Genetic Basis for Persister Cell Formation
Open Access
- Author:
- Kwan, Brian Wai
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 03, 2015
- Committee Members:
- Thomas Keith Wood, Dissertation Advisor/Co-Advisor
Manish Kumar, Committee Member
Howard M Salis, Committee Member
Stephen John Knabel, Committee Member - Keywords:
- persister cells
antibiotic tolerance
toxin/antitoxin system - Abstract:
- The prevalence of bacterial infections poses a significant threat to public health; however, the advent of antibiotics has largely subdued the lethality of infections treated clinically. Nevertheless, antibiotics are ineffective against both resistant and persistent bacteria, which contribute to chronic and recalcitrant infections. Persister cells comprise a small, multi-drug tolerant sub-population of all bacterial cultures, which is a non-hereditary phenotype that occurs due to a state of metabolic dormancy. The low frequency of persister cell formation makes it difficult to isolate and study persisters, leading to a dearth of information regarding the basis for persister cell formation. In this dissertation, a technique is developed to chemically induce persistence via bacteriostatic compounds that mimic toxins from toxin/antitoxin (TA) systems, which have been implicated in persister cell formation. Through these chemical pre-treatments, it is demonstrated that arrested protein synthesis is the key to persister cell formation and that environmental factors contribute to persistence. Investigation of toxin YafQ of the YafQ/DinJ TA system reveals a regulatory mechanism which increases persistence via reduced levels of the intercellular and interkingdom signal indole. Additionally, phosphodiesterase DosP is found to increase persistence through reduction of the global regulator cyclic adenosine monophosphate and consequentially down-regulation of indole levels, corroborating the effect of indole on persistence through a second regulatory pathway. Owing to the relationship between TA systems and persistence, the physiological significance of the MqsR/MqsA TA system was investigated, revealing an important role for this TA system in tolerance to bile stress through regulation of YgiS, a periplasmic protein. MqsR/MqsA is a well-characterized persister cell formation mechanism, so this result reveals bile as an environmental stress that likely influences persistence. Finally, the anti-cancer drug mitomycin C (MMC) was tested for activity against persister cells because MMC crosslinks DNA through a spontaneous, growth-independent mechanism which should be effective in metabolically dormant persisters. MMC was found to eradicate persister cells with efficacy against both planktonic cultures and highly robust biofilm cultures for a broad range of bacterial species, including commensal Escherichia coli K-12 as well as pathogenic species of E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Additionally, MMC was effective in an animal model and in a wound model. Therefore, MMC is the first broad-spectrum compound capable of eliminating persister cells, which can be used clinically against recalcitrant infections. In summary, this dissertation reveals arrested protein synthesis as the basis for persistence, characterizes mechanisms of persister cell formation, and discovers a readily applicable clinical treatment to eradicate persisters via repurposing an FDA-approved anti-cancer drug.