Periodic Reporting for period 4 - EPICut (Molecular mechanisms, evolutionary impacts and applications of prokaryotic epigenetic-targeted immune systems)
Berichtszeitraum: 2023-02-01 bis 2024-07-31
Over billions of years, infection of bacteria by viruses (“phages”) and other mobile genetic elements (MGEs) has led to a coevolutionary arms race: prokaryotes evolved diverse defence systems that can block virus entry, cause cell suicide, or use enzymes to cut the infecting genome. In parallel, phages and MGEs acquired counter-defences, e.g. genome modifications (called “epigenetic modifications”) that block enzyme activity. Characterisation of defence systems led in the 1970s to the discovery of restriction-modification (RM) enzymes critical to gene cloning, and more recently to CRISPR that has supported a new age of gene editing. Defence systems are also significant to our understanding of microbial communities, as they influence horizontal gene transfer (HGT), influencing virulence, pathogenicity and antibiotic resistance. During the project, it has become clearer that CRISPR and RM are the tip of a defence iceberg, with bacteria carrying multiple systems with largely unknown mechanisms. Understanding the wide range of defence mechanisms and their effects on evolutionary dynamics is critical.
EPICut aims to address the mechanism and role in bacterial evolution of classes of enzymes that use and react to DNA modifications. Since phages have evolved metabolic pathways to produce epigenetic modifications that block binding by defence enzymes, bacteria evolved restriction enzymes that can recognise and cut modified DNA. EPICut is a unique interdisciplinary project that combines biophysical analysis of enzyme function with prokaryotic evolutionary ecology to link the molecular mechanisms of prokaryotic defence to individual, population and community-level phenotypes. Diverse systems were to be studied, some of which appear to require interaction with multiple modified sites and some of which require an input of chemical energy (ATP or GTP). Very little was known about these enzymes at a mechanistic level. Additionally, the consequences of defences for the coevolution of prokaryotic-phage communities was almost completely unstudied. It was unclear if and why the presence of these genes in natural environments matters, and how these defences influence trait acquisition, e.g. antibiotic resistance. Deeper analysis of enzyme function will also support reengineering to produce improved lab tools, which are important in human health and disease.
EPICut aims to address the mechanism and role in bacterial evolution of classes of enzymes that use and react to DNA modifications. Since phages have evolved metabolic pathways to produce epigenetic modifications that block binding by defence enzymes, bacteria evolved restriction enzymes that can recognise and cut modified DNA. EPICut is a unique interdisciplinary project that combines biophysical analysis of enzyme function with prokaryotic evolutionary ecology to link the molecular mechanisms of prokaryotic defence to individual, population and community-level phenotypes. Diverse systems were to be studied, some of which appear to require interaction with multiple modified sites and some of which require an input of chemical energy (ATP or GTP). Very little was known about these enzymes at a mechanistic level. Additionally, the consequences of defences for the coevolution of prokaryotic-phage communities was almost completely unstudied. It was unclear if and why the presence of these genes in natural environments matters, and how these defences influence trait acquisition, e.g. antibiotic resistance. Deeper analysis of enzyme function will also support reengineering to produce improved lab tools, which are important in human health and disease.
A key step was to establish “test tube” conditions for protein analysis. Firstly, we developed strategies for purification and modification of defence proteins; e.g. to label the protein with fluorescent dyes to directly follow motion on DNA. Secondly, we designed methods to modify DNA at specific locations with epigenetic markers, allowing precise design for the first time. Thirdly, we developed methods for analysing protein activity by using single-molecule microscopy; fluorescence spectroscopy; and nanopore DNA sequencing. The latter method resulted a new technique called ENDO-pore that can precisely map the breakage of DNA at thousands of sites. Our studies revealed new mechanisms for DNA cleavage by SauUSI and McrBC, enzymes that consume chemical energy in order to cut DNA. For SauUSI we revealed a “molecular motor” that uses chemical energy from ATP to move in one direction while spooling a DNA loop. When the motor stops, it cuts the loop at a random site. For McrBC we showed that the protein is not a motor but instead using chemical energy from GTP to change protein confirmation and bend and distort DNA to allow cleavage at random sites. Both studies revealed a general mechanism of DNA shredding by random DNA cleavage, a potentially important way that bacteria avoid that transferred genes are reassembled via DNA repair. An important aspect of defence systems is the “benefit” in regulating HGT can be offset by the “cost” of carrying the defence. One important cost is auto-immunity, where the defence targets self DNA rather than non-self DNA. We revealed two mechanisms to minimise this. For Type III restriction enzymes we showed the protective DNA modification and destructive DNA cleavage activities are coupled, such that all DNA is methylated but only the non-self DNA is cut. For SauUSI, a second gene is found nearby on the genome that produces a protein that removes methylated DNA building blocks so they don’t accumulate inadvertently in the genome. Using a collection of Escherichia coli strains that carry different defences, we explored whether other bacteria could introduce genes by a method called conjugation. We revealed that restriction enzymes provide a surprisingly weak barrier to horizontal gene transfer. Nonetheless, the dynamics of the process would be influenced and more similar bacteria are more likely to exchange genes than unrelated bacteria. Our work thus reveal a complex dynamics in HGT. Similar results were observed by computer modelling showing that a weak barrier to HGT leads to both a build up of AMR genes and defences. Preliminary experiments to measure the rates of phage-mediated HGT serendipitously showed that antibiotics can alter the host evolutionary response. We determined that CRISPR defences can evolve resistance to phages more rapidly if antibiotics are used that slow growth (rather than the killing activity of bactericidal antibiotics. Finally, to help exploit defence systems as tools in the lab, we used an AI tool called AlphaFold (awarded the Nobel Prize in Chemistry 2024) to predict restriction enzyme structures that undergo a natural change in gene sequence called phase variation. We showed how they fold into a working structure dependent on a structural switch. This switch could be exploited to provide a tuneable restriction system that could be targeted to sites in a user-defined manner.
Our new DNA substrates and biophysical assays provide more strategic methods for testing defence systems than previously employed. The mechanisms of Type IV restriction have been established unambiguously. Single-molecule methods have provided unprecedented details of complex biological processes. We anticipate that our studies will inform on the ever-growing list of defences that are mechanistically undefined. How defences avoid destroying their own genomes (auto-immunity) while destroying non-self-genomes is central to their widespread adoption; an “ideal” system would have minimal auto-immunity while maximising defence activity. Our work on the Type III enzymes revealed an unexpected model to distinguish self from non-self whereby the protective DNA methylation and destructive DNA cleavage activities can occur simultaneously. This model was unexpected and can now be applied to other defences that use DNA modifications. Our streamlined and cost-effective ENDO-pore method provides an accurate high-throughput method for mapping DNA cleavage and can be applied to any system that cuts DNA. Our evolutionary studies have already revealed that antibiotics alter the evolutionary response of bacteria. This will be critical to consider when administering antibiotics in medical/veterinary situations. Furthermore, our work on the link between different defence systems and HGT reveals complex dynamics that depends on levels of HGT and genetic relationships between bacteria. Future work will need to consider the interplay of antibiotics, defences and DNA modifications to fully understand HGT and to be able to make predictions.