A Kinetic Platform to Determine the Fate of Nitric Oxide in Escherichia coli

Abstract

Nitric oxide (NO•) is generated by the innate immune response to neutralize pathogens. NO• and its autoxidation products have an extensive biochemical reaction network that includes reactions with iron-sulfur clusters, DNA, and thiols. The fate of NO• inside a pathogen depends on a kinetic competition among its many targets, and is of critical importance to infection outcomes. Due to the complexity of the NO• biochemical network, where many intermediates are short-lived and at extremely low concentrations, several species can be measured, but stable products are non-unique, and damaged biomolecules are continually repaired or regenerated, kinetic models are required to understand and predict the outcome of NO• treatment. Here, we have constructed a comprehensive kinetic model that encompasses the broad reactivity of NO• in Escherichia coli. The incorporation of spontaneous and enzymatic reactions, as well as damage and repair of biomolecules, allowed for a detailed analysis of how NO• distributes in E. coli cultures. The model was informed with experimental measurements of NO• dynamics, and used to identify control parameters of the NO• distribution. Simulations predicted that NO• dioxygenase (Hmp) functions as a dominant NO• consumption pathway at O₂ concentrations as low as 35 µM (microaerobic), and interestingly, loses utility as the NO• delivery rate increases. We confirmed these predictions experimentally by measuring NO• dynamics in wild-type and mutant cultures at different NO• delivery rates and O₂ concentrations. These data suggest that the kinetics of NO• metabolism must be considered when assessing the importance of cellular components to NO• tolerance, and that models such as the one described here are necessary to rigorously investigate NO• stress in microbes. This model provides a platform to identify novel strategies to potentiate the effects of NO•, and will serve as a template from which analogous models can be generated for other organisms. Nitric oxide (NO•) is a highly reactive metabolite used by immune cells to combat pathogens. Since the biological effects of NO• are governed by its broad reactivity, it is desirable to determine how NO• distributes among its many targets inside a cell. A quantitative understanding of this distribution and how it is controlled will facilitate the development of novel NO•-potentiating therapeutics. Here, we have constructed and experimentally validated a comprehensive kinetic model of NO• biochemistry within Escherichia coli that includes NO• autoxidation, respiratory inhibition, enzymatic detoxification, and damage and repair of biomolecules. Using this model, we investigated the control of NO• dynamics in E. coli cultures, and found that the primary aerobic detoxification system, NO• dioxygenase (Hmp), functions as a dominant NO• consumption pathway under microaerobic conditions (35 µM O₂), and loses utility as the NO• delivery rate increases. We confirmed these predictions experimentally, thereby demonstrating the predictive power of the model. This model will serve as a quantitative platform to study nitrosative stress, provide a template from which models for other organisms can be generated, and facilitate the development of antimicrobials that synergize with host-derived NO•.