Bacterial persisters tolerate antibiotic treatment, and underlie the propensity of biofilm infections to relapse. An improved understanding of persister physiology will lead to the development of more effective therapies against biofilm-utilizing pathogens such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. A critical knowledge gap in our understanding of persisters is their mechanistic response toward antibiotics. We do not yet know how persisters manage antibiotic stress or what aspects of these processes are required for their tolerances. This lack of knowledge largely originates from difficulties associated with measuring persister physiology. Persisters have yet to be isolated, and therefore, fluorescence activated cell sorting (FACS) has become the best technique to quantify persister physiology. We hypothesize that knowledge of persister antibiotic responses and how they compare to those of cells that die can be used to understand persister survival and identify persister biomarkers. Similarities and differences between the antibiotic responses of persisters and dying cells will provide knowledge of how persisters avoid killing, and in addition, the differences will constitute potential biomarkers. To test our hypothesis, we will compare the transcriptional responses of persisters to those of cells that die, and identify features that contribute to persister tolerances and/or can serve as persister biomarkers. To do this, we will measure time-course mRNA from Escherichia coli dying from antibiotics and identify the regulators mediating gene expression changes with Network Component Analysis. These death responses will be used to select promoters for analysis in persisters, because FACS quantification of persister physiology is time- and labor-intensive. Selected promoters will be fluorescently-labelled and studied with sorting, persistence assays, and statistical methods to quantify and compare persister responses to those of dying cells. Similarities and differences will be identified, and both tested for their impacts on persister tolerances through genetic rewiring of the responses at both the promoter and regulatory levels. Results from this proposal will fill fundamental knowledge gaps about the core characteristic of persisters, identify improved biomarkers for persister enrichment, illuminate new avenues for therapeutic intervention through disruption of persister antibiotic stress management, and impact the fields of antibiotic tolerance, microbiology, and infectious disease.

Public Health Relevance

Sixty-five percent of hospital-treated infections are caused by biofilms, and bacterial persisters are responsible for the propensity of these films to cause chronic and recurrent infections. Persisters are bacteria that have a non-inherited ability to tolerate antibiotic treatment, and the physiology of these survivors is ill-defined. The mechanisti responses of persisters to antibiotics have yet to be measured, and the research proposed here will measure and analyze those responses in order to develop novel therapeutics against persisters.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI115075-01A1
Application #
8969012
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Ernst, Nancy Lewis
Project Start
2015-05-11
Project End
2017-04-30
Budget Start
2015-05-11
Budget End
2016-04-30
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Princeton University
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
Aedo, Sandra J; Gelderman, Grant; Brynildsen, Mark P (2017) Tackling host-circuit give and take. Nat Microbiol 2:1584-1585
Orman, Mehmet A; Brynildsen, Mark P (2016) Persister formation in Escherichia coli can be inhibited by treatment with nitric oxide. Free Radic Biol Med 93:145-54
Völzing, Katherine G; Brynildsen, Mark P (2015) Stationary-Phase Persisters to Ofloxacin Sustain DNA Damage and Require Repair Systems Only during Recovery. MBio 6:e00731-15