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Molecular mechanisms, evolutionary impacts and applications of prokaryotic epigenetic-targeted immune systems

Periodic Reporting for period 2 - EPICut (Molecular mechanisms, evolutionary impacts and applications of prokaryotic epigenetic-targeted immune systems)

Reporting period: 2020-02-01 to 2021-07-31

Over billions of years, infection of bacteria and archaea by viruses (bacteriophages, or “phages”) and other mobile genetic elements (MGEs) has led to a coevolutionary arms race: under selective evolutionary pressure, prokaryotes evolved diverse defence systems that can block viral entry, cause cell suicide, or use enzyme activities to cleave the infecting genome or prevent its replication and/or integration. In parallel, phages and MGEs acquired counter-defences, for example, genome modifications (called “epigenetic modifications”) that can block enzyme cleavage. The characterisation and analysis of bacterial defence systems led in the 1970s to the discovery of restriction-modification (RM) enzymes that became critical to gene cloning, and more recently to the CRISPR enzymes that have 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), including acquisition of virulence, pathogenicity or antibiotic resistance genes. The EPICut project aims to address the mechanism and role in bacterial evolution of one class of defence system, the Type IV RM enzymes.

As noted above, a strategy to evade microbial defence is that phages have evolved metabolic pathways to produce epigenetic modifications to block binding by RM and CRIPSR-Cas enzymes. To counter these modifications, bacteria evolved Type IV RM enzymes that can recognise and cut modified DNA. EPICut is a unique interdisciplinary project that combines biophysical and single-molecule analysis of enzyme function with prokaryotic evolutionary ecology to link the molecular mechanisms of prokaryotic defence to individual, population and community-level phenotypes. A diverse range of systems will 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 is known about these enzymes at a mechanistic level. Additionally, the consequences of Type IV enzymes for the coevolution of prokaryotic-phage communities is almost completely unstudied. It is unclear if and why the presence of these genes in natural environments matters, and how these defence strategies affect the acquisition of traits, such as antibiotic resistance. A deeper analysis of Type IV enzyme function will also support reengineering of these systems to produce improved tools for the mapping of eukaryotic epigenetics markers, which are important in human health and disease.
A key step is to establish in vitro (“test tube”) conditions for analysis of protein activity. Firstly, we have developed strategies for purification and modification of Type IV proteins; for example, to label the protein with fluorescent dyes to directly follow protein motion on DNA. Secondly, we needed to design methods to modify DNA substrates at specific locations with the epigenetic markers. For this we turned to Type II RM systems, and have cloned and purified methyltransferase enzymes that can introduce the modification at sites that we can control. For the first time we can design substrates, rather than relying on random modifications. Thirdly, we have developed methods for analysing protein activity by using single molecule microscopy; rapid-mixing, millisecond time resolution fluorescence spectroscopy; and nanopore DNA sequencing.
For GTP-dependent McrBC, using ensemble (in a “test tube”) DNA cleavage assays combined with magnetic tweezers microscopy, we have demonstrated that the DNA cleavage mechanism is distinct from the published dogma and is consistent with a randomly diffusing (“sliding”) enzyme can that produce multiple dsDNA breaks. To properly map the cleavage sites, we developed ENDO-pore, a high-throughput nanopore sequencing method and associated software tools that can map both ends of a DNA cleavage site with single event resolution. We can map exactly where McrBC introduces the dsDNA breaks and how these develop with time. This method will be generally applicable to other DNA cleavage systems, like CRISPR. We are developing single molecule techniques to directly observe how waves of GTP hydrolysis in the McrB ring-shaped protein produce changes in the McrC nuclease to activate it.
For ATP-dependent SauUSI, we have established stepwise motor protein activity using a triplex-displacement method, showing a bidirectional translocation that helps explain the cleavage model where a “pile up” of molecular motors on the DNA results in multiple DNA cleavages that shred a phage genome. We are now mapping these breaks using ENDO-pore. Our next step is to observe the pile up directly using single molecule observation techniques.

Although bacterial innate immune systems are widespread (almost all bacteria carry at least one system), we do not understand how they shape bacterial evolution and ecology. For example, these systems are likely to play important roles in HGT, but experimental tests are limited. We have carried out a comprehensive literature review to summarize our current knowledge and the state-of-the art of the research field that aims at understanding the ecology and evolution of bacterial innate immune systems, and their interplay with bacterial adaptive immune systems (Dimitriu et al Current Biology 2020). We have also carried out large scale bioinformatics analyses to understand how innate and adaptive immune systems shape the acquisition of antimicrobial resistance genes by pathogens. We are supplementing these large scale analyses with focussed studies using human pathogens from the clinic, which we examine in detail using bioinformatics and phenotypic analyses to better understand how bacterial immune systems shape horizontal gene transfer of antibiotics resistance genes. These ‘observational experiments’ will be further complemented with experimental analyses. We have already generated a collection of isogenic E. coli strains that carry different RM types to test the link between host defence and HGT. We expect to start experimental analyses towards the summer of 2021. Preliminary experiments to measure the rates of phage-mediated HGT serendipitously showed that antibiotics can alter the host evolutionary response. We have examined the mechanistic basis for this and are currently working on a manuscript to publish these results.
Our new DNA substrates and biophysical assays provide more strategic methods for testing the hypotheses about Type IV systems than previously employed. We will publish a number of studies on the mechanisms of Type IV enzymes that establish unambiguously their cleavage mechanisms.
Our ENDO-pore sequencing method and Cleavage Site Identifier software workflow provides an accurate high-throughput method for mapping DNA cleavage on normal laboratory DNA plasmids. The method is streamlined and cost-effective, and will be published as a method soon.
Single molecule methods have not been applied to the Type IV enzymes previously. These methods provide unprecedented details of complex biological processes, and we anticipate that our studies will reveal unanticipated mechanisms that have evolved to cleave phage DNA.
Our evolutionary studies have already revealed that antibtiotics and alter the evolutionary response of bacteria, and this study will be published in the coming year. Work will then concentrate on the link between different RM systems and HGT, the first time this has been studied.