L A U B L A B
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
HOWARD HUGHES MEDICAL INSTITUTE
Current projects in our lab fall into three general areas, summarized below. For more information, please see our publications here or on PubMed.
Toxin-antitoxin systems and phage defense
Toxin-antitoxin (TA) systems are prevalent genetic cassettes found in virtually every bacterium, with many species encoding 10-100 individual systems. Typically, a toxin and antitoxin are co-produced from the same operon, binding each other to form an inert complex. If liberated, these toxins can suppress cell growth, but for most systems it has remained mysterious and controversial as to what triggers release of the toxins.
Although commonly postulated to function in a variety of stress responses, we have demonstrated that TA systems are often highly transcribed following stress, but that active toxin is not detectably liberated or released. Instead, we find that many TA systems respond specifically to phage infection, with the released toxin acting to disrupt phage development.
Schematic of a canonical toxin-antitoxin system.
Schematic of phage defense mechanisms, including CRISPR-Cas9, restriction-modification (RM), and TA systems.
We are actively engaged in several lines of study related to these initial findings, including (i) identifying TA systems that protect against phage predation, (ii) elucidating the mechanisms by which phage infection triggers release or activation of toxins, (iii) determining the basis of phage specificity for individual TA systems, and (iv) investigating the molecular means by which toxins disrupt phage replication or development. Finally, we have demonstrated that phage frequently encode inhibitors of TA systems, that may act to either prevent their activation or block toxin activity. Characterization of these mechanisms is revealing the molecular basis of the fierce, ongoing coevolutionary battle between phage and bacteria.
In addition to TA systems, we have recently initiated large-scale screens to identify additional phage defense elements, identifying dozens of new, as-yet uncharacterized immunity mechanisms for future investigation.
All cells harbor a relatively small number of signaling protein families. Through gene duplication and divergence, organisms have dramatically expanded a limited set of signaling proteins, giving rise to large, paralogous protein families that endow cells with sophisticated information processing capabilities. Most bacteria encode dozens to hundreds of two-component signaling systems, usually comprised of a sensor histidine kinase that can respond to an environmental change or stimulus by phosphorylating a cognate response regulator that then triggers changes in cellular physiology or gene expression. We have shown that histidine kinases harbor an intrinsic ability to distinguish their cognate substrate from all possible non-cognate partners rather than relying on auxiliary factors like scaffolds.
(Left) Schematic of a two-component signaling pathway. (Right) Structure of a histidine kinase and its cognate response regulator highlighting the coevolving residues that dictate interaction specificity.
By studying patterns of amino-acid coevolution in cognate kinase-substrate pairs, we have identified the key specificity-determining residues. This work has guided the rational rewiring of two-component signaling pathways and forms the foundation for current studies in three areas. (1) Protein evolution. We are studying the mechanisms by which two-component pathways diverge after duplication to create novel signaling pathways that are insulated from existing pathways. We use a combination of genetics, biochemistry, and phylogenetic studies, including ancestral reconstructions, to gain insight into the mutational trajectories that give rise to new signaling proteins. (2) Molecular recognition. Because partner specificity relies on only a handful of residues in each protein, two-component signaling pathways are amenable to systematic investigations into molecular recognition. By building large, comprehensive mutant libraries and with deep-sequencing as a readout, we are generating maps of the sequence space that underlies the kinase-substrate interaction in two-component pathways. The results are providing insight into the relationship of protein structure, function, and evolution. (3) Synthetic biology. The ability to rationally reprogram kinase and substrate specificity in two-component pathways is enabling new efforts in the design of synthetic signaling circuits in vivo.
Finally, we also study the specificity and evolution of toxin-antitoxin systems. As with two-component signaling proteins, we have used analyses of amino-acid coevolution in cognate toxin-antitoxin pairs to pinpoint the residues crucial to interaction specificity. We are using these studies to map the sequence space underlying toxin-antitoxin systems and to elucidate fundamental principles of how protein-protein interactions evolve.
The nucleotide ppGpp (along with the related nucleotide pppGpp) is a universally conserved regulator of growth rate in bacteria. ppGpp can accumulate very rapidly in response to a wide range of stresses, including amino-acid starvation, and shut down cell growth. Although often asserted to primarily target transcription by binding RNA polymerase, ppGpp actually targets many critical cellular processes to orchestrate a slow down of cell growth. The precise binding targets of ppGpp have remained elusive for decades. We recently developed a capture compound, mass spectrometry approach to rapidly and systematically identify the direct targets of ppGpp. We use a combination of genetics, biochemistry, and structural biology to characterize these targets and elucidate how the regulation of each contributes to the overall control of cell growth by ppGpp. We have also recently identified a toxin in P. aeruginosa that produces ppApp, the first physiologically relevant example of ppApp. Finally, we have recently begun to identify and characterizing predicted ppGpp/ppApp synthases that function in phage defense.
(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.