Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADHdehydrogenasesisrefractorytotheearlyNADPHoxidasedependent antimicrobial activity of IFN␥-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO ؊ ) and hydrogen peroxide (H 2 O 2 ). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H 2 O 2 . NADH antagonizes the hydroxyl radical (OH ⅐ ) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation.Oxidative stress engendered by the sustained synthesis of NO mediates cytotoxicity against a variety of eukaryotic and prokaryotic cells (1-3). Because of its unpaired electron, NO directly reacts with metal prosthetic groups of cytochromes in the electron transport chain (ETC) 2 and [Fe-S] clusters of dehydratases (4, 5). Alternatively, reactive nitrogen species (RNS) generated through the interaction of NO with O 2 and superoxide (O 2 . ) indirectly mediate cytotoxicity of this diatomic radical.

By remodeling the phagosomal membrane, the type III secretion system encoded within the Salmonella pathogenicity island-2 (SPI2) helps Salmonella thrive within professional phagocytes. We report here that nitric oxide (NO) generated by IFNγ-activated macrophages abrogates the intracellular survival advantage associated with a functional SPI2 type III secretion system. NO congeners inhibit overall expression of SPI2 effectors encoded both inside and outside the SPI2 gene cluster, reflecting a reduced transcript level of the sensor kinase SsrA that governs overall SPI2 transcription. Down-regulation of SPI2 expression in IFNγ-treated macrophages does not seem to be the result of global NO cytotoxicity, because transcription of the housekeeping rpoD sigma factor remains unchanged, whereas the expression of the hmpA-encoded, NO-metabolizing flavohemoprotein is stimulated. Because of the reduced SPI2 expression, Salmonella-containing vacuoles interact more efficiently with compartments of the late endosomal/lysosomal system in NO-producing, IFNγ-treated macrophages. These findings demonstrate that inhibition of intracellular SPI2 transcription by NO promotes the interaction of Salmonella phagosomes with the degradative compartments required for enhanced antimicrobial activity. Transcriptional repression of a type III secretion system that blocks phagolysosome biogenesis represents a novel mechanism by which NO mediates resistance of IFNγ-activated phagocytes to an intracellular pathogen.

Our investigations have identified a mechanism by which exogenous production of nitric oxide (NO) induces resistance of Gram-positive and -negative bacteria to aminoglycosides. An NO donor was found to protect Salmonella spp. against structurally diverse classes of aminoglycosides of the 4,6-disubstituted 2-deoxystreptamine group. Likewise, NO generated enzymatically by inducible NO synthase of gamma interferon-primed macrophages protected intracellular Salmonella against the cytotoxicity of gentamicin. NO levels that elicited protection against aminoglycosides repressed Salmonella respiratory activity. NO nitrosylated terminal quinol cytochrome oxidases, without exerting long-lasting inhibition of NADH dehydrogenases of the electron transport chain. The NO-mediated repression of respiratory activity blocked both energy-dependent phases I and II of aminoglycoside uptake but not the initial electrostatic interaction of the drug with the bacterial cell envelope. As seen in Salmonella, the NO-dependent inhibition of the electron transport chain also afforded aminoglycoside resistance to the clinically important pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Together, these findings provide evidence for a model in which repression of aerobic respiration by NO fluxes associated with host inflammatory responses can reduce drug uptake, thus promoting resistance to several members of the aminoglycoside family in phylogenetically diverse bacteria.