and combat viral infection. Bacteria have more than 100 different defence systems, some of which are conserved right through to humans. One example is the use of signalling molecules formed from nucleotides by a cyclase enzyme, which bind and activate effector proteins to provide defence. In bacteria, these have the collective term CBASS (Cyclic nucleotide based antiphage signalling system). It is important that we study and understand these systems, as they can provide useful information on the evolution and mechanism of defence against viruses. Furthermore, the use of bacterial viruses (phages) to combat drug resistant bacteria necessitates a complete understanding of bacterial immune systems.
The objectives of this project are to work out the mechanistic details of CBASS defence systems operating via cyclic nucleotide signalling and to investigate the virus counter-measures to this defence. The project focuses on how the nucleotide cyclases are regulated and on the molecular details of effector protein activation. Furthermore, we aim to discover the ways in which viruses overcome host defence by neutralising the signalling pathways. The approach taken is a combination of molecular microbiology, bioinformatics, biochemistry and structural biology.
The objectives of this project are to work out the mechanistic details of CBASS defence systems operating via cyclic nucleotide signalling and to investigate the virus counter-measures to this defence. The project focuses on how the nucleotide cyclases are regulated and on the molecular details of effector protein activation. Furthermore, we aim to discover the ways in which viruses overcome host defence by neutralising the signalling pathways. The approach taken is a combination of molecular microbiology, bioinformatics, biochemistry and structural biology.
Progress so far has focussed on two main areas: the regulation of nucleotide cyclase activity and the mechanism of effector function by cyclic nucleotides. We have uncovered key new molecular details regarding the activation of a particular type of CBASS system which requires specialised cellular machinery to couple cyclases to other proteins – analogous to the ubiquitin protein conjugation system. By studying CBASS defence in the model bacterium Bacillus subtilis, we have identified the specific protein that is joined to the CBASS cyclase – a membrane protein known as Phage Shock Protein A (PspA). Coupling of cyclase to PspA appears to be a mechanism to hold the cyclase in an inactive state, and slow down its activation on virus infection. We have also focussed on the mechanism of activation of anti-viral effectors by cyclic nucleotides. Firstly, revealing key details of structural reorganisation of a “TIR-SAVED” effector, which forms a long helical filament and degrades the key cellular molecule NAD+. Secondly, investigating how effector nucleases and proteases are activated, and the cellular consequences. Recently, we identified a new class of antiviral signalling molecule formed by joining two common cofactors, ATP and SAM, together in a novel way, generating “SAM-AMP” – a molecule that activates antiviral defence by binding to a membrane protein.
Our work has already advanced the state of the art in the field of antiviral nucleotide signalling pathways, in several respects. In particular, we have identified the first specific partner for CBASS cyclase conjugation and defined a new class of immune signalling molecule, SAM-AMP.
In the remaining period of the project we aim to uncover new effectors, delineate their mechanisms of action and understand how viruses counteract cellular defences by interrupting immune signalling.
In the remaining period of the project we aim to uncover new effectors, delineate their mechanisms of action and understand how viruses counteract cellular defences by interrupting immune signalling.