Brynildsen, Mark P. Princeton University
Antibiotic resistance is a growing public health threat that is worsened by a declining antibiotic pipeline. Strains resistant to 'last line of defense' antibiotics have appeared and the danger of our antibiotic arsenal becoming obsolete is quite real. Since conventional antibiotic discovery has failed to keep pace with the rise of resistance, novel methodologies are required to address this looming crisis. Antivirulence therapies comprise a novel class of anti-infectives that target host-pathogen interactions required for infection. Since antibiotics exert selective pressure regardless of their location (e.g., inside humans, livestock, sewage, soil) and antivirulence therapies exert selective pressure only at infection sites within the host, antivirulence therapies are projected to be less prone to resistance development than are antibiotics. Further, due to their pathogen-specific nature, they are also predicted to be less harmful to commensal bacteria than are antibiotics. Nitric oxide (NO) is an antimicrobial generated by immune cells, and its importance to inhibiting pathogenesis is highlighted by the many bacteria that require NO defense systems to establish or sustain an infection. Disabling pathogen NO defenses constitutes a promising antivirulence strategy; however, inhibitors of the known elements of these systems are either toxic to humans or suffer from poor transport into bacterial cells. The overall goal of this project is to develop a detailed, quantitative understanding of NO stress in multiple bacterial pathogens in order to identify novel antivirulence strategies that target NO defense systems. The challenge of potentiating NO toxicity in pathogens will be approached as a metabolic engineering problem, which is a paradigm shift from other antivirulence research, and this work will fill fundamental knowledge gaps in understanding of bacterial NO stress under rarely studied, but physiologically important conditions. Results from the proposed research are expected to lead to antivirulence therapies that address the public health crisis of antibiotic resistance, application of metabolic engineering approaches to diverse NO-based phenomena (e.g., symbiosis), and stimulation of interest in bioengineering in a diverse group of individuals (4th-12th graders, community college and undergraduate students, under-represented minority students, and the general public).
Since the biological outcome of NO exposure is dictated by a complex kinetic competition, the challenge of potentiating NO toxicity can be reduced to a metabolic engineering problem: How can NO flux be directed away from detoxification systems and toward damaging reactions? In this proposal, metabolic engineering techniques will be applied to identify and understand weaknesses within bacterial NO defenses under physiological conditions (e.g., microaerobic, acidic pH). First, quantitative, experimentally-validated kinetic models of NO stress in three bacterial species (one model organism, two pathogens) under physiological conditions will be developed. Second, novel participants in NO defense under those conditions will be identified through the use of genome-scale mutant libraries, competition assays, and next-generation DNA sequencing. Since mechanistic insight can illuminate emergent strategies to increase NO toxicity (e.g., synergistic effects), the mechanisms by which novel participants alter NO defenses will be identified with the use of an ensemble modeling approach. To complement these research activities and inspire individuals to pursue careers in bioengineering, one of the NO models will form the basis of a host-pathogen web-game; elements of the proposed research will be performed by community college, high school, and undergraduate students; and results from the research will be incorporated into an innovative suite of activities in a metabolic engineering elective. Further, community college and high school students will present their work at public forums to educate and inspire 4th-12th graders, under-represented minorities, and the general public. Successful achievement of these goals will provide targets for the development of antivirulence therapies, improve understanding of both bacterial pathogenesis and NO stress under physiological conditions, and motivate students and the general public to pursue careers in bioengineering.
Due to the interdisciplinary nature of the project, this CAREER award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.
