Katrina M Miranda

Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.

PMID: 12177417;PMCID: PMC123192;Abstract:

A potential of about-0.8 (±0.2) V (at 1 M versus normal hydrogen electrode) for the reduction of nitric oxide (NO) to its one-electron reduced species, nitroxyl anion (³NO^-) has been determined by a combination of quantum mechanical calculations, cyclic voltammetry measurements, and chemical reduction experiments. This value is in accord with some, but not the most commonly accepted, previous electrochemical measurements involving NO. Reduction of NO to ¹NO^- is highly unfavorable, with a predicted reduction potential of about -1.7 (±0.2) V at 1 M versus normal hydrogen electrode. These results represent a substantial revision of the derived and widely cited values of +0.39 V and -0.35 V for the NO/³NO^-and NO/¹NO^- couples, respectively, and provide support for previous measurements obtained by electrochemical and photoelectrochemical means. With such highly negative reduction potentials, NO is inert to reduction compared with physiological events that reduce molecular oxygen to superoxide. From these reduction potentials, the pKa of ³NO^- has been reevaluated as 11.6 (±3.4). Thus, nitroxyl exists almost exclusively in its protonated form, HNO, under physiological conditions. The singlet state of nitroxyl anion, ¹NO^-, is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and O2 under reductive conditions is discussed.

PMID: 11165873;Abstract:

The physiological function of nitric oxide (NO) in the defense against pathogens is multifaceted. The exact chemistry by which NO combats intracellular pathogens such as Listeria monocytogenes is yet unresolved. We examined the effects of NO exposure, either delivered by NO donors or generated in situ within ANA-1 murine macrophages, on L. monocytogenes growth. Production of NO by the two NONOate compounds PAPA/NO (NH2(C3H6) (N[N(O)NO]C3H7)) and DEA/NO (Na(C2H5)2N[N(O)NO]) resulted in L. monocytogenes cytostasis with minimal cytotoxicity. Reactive oxygen species generated from xanthine oxidase/hypoxanthine were neither bactericidal nor cytostatic and did not alter the action of NO. L. monocytogenes growth was also suppressed upon internalization into ANA-1 murine macrophages primed with interferon-γ (INF-γ) + tumor necrosis factor-α (TNF-α or INF-γ + lipid polysaccharide (LPS). Growth suppression correlated with nitrite formation and nitrosation of 2,3-diaminonaphthalene elicited by stimulated murine macrophages. This nitrosative chemistry was not dependent upon nor mediated by interaction with reactive oxygen species (ROS), but resulted solely from NO and intermediates related to nitrosative stress. The role of nitrosation in controlling L. monocytogenes was further examined by monitoring the effects of exposure to NO on an important virulence factor, Listeriolysin O, which was inhibited under nitrosative conditions. These results suggest that nitrosative stress mediated by macrophages is an important component of the immunological arsenal in controlling L. monocytogenes infections. © 2001 Elsevier Science Inc.

PMID: 10830872;Abstract:

Nitric oxide (NO) has been shown to be a key bioregulatory agent in a wide variety of biological processes, yet cytotoxic properties have been reported as well. This dichotomy has raised the question of how this potentially toxic species can be involved in so many fundamental physiological processes. We have investigated the effects of NO on a variety of toxic agents and correlated how its chemistry might pertain to the observed biology. The results generate a scheme termed the chemical biology of NO in which the pertinent reactions can be categorized into direct and indirect effects. The former involves the direct reaction of NO with its biological targets generally at low fluxes of NO. Indirect effects are reactions mediated by reactive nitrogen oxide species, such as those generated from the NO/O2 and NO/O2^- reactions, which can lead to cellular damage via nitrosation or oxidation of biological components. This report discusses several examples of cytotoxicity involved with these stresses. (C) 2000 Elsevier Science Inc.

Donors of nitroxyl (HNO) exhibit pharmacological properties that are potentially favorable for treatment of a variety of diseases. To fully evaluate the pharmacological utility of HNO, it is therefore important to understand its chemistry, particularly involvement in deleterious biological reactions. Of particular note is the cytotoxic species formed from HNO autoxidation that is capable of inducing double strand DNA breaks. The identity of this species remains elusive, but a conceivable product is peroxynitrous acid. However, chemical comparison studies have demonstrated that HNO autoxidation leads to a unique reactive nitrogen oxide species to that of synthetic peroxynitrite. Here, we extend the analysis to include a new preparation of peroxynitrite formed via autoxidation of nitroxyl anion (NO(-)). Both peroxynitrite preparations exhibited similar chemical profiles, although autoxidation of NO(-) provided a more reliable sample of peroxynitrite. Furthermore, the observed dissimilarities to the HNO donor Angeli's salt substantiate that HNO autoxidation produces a unique intermediate from peroxynitrite.