Current projects in our lab fall into three general areas, summarized below. For more information, please see our publications here or on PubMed.
Identification of new anti-phage defense systems
Bacteria have evolved numerous immunity mechanisms to defend themselves against phage infection. This includes restriction-modification and CRISPR-Cas systems, but in recent years it has become clear that there are many additional systems. Some have been found through bioinformatics, leveraging the observation that some defense systems cluster in so-called defense islands within bacterial genomes. But not all systems are necessarily in islands or identifiable through such associations. To more comprehensively identify new defense systems, we developed a powerful functional selection approach that led to the identification of 21 new anti-phage defense systems in a diverse set of E. coli strains. We continue to use and modify this functional selection to identify yet other new systems, both in E. coli and other bacterial species. Further, we have developed new computational methods for rapidly predicting new anti-phage defense systems. Collectively, these studies are providing a wealth immunity mechanisms to investigate, as described below, and providing new insights into the overall landscape, composition, and evolution of anti-phage immunity in bacteria.
Functional screening approach for identifying new anti-phage defense systems.
Mechanistic studies of anti-phage defense
For the systems we have identified through our functional selections, as well as others identified computationally, we investigate the mechanistic basis of anti-phage defense. For each system, we focus on understanding how the system is triggered, or activated, in response to phage infection and how the effector components of a system then block or disrupt phage replication. We use a combination of genetic, biochemical, genomic, and structural approaches to tackle these questions, with the aim of revealing how anti-phage defense systems work at a very detailed molecular level. We have a particular interest in dissecting toxin-antitoxin (TA) systems that function in anti-phage defense, including novel TA systems isolated in our recent screening efforts. But we also investigate a range of other new defense systems featuring unusual effector mechanisms or those that are amenable to understanding how bacteria sense phage infection.
Schematic of anti-phage defense mechanisms, including CRISPR-Cas, restriction-modification (RM), and TA systems.
Structures of the anti-phage defense protein CapRel in the closed (left) and open (right) states, with the activator Gp57 from phage SECphi27 bound to and stabilizing the open state.
We study the tempo and mechanisms of bacterial and phage evolution. We routinely employ experimental evolution to examine how phages can overcome various anti-phage defense systems and how, in turn, bacteria coevolve in the face of such counter-defense mechanisms. We use deep sequencing both to identify the outcomes of such experimental evolutions and to assess the patterns and types of mutation that arise, including insertion/deletions, point mutations, rearrangements, recombination, and more. Additionally, we use phylogenetic and ancestral reconstructions to elucidate, retrospectively, the coevolution of bacteria and phage. Finally, in work that builds off our long-standing studies of protein coevolution done in the context of two-component signaling and toxin-antitoxin systems, we employ deep mutational scanning to systematically interrogate mutational trajectories and the constraints that influence coevolving proteins relevant to bacteria-phage conflicts. We aim to elucidate general principles and mechanisms underlying the Red Queen dynamics of host-pathogen conflicts.
Schematic of experimental evolution approach (top) and example of genomic data revealing deletions and insertions enabling T4 phage to evade the toxIN defense system.
(Left) Chemical structure of ppGpp. (Center) Schematic of mass spectrometry-based approach for identifying ppGpp binding targets. (Right) Zoomed in view of ppGpp bound to PurF, a newly identified target in E. coli.