Metabolic Strategies to Enhance the Toxicity of Nitric Oxide in Pathogens

Title: Metabolic Strategies to Enhance the Toxicity of Nitric Oxide in Pathogens
Author: Mark P. Brynildsen
Nitric oxide (NO•) is an antimicrobial used by immunity to neutralize pathogens. The importance of NO• to immune function is evidenced by the many pathogens, including Mycobacterium tuberculosis, Neisseria meningitides, Vibrio cholerae, Salmonella enterica, and enterohemorrhagic Escherichia coli (EHEC), that depend on NO• detoxification to establish an infection1-7. Inhibitors of NO• defense systems are under investigation as next-generation antibiotics8,9, and direct delivery of NO• has shown potential for treating infections when antibiotics fail10-17. However, these efforts have been hampered by a narrow concentration window within which bacteria are neutralized and host cells remain unharmed, and a lack of effective agents that enhance NO• toxicity in pathogens9,14. A quantitative understanding of NO• cytotoxicity, and the adaptive responses mounted by bacteria would aid in identifying targets to sensitize pathogens toward host- or therapeutic-derived NO•. Due to the complexity of the NO• biochemical reaction network, where NO• directly reacts with Fe-S clusters, O2, and O2•-, and its autoxidation products (e.g., N2O3, ONOO-) damage thiols, tyrosine residues, and DNA bases18-
20, coupled with the metabolic requirements of NO• defense systems (e.g., NADH, ATP), computational approaches are required to understand how bacteria process and respond to NO• stress.

Here I will discuss our work on the construction, experimental validation, and systems-level exploration of a detailed kinetic model of NO• metabolism and stress in E. coli21. This model has provided accurate predictions of NO• distributions among its reactions pathways under both aerobic and microaerobic conditions, enabled the discovery of a novel kinetic dependency of a major NO• detoxification system, and most recently been used to systematically identify the mechanism by which deletion of a protease produces major defects in NO• detoxification.

Further, I will discuss how we have translated the model to EHEC, and used it to investigate NO•
control of virulence factor expression in this dangerous food-borne pathogen. These results demonstrate the utility of quantitative metabolic modeling to the study of NO• stress in bacteria, and further, identify novel targets that when inhibited sensitize bacteria toward NO•.
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