Nitric Oxide Inhibition of Rickettsia rickettsii.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is an enzootic, obligate, intracellular bacterial pathogen. Nitric oxide (NO) synthesized by the inducible NO synthase (iNOS) is a potent antimicrobial component of innate immunity and has been implicated in the control of virulent Rickettsia spp. in diverse cell types. In this study, we examined the antibacterial role of NO on R. rickettsii. Our results indicate that NO challenge dramatically reduces R. rickettsii adhesion through the disruption of bacterial energetics. Additionally, NO-treated R. rickettsii cells were unable to synthesize protein or replicate in permissive cells. Activated, NO-producing macrophages restricted R. rickettsii infections, but inhibition of iNOS ablated the inhibition of bacterial growth. These data indicate that NO is a potent antirickettsial effector of innate immunity that targets energy generation in these pathogenic bacteria to prevent growth and subversion of infected host cells.

Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism.

Cytostasis is the most salient manifestation of the potent antimicrobial activity of nitric oxide (NO), yet the mechanism by which NO disrupts bacterial cell division is unknown. Here, we show that in respiring Escherichia coli, Salmonella, and Bacillus subtilis, NO arrests the first step in division, namely, the GTP-dependent assembly of the bacterial tubulin homolog FtsZ into a cytokinetic ring. FtsZ assembly fails in respiring cells because NO inactivates inosine 5'-monophosphate dehydrogenase in de novo purine nucleotide biosynthesis and quinol oxidases in the electron transport chain, leading to drastic depletion of nucleoside triphosphates, including the GTP needed for the polymerization of FtsZ. Despite inhibiting respiration and dissipating proton motive force, NO does not destroy Z ring formation and only modestly decreases nucleoside triphosphates in glycolytic cells, which obtain much of their ATP by substrate-level phosphorylation and overexpress inosine 5'-monophosphate dehydrogenase. Purine metabolism dictates the susceptibility of early morphogenic steps in cytokinesis to NO toxicity.

SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.

DksA-DnaJ redox interactions provide a signal for the activation of bacterial RNA polymerase.

RNA polymerase is the only known protein partner of the transcriptional regulator DksA. Herein, we demonstrate that the chaperone DnaJ establishes direct, redox-based interactions with oxidized DksA. Cysteine residues in the zinc finger of DksA become oxidized in Salmonella exposed to low concentrations of hydrogen peroxide (H2O2). The resulting disulfide bonds unfold the globular domain of DksA, signaling high-affinity interaction of the C-terminal α-helix to DnaJ. Oxidoreductase and chaperone activities of DnaJ reduce the disulfide bonds of its client and promote productive interactions between DksA and RNA polymerase. Simultaneously, guanosine tetraphosphate (ppGpp), which is synthesized by RelA in response to low concentrations of H2O2, binds at site 2 formed at the interface of DksA and RNA polymerase and synergizes with the DksA/DnaJ redox couple, thus activating the transcription of genes involved in amino acid biosynthesis and transport. However, the high concentrations of ppGpp produced by Salmonella experiencing oxidative stress oppose DksA/DnaJ-dependent transcription. Cumulatively, the interplay of DksA, DnaJ, and ppGpp on RNA polymerase protects Salmonella from the antimicrobial activity of the NADPH phagocyte oxidase. Our research has identified redox-based signaling that activates the transcriptional activity of the RNA polymerase regulator DksA.

Salmonella Reprograms Nucleotide Metabolism in Its Adaptation to Nitrosative Stress.

The adaptations that protect pathogenic microorganisms against the cytotoxicity of nitric oxide (NO) engendered in the immune response are incompletely understood. We show here that salmonellae experiencing nitrosative stress suffer dramatic losses of the nucleoside triphosphates ATP, GTP, CTP, and UTP while simultaneously generating a massive burst of the alarmone nucleotide guanosine tetraphosphate. RelA proteins associated with ribosomes overwhelmingly synthesize guanosine tetraphosphate in response to NO as a feedback mechanism to transient branched-chain amino acid auxotrophies. Guanosine tetraphosphate activates the transcription of valine biosynthetic genes, thereby reestablishing branched-chain amino acid biosynthesis that enables the translation of the NO-consuming flavohemoglobin Hmp. Guanosine tetraphosphate synthesized by RelA protects salmonellae from the metabolic stress inflicted by reactive nitrogen species generated in the mammalian host response. This research illustrates the importance of nucleotide metabolism in the adaptation of salmonellae to the nutritional stress imposed by NO released in the innate host response.IMPORTANCE Nitric oxide triggers dramatic drops in nucleoside triphosphates, the building blocks that power DNA replication; RNA transcription; translation; cell division; and the biosynthesis of fatty acids, lipopolysaccharide, and peptidoglycan. Concomitantly, this diatomic gas stimulates a burst of guanosine tetraphosphate. Global changes in nucleotide metabolism may contribute to the potent bacteriostatic activity of nitric oxide. In addition to inhibiting numerous growth-dependent processes, guanosine tetraphosphate positively regulates the transcription of branched-chain amino acid biosynthesis genes, thereby facilitating the translation of antinitrosative defenses that mediate recovery from nitrosative stress.

Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content.

UNLABELLED: The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments. IMPORTANCE: In order to cause disease, pathogenic bacteria must rapidly sense and respond to antimicrobial pressures encountered within the host. Prominent among these stresses, and of particular relevance to intracellular pathogens such as Salmonella, are nutritional restriction and the enzymatic generation of reactive oxygen and nitrogen species. The conserved transcriptional regulator DksA controls adaptive responses to nutritional limitation, as well as to oxidative and nitrosative stress. Here, we demonstrate that each of these functions contributes to bacterial pathogenesis. Our observations highlight the importance of metabolic adaptation in bacterial pathogenesis and show the mechanism by which DksA orthologues are optimized to sense the levels of oxidative and nitrosative stress encountered in their natural habitats. An improved understanding of the conserved processes used by bacteria to sense, respond to, and limit host defense will inform the development of novel strategies to treat infections caused by pathogenic, potentially multidrug-resistant bacteria.

Characterization of the GbdR regulon in Pseudomonas aeruginosa.

Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa's response to the host, particularly changes regulated by the host-derived quaternary amines choline and glycine betaine (GB). We previously identified GbdR as an AraC family transcription factor that directly regulates choline acquisition from host phospholipids (via binding to plcH and pchP promoters), is required for catabolism of the choline metabolite GB, and is an activator that induces transcription in response to GB or dimethylglycine. Our goal was to characterize the GbdR regulon in P. aeruginosa by using genetics and chemical biology in combination with transcriptomics and in vitro DNA-binding assays. Here we show that GbdR activation regulates transcription of 26 genes from 12 promoters, 11 of which have measureable binding to GbdR in vitro. The GbdR regulon includes the genes encoding GB, dimethylglycine, sarcosine, glycine, and serine catabolic enzymes and the BetX and CbcXWV quaternary amine transport proteins. We characterized the GbdR consensus binding site and used it to identify that the recently characterized acetylcholine esterase gene, choE (PA4921), is also regulated by GbdR. The regulon member not directly controlled by GbdR is the secreted lipase gene lipA, which was also the only regulon member repressed under GbdR-activating conditions. Determination of the GbdR regulon provides deeper understanding of how GbdR links bacterial metabolism and virulence. Additionally, identification of two uncharacterized regulon members suggests roles for these proteins in response to choline metabolites.

Pseudomonas aeruginosa AlgR phosphorylation modulates rhamnolipid production and motility.

AlgR is a key Pseudomonas aeruginosa transcriptional response regulator required for virulence. AlgR activates alginate production and twitching motility but represses the Rhl quorum-sensing (QS) system, including rhamnolipid production. The role of AlgR phosphorylation is enigmatic, since phosphorylated AlgR (AlgR-P) is required for twitching motility through the fimU promoter but is not required for the activation of alginate production. In order to examine the role of AlgR phosphorylation in vivo, a PAO1 algRD54E strain (with algR encoding a D-to-E change at position 54), which constitutively activates fimU transcription and exhibits twitching motility, was created. A corresponding PAO1 algRD54N strain (with algR encoding a D-to-N change at position 54) that does not activate fimU or twitching motility was compared to PAO1, PAO1 algRD54E, PAO1 ΔalgZ (deletion of the algZ [fimS] gene, encoding a putative histidine kinase), and PAO1 ΔalgR for swarming motility, rhamnolipid production, and rhlA transcription. PAO1 and PAO1 algRD54E produced approximately 2-fold-higher levels of rhamnolipids than PAO1 algRD54N and PAO1 ΔalgZ, thereby indicating that phosphorylated AlgR is required for normal rhamnolipid production. Examination of purified AlgR, AlgR-P, AlgR D54N, and AlgR D54E showed that AlgR-P and AlgR D54E bound preferentially to the fimU and rhlA promoters. Additionally, AlgR-P bound specifically to two sites within the rhlA promoter that were not bound by unphosphorylated AlgR. Taken together, these results indicate that phosphorylated AlgR-P has increased affinity for the rhlA promoter and is required for the coordinate activation of twitching motility, rhamnolipid production, and swarming motility in P. aeruginosa.

Cellular choline and glycine betaine pools impact osmoprotection and phospholipase C production in Pseudomonas aeruginosa.

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used (13)C nuclear magnetic resonance ((13)C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology.

Small-molecule inhibition of choline catabolism in Pseudomonas aeruginosa and other aerobic choline-catabolizing bacteria.

Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.