Maroof Husain

Nitric oxide evokes an adaptive response to oxidative stress by arresting respiration

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 NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNγ-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 (H2O2). 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 H2O2. NADH antagonizes the hydroxyl radical (OH·) 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 Salmonellas 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.

Nitric Oxide Protects Bacteria from Aminoglycosides by Blocking the Energy-Dependent Phases of Drug Uptake

ABSTRACT 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.

Redox sensor SsrB Cys203 enhances Salmonella fitness against nitric oxide generated in the host immune response to oral infection

We show herein that the Salmonella pathogenicity island 2 (SPI2) response regulator SsrB undergoes S-nitrosylation upon exposure of Salmonella to acidified nitrite, a signal encountered by this enteropathogen in phagosomes of macrophages. Mutational analysis has identified Cys203 in the C-terminal dimerization domain of SsrB as the redox-active residue responding to nitric oxide (NO) congeners generated in the acidification of nitrite. Peroxynitrite and products of the autooxidation of NO in the presence of oxygen, but not hydrogen peroxide, inhibit the DNA-binding capacity of SsrB, demonstrating the selectivity of the reaction of Cys203 with reactive nitrogen species (RNS). These findings identify the two-component response regulator SsrB Cys203 as a thiol-based redox sensor. A C203S substitution protects SsrB against the attack of RNS while preserving its DNA-binding capacity. When exposed to SPI2-inducing conditions, Salmonella expressing the wild-type ssrB allele or the ssrB C203S variant sustain transcription of the sifA, sspH2, and srfJ effector genes. Nonetheless, compared with the strain expressing a redox-resistant SsrB C203S variant, wild-type Salmonella bearing the NO-responsive allele exhibit increased fitness when exposed to RNS in an NRAMPR, C3H/HeN murine model of acute oral infection. Given the widespread occurrence of the wild-type allele in Salmonella enterica, these findings indicate that SsrB Cys203 increases Salmonella virulence by serving as a redox sensor of NO resulting from the host immune response to oral infection.

Low‐molecular‐weight thiol‐dependent antioxidant and antinitrosative defences in Salmonella pathogenesis

We found herein that the intracytoplasmic pool of the low‐molecular‐weight (LMW) thiol glutathione (GSH) is readily oxidized in Salmonella exposed to nitric oxide (NO). The hypersusceptibility of gshA and gshB mutants lacking γ‐glutamylcysteine and glutathione synthetases to NO and S‐nitrosoglutathione indicates that GSH antagonizes the bacteriostatic activity of reactive nitrogen species. Metabolites of the GSH biosynthetic pathway do not affect the enzymatic activity of classical NO targets such as quinol oxidases. In contrast, LMW thiols diminish the nitrosative stress experienced by enzymes, such as glutamine oxoglutarate amidotransferase, that contain redox active cysteines. LMW thiols also preserve the transcription of Salmonella pathogenicity island 2 gene targets from the inhibitory activity of nitrogen oxides. These findings are consistent with the idea that GSH scavenges reactive nitrogen species (RNS) other than NO. Compared with the adaptive response afforded by inducible systems such as the hmp‐encoded flavohaemoprotein, gshA, encoding the first step of GSH biosynthesis, is constitutively expressed in Salmonella. An acute model of salmonellosis has revealed that the antioxidant and antinitrosative properties associated with the GSH biosynthetic pathway represent a first line of Salmonella resistance against reactive oxygen and nitrogen species engendered in the context of a functional NRAMP1R divalent metal transporter.

The 4‐cysteine zinc‐finger motif of the RNA polymerase regulator DksA serves as a thiol switch for sensing oxidative and nitrosative stress

We show that thiols in the 4‐cysteine zinc‐finger motif of DksA, an RNA polymerase accessory protein known to regulate the stringent response, sense oxidative and nitrosative stress. Hydrogen peroxide‐ or nitric oxide (NO)‐mediated modifications of thiols in the DksA 4‐cysteine zinc‐finger motif release the metal cofactor and drive reversible changes in the α‐helicity of the protein. Wild‐type and relA spoT mutant Salmonella, but not isogenic dksA‐deficient bacteria, experience the downregulation of r‐protein and amino acid transport expression after NO treatment, suggesting that DksA can regulate gene expression in response to NO congeners independently of the ppGpp alarmone. Oxidative stress enhances the DksA‐dependent repression of rpsM, while preventing the activation of livJ and hisG gene transcription that is supported by reduced, zinc‐bound DksA. The inhibitory effects of oxidized DksA on transcription are reversible with dithiothreitol. Our investigations indicate that sensing of reactive species by DksA redox active thiols fine‐tunes the expression of translational machinery and amino acid assimilation and biosynthesis in accord with the metabolic stress imposed by oxidative and nitrosative stress. Given the conservation of Cys114, and neighbouring hydrophobic and charged amino acids in DksA orthologues, phylogenetically diverse microorganisms may use the DksA thiol switch to regulate transcriptional responses to oxidative and nitrosative stress.

Synergistic Activities of Moxifloxacin Combined with Piperacillin-Tazobactam or Cefepime against Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii Clinical Isolates

ABSTRACT The bactericidal activity of moxifloxacin alone and in combination with cefepime or piperacillin-tazobactam against clinical isolates of Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii was evaluated by using time-kill methods and antimicrobial concentrations of one-half and one times the MIC. Synergy was observed in 58 to 88% of the strains and resulted in bactericidal activity against 60 to 100% of the strains. Combinations including moxifloxacin demonstrated enhanced bactericidal activity compared with that of either agent tested alone.

Cytochrome bd-Dependent Bioenergetics and Antinitrosative Defenses in Salmonella Pathogenesis.

ABSTRACT In the course of an infection, Salmonella enterica occupies diverse anatomical sites with various concentrations of oxygen (O2) and nitric oxide (NO). These diatomic gases compete for binding to catalytic metal groups of quinol oxidases. Enterobacteriaceae express two evolutionarily distinct classes of quinol oxidases that differ in affinity for O2 and NO as well as stoichiometry of H+ translocated across the cytoplasmic membrane. The investigations presented here show that the dual function of bacterial cytochrome bd in bioenergetics and antinitrosative defense enhances Salmonella virulence. The high affinity of cytochrome bd for O2 optimizes respiratory rates in hypoxic cultures, and thus, this quinol oxidase maximizes bacterial growth under O2-limiting conditions. Our investigations also indicate that cytochrome bd, rather than cytochrome bo, is an intrinsic component of the adaptive antinitrosative toolbox of Salmonella. Accordingly, induction of cytochrome bd helps Salmonella grow and respire in the presence of inhibitory NO. The combined antinitrosative defenses of cytochrome bd and the flavohemoglobin Hmp account for a great part of the adaptations that help Salmonella recover from the antimicrobial activity of NO. Moreover, the antinitrosative defenses of cytochrome bd and flavohemoglobin Hmp synergize to promote Salmonella growth in systemic tissues. Collectively, our investigations indicate that cytochrome bd is a critical means by which Salmonella resists the nitrosative stress that is engendered in the innate response of mammalian hosts while it concomitantly allows for proper O2 utilization in tissue hypoxia. IMPORTANCE It is becoming quite apparent that metabolism is critically important to the virulence potential of pathogenic microorganisms. Bacterial cells use a variety of terminal electron acceptors to power electron transport chains and metabolic processes. Of all the electron acceptors available to bacteria, utilization of O2 yields the most energy while diversifying the type of substrates that a pathogen can use. Recent investigations have demonstrated important roles for bd-type quinol oxidases with high affinity for O2 in bacterial pathogenesis. The investigations presented here have revealed that cytochrome bd potentiates virulence of a clinically relevant bacterial pathogen by fueling bioenergetics of prokaryotic cells while protecting the respiratory chain against NO toxicity. The adaptive antinitrosative defenses afforded by cytochrome bd synergize with other NO-detoxifying systems to preserve cellular bioenergetics, thereby promoting bacterial virulence in tissue hypoxia. It is becoming quite apparent that metabolism is critically important to the virulence potential of pathogenic microorganisms. Bacterial cells use a variety of terminal electron acceptors to power electron transport chains and metabolic processes. Of all the electron acceptors available to bacteria, utilization of O2 yields the most energy while diversifying the type of substrates that a pathogen can use. Recent investigations have demonstrated important roles for bd-type quinol oxidases with high affinity for O2 in bacterial pathogenesis. The investigations presented here have revealed that cytochrome bd potentiates virulence of a clinically relevant bacterial pathogen by fueling bioenergetics of prokaryotic cells while protecting the respiratory chain against NO toxicity. The adaptive antinitrosative defenses afforded by cytochrome bd synergize with other NO-detoxifying systems to preserve cellular bioenergetics, thereby promoting bacterial virulence in tissue hypoxia.

Ferric Uptake Regulator-Dependent Antinitrosative Defenses in Salmonella enterica Serovar Typhimurium Pathogenesis

ABSTRACT Herein we report an important role for the ferric uptake regulator (Fur) in the resistance of Salmonella enterica serovar Typhimurium to the reactive nitrogen species produced by inducible nitric oxide (NO) synthase in an NRAMP1r murine model of acute systemic infection. The expression of fur protected Salmonella grown under normoxic and hypoxic conditions against the bacteriostatic activity of NO. The hypersusceptibility of fur-deficient Salmonella to the cytotoxic actions of NO coincides with a marked repression of respiratory activity and the reduced ability of the bacteria to detoxify NO. A fur mutant Salmonella strain contained reduced levels of the terminal quinol oxidases of the electron transport chain. Addition of the heme precursor δ-aminolevulinic acid restored the cytochrome content, respiratory activity, NO consumption, and wild-type growth in bacteria undergoing nitrosative stress. The innate antinitrosative defenses regulated by Fur added to the adaptive response associated with the NO-detoxifying activity of the flavohemoprotein Hmp. Our investigations indicate that, in addition to playing a critical role in iron homeostasis, Fur is an important antinitrosative determinant of Salmonella pathogenesis.

Magnesium homeostasis protects Salmonella against nitrooxidative stress

The PhoPQ two-component regulatory system coordinates the response of Salmonella enterica serovar Typhimurium to diverse environmental challenges encountered during infection of hosts, including changes in Mg2+ concentrations, pH, and antimicrobial peptides. Moreover, PhoPQ-dependent regulation of gene expression promotes intracellular survival of Salmonella in macrophages, and contributes to the resistance of this pathogen to reactive nitrogen species (RNS) generated from the nitric oxide produced by the inducible nitric oxide (NO) synthase of macrophages. We report here that Salmonella strains with mutations of phoPQ are hypersensitive to killing by RNS generated in vitro. The increased susceptibility of ∆phoQ Salmonella to RNS requires molecular O2 and coincides with the nitrotyrosine formation, the oxidation of [4Fe-4S] clusters of dehydratases, and DNA damage. Mutations of respiratory NADH dehydrogenases prevent nitrotyrosine formation and abrogate the cytotoxicity of RNS against ∆phoQ Salmonella, presumably by limiting the formation of peroxynitrite (ONOO−) arising from the diffusion-limited reaction of exogenous NO and endogenous superoxide (O2•−) produced in the electron transport chain. The mechanism underlying PhoPQ-mediated resistance to RNS is linked to the coordination of Mg2+ homeostasis through the PhoPQ-regulated MgtA transporter. Collectively, our investigations are consistent with a model in which PhoPQ-dependent Mg2+ homeostasis protects Salmonella against nitrooxidative stress.

Acute pulmonary effects of aerosolized nicotine

Nicotine is a highly addictive principal component of both tobacco and electronic cigarette that is readily absorbed in blood. Nicotine-containing electronic cigarettes are promoted as a safe alternative to cigarette smoking. However, the isolated effects of inhaled nicotine are largely unknown. Here we report a novel rat model of aerosolized nicotine with a particle size (~1 μm) in the respirable diameter range. Acute nicotine inhalation caused increased pulmonary edema and lung injury as measured by enhanced bronchoalveolar lavage fluid protein, IgM, lung wet-to-dry weight ratio, and high-mobility group box 1 (HMGB1) protein and decreased lung E-cadherin protein. Immunohistochemical analysis revealed congested blood vessels and increased neutrophil infiltration. Lung myeloperoxidase mRNA and protein increased in the nicotine-exposed rats. Complete blood counts also showed an increase in neutrophils, white blood cells, eosinophils, and basophils. Arterial blood gas measurements showed an increase in lactate. Lungs of nicotine-inhaling animals revealed increased mRNA levels of IL-1A and CXCL1. There was also an increase in IL-1α protein. In in vitro air-liquid interface cultures of airway epithelial cells, there was a dose dependent increase in HMGB1 release with nicotine treatment. Air-liquid cultures exposed to nicotine also resulted in a dose-dependent loss of barrier as measured by transepithelial electrical resistance and a decrease in E-cadherin expression. Nicotine also caused a dose-dependent increase in epithelial cell death and an increase in caspase-3/7 activities. These results show that the nicotine content of electronic cigarettes may have adverse pulmonary and systemic effects.