INVESTIGATION OF BACTERIAL NITRIC OXIDE DEFENSES FOR THERAPEUTIC AND BIOTECHNOLOGY APPLICATIONS

Abstract

Antimicrobial resistance is a growing public health threat that has diminished the efficacyof antibiotics, which we rely on to treat and prevent bacterial infections. Anti-virulence strategies that target the abilities of pathogens to infect hosts are promising alternatives to traditional antibiotics. One anti-virulence strategy involves sensitizing bacteria to immune-derived nitric oxide (·NO). While phagocytes use ·NO and other stresses against phagocytized bacteria, pathogens have evolved countermeasures to survive in or escape from phagosomes. To understand the mechanisms bacteria use to survive such multi-stress environments, we employed a bottom-up approach, in which we studied the effects of phagosomal stressors on bacteria in simplified systems. In this doctoral thesis, we examined bacterial ·NO detoxification in different nutrient environments and a potential biotechnology application for the main ·NO defense enzyme, Hmp. First, we investigated how bacteria cope with ·NO while starved for nitrogen, which can be limited in phagosomes. We discovered that ·NO detoxification by Escherichia coli was robust to nitrogen starvation due to enhanced transcription of hmp, which required RelA. Next, we investigated the impact of amino acid-replete conditions on E. coli ·NO defenses. Surprisingly, we found that ·NO detoxification was impaired by an abundance of amino acids due to precipitous depletion of ATP by amino acid import. Further, the stringent response was activated by ·NO in amino acid-replete conditions and it served to enhance ·NO detoxification. In addition, while investigating the ·NO detoxification enzyme Hmp, we observed that translational fusions to its C terminus increased heterologous protein expression, which suggested that it could be useful as a protein fusion tag in the biotechnology industry. The dissertation then concludes by summarizing the potential impact of this work on the antibiotic resistance crisis and identifying interesting future directions.