Research in the O'Toole Lab

Much of the work in my lab focuses on the study of surface-attached microbial communities known as biofilms. These surface attached communities can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Biofilm microbes are typically surrounded by an extracellular matrix, which provides structure and protection to the community. Biofilm-grown bacteria are notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics. Key goals of my lab are to understand how biofilms form on both living (airway cells) and non-living (medical implant) surfaces. We are also interested in the role of biofilms in host-pathogen interactions and resistance to antibiotic therapy.

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A central interest in the lab is the understanding of how bacteria transition from a planktonic (free-swimming) mode of growth to life in a biofilm. Using the model organism Pseudomonas aeruginosa, an opportunistic human pathogen that causes a variety of diseases in immune compromised patients, we have asked the question, "Can bacteria sense a surface?" We think the answer is yes! Our work has shown that "surface sensing" requires the bacterial pilus and two key second messengers: cAMP and c-di-GMP. For these studies, we work with a biophysicist, Gerard Wong at UCLA, and theoretical physicist, Ramin Golestanian at Oxford, to combine molecular genetics, single cell tracking and theoretical frameworks to understand how microbes engage with and attach to surfaces (check out this and this as examples of our collaborative studies). We continue to explore the mechanisms of surface sensing with our trans-disciplinary, international team.

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How do microbes regulate biofilm formation via the c-di-GMP network and a large cell surface adhesin? The second messenger cyclic-di-GMP regulates biofilm formation by the soil microbe Pseudomonas fluorescens. This soil microbe uses cyclic-di-GMP to regulate the localization of a large surface adhesion called LapA, which is critical for biofilm formation. Our work has uncovered a novel "inside-out" signal transduction pathway that allows the cell to control localization of this adhesin in response to changing pools of cyclic-di-GMP inside the cell. We work closely with Holger Sondermann, a structural biologist and biochemist at Cornell, to understand the structural basis of this signaling pathway. We have also employed atomic force microscopy to understand how the large adhesin LapA allows this microbe to initiate biofilm formation. Recent studies have focused on which parts of LapA contribute to attachment, and how this protein is anchored to the cell surface.

We also are exploring the c-di-GMP regulatory network in this organism, as there are 50+ proteins that make, break or bind this second messenger. A key goal in the field is to understand how the cell can coordinate the action of all of these players. Towards this end, we reported a network analysis that combined assaying 50+ mutants for biofilm formation under ~180 growth conditions, testing the expression profiles of these genes in ~50 growth conditions, and performing 2000+ bacterial two hybrid assays. This study suggested that the cell uses a combination of regulatory mechanisms to control the network output.

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The microbiome and polymicrobial interactions: in the lab and in the clinic. In recent years we have begun to explore the polymicrobial communities associated with the disease cystic fibrosis (CF). A key impact of CF disease is the chronic airway infections, but this is a systemic disease that also impacts patient nutrition, including the gut microbiota. We have taken two different approaches to tackle the question of how microbial communities impact the host.

First, we have studied the microbiome of the airway and intestine of infants and children, teens and adults with CF. Our studies of the airway microbiota of teens and adult patients with CF have helped us rethink how the polymicrobial infections impact patient health. I reviewed this literature in 2017. I also work closely with Juliette Madan, a physician scientist at Dartmouth, who has tracked a cohort of infants and children with CF from birth to seven years of age. Her work has uncovered a striking and surprising finding — the best predictor of airway disease outcome is the microbial communities found in the gut. Our ongoing microbiome studies are geared towards a better understanding of the link between gut and airway communities, and their impacts on patient health.

Second, we have investigated polymicrobial interactions in the context of in vitro laboratory models, including biofilms grown on CF-derived airway cells. These studies have been performed with Pseudomonas aeruginosa and Staphylococcus aureus, and well as P. aeruginosa and Streptooccus spp. In our published studies, we have found that non-mucoid strains can, in some conditions, kill S. aureus and in other conditions protect S. aureus from antibiotic treatment. We have also found that mucoid strains of P. aeruginosa can coexist with S. aureus. Thus the interactions between these microbes are impacted by their environment and are not easily predicted from their behavior in monoculture. Interactions between P. aeruginosa and Streptooccus spp. can be equally complex. We have also reviewed the complex nature of cystic fibrosis infections. We are now exploring the nature of polymicrobial interactions in the context of gut microbiota relevant to CF.