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.

Discovery and dissection of metabolic oscillations in the microaerobic nitric oxide response network ofEscherichia coli

“…The E. coli NO · model was constructed (37) and further developed (36,38) in previous studies. Briefly, the model is comprised of a system of ordinary differential equations (ODEs) describing the change in concentration of biochemical species (C) as a function of intensive reaction rates (r I ) and reaction stoichiometries (Ŝ):…”

An ensemble-guided approach identifies ClpP as a major regulator of transcript levels in nitric oxide-stressed Escherichia coli

“…The promiscuity and reactivity of NO• and its reaction products give rise to a broad, complex biochemical reaction network that can be difficult to interrogate without the use of mathematical models [3,14,29] The analysis identified seven parameters whose adjustment could largely reduce the error (quantified as the sum of the squared residuals; SSR) between the model and ΔclpP experimental data ( Fig. 2B), as well as many parameters that could not.…”

Design, synthesis, nitric oxide release and antibacterial evaluation of novel nitrated ocotillol-type derivatives

“…As NO is known to affect bacterial and mammalian membranes [3], the cytotoxicity of NO donor lead compounds should be considered in drug discovery. We have tested the cytotoxicity of 17c against human breast cancer (MCF-7), gastric cancer (MKN-45) and proximal tubule epithelial (HK-2) cell lines as shown in Table 3.…”

“…In addition to serving as a powerful vasodilator and neurotransmitter, NO also takes part in the human immune system, in which phagocytes generate NO through inducible nitric oxide synthase (iNOS) [2]. As an immunological response, the resulting free radicals together with the auto-oxidation products are toxic to bacteria damaging their lipid membrane and DNA [3], as well as affecting biofilm formation [4].…”

“…Exogenous gaseous NO (gNO) was demonstrated to be bacteriostatic at 1.9 ppm [5], and bactericidal at relatively high concentration (120e200 ppm) [6,7], which suggested a high level of NO production close to a membrane is critical for its therapeutic potential for use against bacterial infections [3]. It has also been shown that protein bound and nanoparticle fixed NO donors displayed antibacterial activity [8,9].…”