Exploration of Bacterial Nitric Oxide Stress Responses as a Source of Antivirulence Targets

Abstract

Nitric oxide (NO•) is a potent antimicrobial produced by the immune system to neutralize pathogens. Many pathogens, such as Mycobacterium tuberculosis, Salmonella enterica, and pathogenic Escherichia coli, harbor NO• defenses that have been linked to their virulence, and these defenses therefore present an attractive target toward which novel anti-infective therapies could be designed. Given that the few compounds known to inhibit major NO• detoxification systems in bacteria suffer from poor intracellular transport or toxicity toward humans, we sought to investigate bacterial NO• defense networks to identify alternate targets. To account for the broad reactivity of NO• and its metabolites, as well as the systems-level response NO• elicits in microbes, a computational approach was required. We constructed a kinetic model of the E. coli NO• biochemical network, including relevant processes such as NO• autoxidation, iron-sulfur cluster nitrosylation, DNA deamination, and transcriptional regulation. This model enabled discovery of a novel kinetic regime where the major aerobic NO• detoxification system (NO• dioxygenase; Hmp) was rendered unimportant, and facilitated characterization of the complex relationship between NO• delivery rate, payload, and bacterial cytotoxicity. In addition, we found that an E. coli mutant lacking ClpP protease exhibited increased NO• sensitivity, and used the model with an ensemble-guided approach to elucidate the underlying mechanism. Further, we investigated NO• detoxification under microaerobic environments, and discovered a regime in which E. coli NO• defenses were severely compromised, as well as conditions that exhibited oscillations in the concentration of NO•. We identified that NO• detoxification was strongly impaired at low O2 due to a combination of its inhibitory effects on NO• reductase (NorV), Hmp, and translational activities, whereas oscillations were found to result from a competition for O2 between Hmp and respiratory cytochromes. Finally, the versatility of our approach was demonstrated by adapting the model to enterohemorrhagic E. coli, where the effects of genetic perturbations in environments with different levels of oxygenation were accurately predicted. The integrated computational and experimental approaches developed in this dissertation are valuable tools for the study of bacterial NO• defense networks and the discovery of novel targets for the development of next-generation anti-infectives.