BREX phage defence: expanding the role of cyclic nucleotide signalling in the prokaryotic immune system
Lead Research Organisation:
Durham University
Department Name: Biosciences
Abstract
"Gene-editing" has transferred from the realms of sci-fi to mainstream biotechnology. We have the ability to edit the genes of organisms in order to manipulate how they look, grow, behave, and to cure disease. This has been made possible through "CRISPR-cas", a tool that now regularly gets name-checked in newspapers and even popular TV and streaming shows. What is not often mentioned is that CRISPR-cas is based upon the weaponry of an ancient and ongoing war that surrounds our everyday lives.
The Earth is home to truly astronomical numbers of bacteria, and they are outnumbered 10-to-1 by viruses called bacteriophages. Bacteria and bacteriophages have been co-evolving for millions of years. These bacteriophages thankfully don't harm humans. But in the same way that our immune system responds to infections, bacteria have been forced to evolve systems that protect from bacteriophages. These defence systems often have specific activities, such as cutting DNA. As a result, many of our biotechnologies have arisen from the applications of these systems, including CRISPR-cas.
There has been a recent explosion in the discovery of systems bacteria use for defence against bacteriophages. As a result, the analogy comparing our own immune system with that of bacteria has become reality. There are now proven evolutionary and functional links between the antiviral response in mammals and bacteriophage defence in bacteria. Furthermore, we can reverse this relationship, using the discovery of new systems in bacteria to predict forward and identify new systems in the mammalian immune system!
This finding suggests that perhaps we should consider bacteriophage defence in bacteria as an "immune system". As such, the varied defence systems would be expected to communicate and be co-regulated. Work in the Blower lab has already identified proteins that co-regulate diverse defence systems. Work in other labs has identified a series of molecules called cyclic nucleotides that move between component parts of defence systems and switch them on or off. Going back to our analogy, we know that these cyclic nucleotides are used in humans to control networks of immunity. It follows that cyclic nucleotides might also therefore be key to control within the bacterial immune system.
We have identified a defence protein that degrades cyclic nucleotides as part of a defence mechanism called Bacteriophage Exclusion (BREX). It is not currently understood why this activity is present, and how it impacts BREX. The overall mechanism for BREX is also not understood.
Our objective is to investigate cyclic nucleotide usage in the context of BREX. We will characterise the activity of the protein that degrades cyclic nucleotides, and which forms of nucleotides it prefers to target. We will look at how the varied proteins that make up BREX (there are six), interact, and how the presence of cyclic nucleotides might alter these interactions. We will then look at how to use BREX biotechnologically by understanding how to engineer it to make modifications in DNA. BREX adds a modification that is important for controlling use of DNA in processes such as aging and disease. Having a targeted method of modification will provide another tool for biomedical research.
The overall outputs will be to provide further evidence towards a cohesive bacterial immune system and progress our understanding of BREX towards potential biotechnological exploitation.
The Earth is home to truly astronomical numbers of bacteria, and they are outnumbered 10-to-1 by viruses called bacteriophages. Bacteria and bacteriophages have been co-evolving for millions of years. These bacteriophages thankfully don't harm humans. But in the same way that our immune system responds to infections, bacteria have been forced to evolve systems that protect from bacteriophages. These defence systems often have specific activities, such as cutting DNA. As a result, many of our biotechnologies have arisen from the applications of these systems, including CRISPR-cas.
There has been a recent explosion in the discovery of systems bacteria use for defence against bacteriophages. As a result, the analogy comparing our own immune system with that of bacteria has become reality. There are now proven evolutionary and functional links between the antiviral response in mammals and bacteriophage defence in bacteria. Furthermore, we can reverse this relationship, using the discovery of new systems in bacteria to predict forward and identify new systems in the mammalian immune system!
This finding suggests that perhaps we should consider bacteriophage defence in bacteria as an "immune system". As such, the varied defence systems would be expected to communicate and be co-regulated. Work in the Blower lab has already identified proteins that co-regulate diverse defence systems. Work in other labs has identified a series of molecules called cyclic nucleotides that move between component parts of defence systems and switch them on or off. Going back to our analogy, we know that these cyclic nucleotides are used in humans to control networks of immunity. It follows that cyclic nucleotides might also therefore be key to control within the bacterial immune system.
We have identified a defence protein that degrades cyclic nucleotides as part of a defence mechanism called Bacteriophage Exclusion (BREX). It is not currently understood why this activity is present, and how it impacts BREX. The overall mechanism for BREX is also not understood.
Our objective is to investigate cyclic nucleotide usage in the context of BREX. We will characterise the activity of the protein that degrades cyclic nucleotides, and which forms of nucleotides it prefers to target. We will look at how the varied proteins that make up BREX (there are six), interact, and how the presence of cyclic nucleotides might alter these interactions. We will then look at how to use BREX biotechnologically by understanding how to engineer it to make modifications in DNA. BREX adds a modification that is important for controlling use of DNA in processes such as aging and disease. Having a targeted method of modification will provide another tool for biomedical research.
The overall outputs will be to provide further evidence towards a cohesive bacterial immune system and progress our understanding of BREX towards potential biotechnological exploitation.
Technical Summary
Our objective is to investigate cyclic nucleotide usage by Bacteriophage Exclusion (BREX) bacterial phage defence systems.
Recent work on phage defence systems has shown evolutionary and functional links between prokaryotic viral immunity and human viral immunity. Phage defence systems are often clustered into defence islands, and we have identified the BrxR family of transcriptional regulators that co-regulate gathered defence systems. In CBASS phage defence, cyclic nucleotides are synthesised in response to phage infection and activate effector proteins that stop the infection. These same cyclic nucleotides have been noted to allow cross-talk between CBASS and CRISPR-Cas. This wealth of evidence shows that phage defence systems do not operate as disparate systems. Instead, phage defence systems can work in a co-ordinated manner. Phage defence is starting to be considered a global "prokaryotic immune system".
BREX is one of the most common and widespread phage defence systems. Nevertheless, the BREX mechanism is unknown. We have shown that BREX protein PglZ cleaves cyclic nucleotides. Therefore, PglZ activity indicates cyclic nucleotide signalling impacts BREX defence. PglZ activity provides an opportunity to understand BREX and expand our knowledge of how cyclic nucleotides are used in phage defence. Knowledge of defence system biochemistry is also essential for biotechnological exploitation. We will use a range of microbiological, genetic, and biochemical assays (including our new Dianthus high-throughput affinity platform) to study BREX interactions and activities.
Demonstrating that cyclic nucleotide signalling is used by BREX will have implications for expanding the role of cyclic nucleotides across a greater diversity of defence systems. The results will have impacts towards 1) developing a working model for a cohesive, inter-linked, prokaryotic immune system and 2) building a greater arsenal of biotechnological tools.
Recent work on phage defence systems has shown evolutionary and functional links between prokaryotic viral immunity and human viral immunity. Phage defence systems are often clustered into defence islands, and we have identified the BrxR family of transcriptional regulators that co-regulate gathered defence systems. In CBASS phage defence, cyclic nucleotides are synthesised in response to phage infection and activate effector proteins that stop the infection. These same cyclic nucleotides have been noted to allow cross-talk between CBASS and CRISPR-Cas. This wealth of evidence shows that phage defence systems do not operate as disparate systems. Instead, phage defence systems can work in a co-ordinated manner. Phage defence is starting to be considered a global "prokaryotic immune system".
BREX is one of the most common and widespread phage defence systems. Nevertheless, the BREX mechanism is unknown. We have shown that BREX protein PglZ cleaves cyclic nucleotides. Therefore, PglZ activity indicates cyclic nucleotide signalling impacts BREX defence. PglZ activity provides an opportunity to understand BREX and expand our knowledge of how cyclic nucleotides are used in phage defence. Knowledge of defence system biochemistry is also essential for biotechnological exploitation. We will use a range of microbiological, genetic, and biochemical assays (including our new Dianthus high-throughput affinity platform) to study BREX interactions and activities.
Demonstrating that cyclic nucleotide signalling is used by BREX will have implications for expanding the role of cyclic nucleotides across a greater diversity of defence systems. The results will have impacts towards 1) developing a working model for a cohesive, inter-linked, prokaryotic immune system and 2) building a greater arsenal of biotechnological tools.
