The
Research
Current Projects in the Dennehy Lab
1. Control of Cellular Event Timing
The inherent probabilistic nature of biochemical reactions and low copy numbers of molecules involved results in significant random fluctuations (noise) in mRNA/protein levels inside individual cells. Such stochastic expression is an unavoidable aspect of life at the single-cell level and creates considerable variation in gene product levels across isogenic cells exposed to the same environment. Increasing evidence shows that stochastic expression affects biological functions ranging from driving genetically identical cells to different fates to corrupting information processing by cells. While the origins of stochastic expression have been extensively studied across organisms, how noisy expression of key regulatory proteins impacts the timing of intracellular events is not well understood. Characterization of control strategies that buffer stochasticity in event timing is critically needed to understand the reliable functioning of diverse cellular processes that rely on precise temporal triggering of events.We use the highly malleable bacteriophage λ as a model system for studying event timing in individual cells. Here, an easily observable event (cell lysis) is the result of expression and accumulation of a single protein (holin) in the E. coli cell membrane up to a threshold level. Preliminary data reveals precision in timing: lysis occurs on average at 65 min with a coefficient of variation of less than 5%. Intriguingly, mutations in the holin coding sequence can increase lysis time variation while keeping the mean fixed, illustrating independent tuning of noise in λ’s lysis system. We mathematically model timing of intracellular events as a first-passage time problem, where an event is triggered when a stochastic process (holin level) hits a threshold for the first time. Theoretical predictions are integrated with single-cell lysis time measurements in wild-type λ and strains containing mutations in regulatory regions controlling holin expression. Our preliminary results are promising and indicate that λ uses various mechanisms for buffering noise in order to schedule lysis at an optimal time. Recent publications under this theme include “Optimum threshold minimizes noise in timing of intracellular events” and “First-passage time approach to controlling noise in timing of intra-cellular events“
2. Novel Strategies for Treating Biofilm-Forming Pathogens with Phage Therapy
The growing antibiotic resistance problem requires that we urgently develop and test new approaches to controlling bacterial infections. Phage therapy approaches offer great promise, but significant knowledge gaps currently exist that limit their application. Two issues that currently limit the broader use of phage therapy are the problem of curating specific lytic phage strains for each infection, and the difficulty of delivering phages directly to the infection sites where they are needed. We hypothesize that bacterial pathways that typically promote biofilm-associated growth can be decoupled from the stress-response pathways capable of inducing resident prophages. By separating these cellular responses, we propose to stabilize biofilms while also inducing temperate phages already present in the bacterial genome to enter lytic phase and kill the host cell. In so doing, we effectively bypass the limitations imposed by finding and delivering strain matched lytic phages to infections, while also developing approaches that make use of temperate phage therapy approaches. Our first goal is to determine biofilm dispersal rates and ascertain how biofilm-associated growth can be manipulated using cellular pathways. Our second goal is to we analyze mechanisms by which lysogens can be induced into lytic phase to kill their hosts without spreading virulence factors beyond the biofilm. Our final goal is to reinforce the approaches of the previous aims with an optimized lytic phage ambush of any escaping dispersers. The overall goal of this work is to develop an innovative, multi-pronged approach to phage therapy for biofilm-associated bacteria while also increasing overall knowledge of the limits and applicability of phage therapy.
3. Characteristics and Consequences of Rotavirus Exploitation of Extracellular Vesicles
Although non-enveloped viruses like rotaviruses were believed to exit host cells only via lysis, recent evidence shows they can exit non-lytically in extracellular vesicles, which are then used to infect new host cells. This new infection mode allows collective infection of new host cells and has implications for viral biology, evolution, and pathogenesis. The long-term goal of our research is to uncover new treatments that target this transmission mechanism. However, many basic questions remain regarding MV use by viruses. Our main objective is to characterize microvesicle (MV)-facilitated rotavirus infections and assess their evolutionary consequences. We will pursue these aims using an innovative combination of analytical and manipulative techniques drawn from fields ranging from molecular to evolutionary biology. Our first goal is to characterize basic life history parameters relevant to MV-facilitated rotavirus infections, since quantifying essential parameters will aid in the development of mathematical models and simulations. The rationale of these experiments is to better understand the consequences of infections generated by single virions versus those generated by collectively infecting viruses. Our second aim is to examine the evolutionary consequences of collective infection. The rationale for this goal is to determine if MV-facilitated transmission can be targeted to slow down the acquisition of antiviral drug/vaccine resistance.
4. Developing Resources for Bacteriophage Therapy
The growing threat of antibiotic resistance poses a dire global health crisis, with projections indicating that bacterial infections resistant to antibiotics could cause over 39 million deaths by 2050—a 70% annual increase compared to 2021. Without intervention, annual deaths directly linked to antibiotic resistance may reach 1.91 million by 2050, with an additional 8 million deaths indirectly attributed to resistant infections. This escalation stems from factors like antibiotic overuse in healthcare and agriculture, poor infection control, and the slow development of new antibiotics. The economic toll could exceed $1 trillion annually by 2030. Bacteriophage (phage) therapy emerges as a promising alternative, leveraging viruses that specifically target and kill bacteria without affecting human cells. Unlike broad-spectrum antibiotics, phages are highly specific, reducing collateral damage to beneficial microbiota and lowering the risk of resistance spread via horizontal gene transfer. The Dennehy Lab is actively isolating and characterizing bacteriophages that can be used as a therapeutic treatment for several of the worst bacterial pathogens, including Mycobacterium tuberculosis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Mycobacterium intracellulare. Additionally, we are collecting bacteriophages active against Bacteroides species, a common inhabitant of the human gut that may be linked to negative health outcomes. The overall goal of this work is to develop a repository of phages that can be used to treat antibiotic resistant infections or to perform precision microbiome editing.