Research
We take a systems biology approach to phage-host interactions, focusing particularly on small, fast-evolving, non-essential genes encoded by phages.
Using functional metaviromics to characterize the most salient antivirus defenses in wild strains of bacteria, we develop strategies to rationally design and augment phage-based therapies. A single screen reports phage host-range, and identifies phage genes that activate or inhibit host immunity.
By elucidating the complex relationships between bacteria and their viruses, we aim to build a platform for rational design of phage therapy.
Functional Metaviromics and Phage Therapy
Unlike small molecules, phages are constantly evolving new counter-measures to circumvent bacterial defenses as a part of their perpetual “arms race”. In any bacterial niche, a phage must simultaneously evade host immune systems that recognize specific phage patterns (e.g. Restriction-Modification (R-M) and CRISPR), as well as overcome barriers posed by host cell biology, such as safeguards against translational takeover or cell-surface modifications that block access to phage receptors. Different strains of the same species of bacteria can also mount various combinations of defenses, narrowing the host-range of phages. A reliable phage therapy against a given pathogen must account for all such antivirus strategies in any niche.
At present, our understanding of these defenses is clouded by several challenges. First, most defense systems are rare, unevenly distributed, and difficult to deconvolute in clinically relevant strains of bacteria. Second, immune systems sense highly specific (but mostly unknown) phage components, so immune responses cannot be predicted for most phages. Third, phages encoding the same immune-activators can still produce differing infection outcomes since closely related phages can deploy substantially variable immune-evasive strategies. Fourth, there is no modality for determining how various antivirus defenses interact and complement each other, as they have mostly been studied in laboratory strains of bacteria, stripped of their native context. A holistic view of the architecture of anti-virus immunity in bacteria is still lacking.
We developed an algorithm to detect rapidly evolving genes of unknown function, called accessory genes (AGs). To test AG function, we built a high-throughput screening platform to assay which AGs can weaken cellular defenses during infection of wild bacterial strains by various phages.
To enable rational design of phage therapy, we have built an integrated computational and experimental framework to systematically characterize phage-encoded immune activators and inhibitors in clinically relevant bacteria. This platform combines a pipeline to identify phage accessory genes (AGs) that are most likely to interact with host biology, and a series of pooled screens to determine how they modulate infectivity. Crucially, we perform our assays in many wild bacterial strains in parallel, where anti-virus defense systems are natively regulated. In this way, we can identify the most salient barriers to phage infection by determining which defenses the phages have evolved countermeasures against. We are working on scaling up these screens and deploying our libraries in various bacterial species.
Read more about the questions we hope to answer using our phage-independent and phage-dependent screens below, and contact us to learn how to collaborate with us and use our AG libraries to find phage genes that interface with your favorite bacterial process or pathway.
How Bacteria Sense Virus Infection
How specific phage factors activate bacterial immunity remains a principal unanswered question. It has been challenging to identify phage factors that trigger immune activation for any defense system, especially when the effector causes programmed cell death (PCD). Our phage-independent AG screens turn this weakness into a strength. Expressing thousands of phage proteins in closely related wild strains of bacteria will allow us to investigate every case where an AG selectively triggers PCD in some strains but not others. Follow-up transposon suppressor screens can then identify the PCD-causing immune loci.
Phage-independent screens to detect AGs that activate PCD systems. (1) Some AGs kill specific host strains, potentially by triggering PCD. (2) Transposons interrupt abortive loci and permit cell survival despite expression of the trigger AG. (3) Various abortive systems and their triggers identified from a pilot screen with 200 AGs (10x dilutions of cells with abortive system and AG).
Comprehensive mapping of triggers to sensors will reveal: (1) commonalities between phage proteins sensed as hallmarks of infection by different immune systems, (2) the distribution of different kinds of triggers that may be recognized by the same sensors, and (3) how robust phage-sensing is to mutation/loss of the trigger. Unlike core phage components (e.g. capsid proteins) that were classically thought to function as immune triggers, AGs can easily be mutated or lost, seemingly allowing for facile immune evasion by viruses. We hypothesize that AG-loss might be the intended outcome of defenses that sense phage AGs, since AGs often perform useful functions for the phage such as immune suppression or translational takeover. This places the phage in a lose-lose scenario, where it must either lose AG function or expose itself to restriction. We are investigating how this strategy constrains phage evolution, whether it is a general feature of the phage-host arms race, and how phages might escape this double-bind.
How Antivirus Defenses Are Integrated With Bacterial Cell Biology
In our phage-dependent AG screens, several AGs exhibited “broad spectrum” anti-defense phenotypes, promoting infection by multiple phages in multiple strains of bacteria. We hypothesize that these AGs can modify the cell surface by interfering with core metabolic processes and rewiring transcriptional circuits. We are creating a catalog of broad-spectrum AGs for phage therapy, and using high-throughput mass spectrometry to identify their targets in bacterial cells.
Phage-dependent screens identify AGs that interfere with core metabolic processes. (1) Some AGs have broad-spectrum anti-defense phenotypes. (2) Transposons interrupt the defense system and allow the virus to kill the host. (3-5) Host targets can be identified by affinity purification of the AG expressed in many wild host strains in parallel, followed by AP-MS to identify co-purifying bacterial factors.
AGs that target canonical defense systems (like R-M, or CRISPR) produce “narrow spectrum” phenotypes. Even when a specific phage is targeted by multiple defenses in the same host strain, AG screening can tease apart the redundancy, since an anti-defense AG paired with its cognate immune target in the correct strain still partially weakens host immunity. Once a phage-sensitizing strain-AG combination is identified, successive transposon screens to peel apart bacterial defenses layer-by-layer can identify salient immune systems in the phage-host pair. We have many such projects ready for mechanistic dissection and suitable for graduate and rotation students.
Phage AGs That Modulate Other Host Processes
AG screening might reveal immune-supporting functions of core host factors, such as those involved in translational arrest in response to phage infection. We are also interested in screening for phage AGs that interact with essential cellular pathways by selecting for synthetic-lethal interactions with various stressors (e.g. antibiotics, detergents, heat, desiccation), and are actively seeking postdocs/collaborators in this area. Please contact me directly if you are interested in working with us and using our libraries in your workflows.