Staphylococcus aureus lactate- and malate-quinone oxidoreductases contribute to nitric oxide resistance and virulence.
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ABSTRACT:
SUBMITTER: Spahich NA
PROVIDER: S-EPMC4894658 | biostudies-literature | 2016 Jun
REPOSITORIES: biostudies-literature
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Project description:Staphylococcus aureus exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). S. aureus NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for S. aureus NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for S. aureus to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F1F0 ATPase, have not been previously reported to contribute to S. aureus NO· resistance. The most striking finding was that loss of any genes encoding components of the F1F0 ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in S. aureus, the F1F0 ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the ?atpG mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ? 7.0 and the ?atpG mutant cannot thrive. Thus, S. aureus NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F1F0 ATPase enzyme complex.
| S-EPMC5884563 | biostudies-literature
Project description:Recent studies have indicated that Staphylococcus aureus can survive the nitrosative stress (caused by the radical nitric oxide; NO.) mounted by the immune system of the infected host. It does this by expressing a nitric oxide-inducible L-lactate dehydrogenase (Sa-LDH-1). Therefore, if efficient inhibitors of Sa-LDH-1 can be designed then Sa-LDH-1 could be a potential drug target against the pathogen S. aureus. For this purpose, the nitric acid-inducible LDH-1 from S. aureus COL strain has been cloned into the expression vector pET-28a(+) and the protein has been expressed, purified and crystallized. The Sa-LDH-1 crystal diffracted to 2.4 A resolution at a home X-ray source and belonged to space group C2, with unit-cell parameters a = 131.4, b = 74.4, c = 103.2 A, beta = 133.4 degrees .
| S-EPMC2765899 | biostudies-literature
Project description:Staphylococcus aureus is a prolific human pathogen capable of causing severe invasive disease with a myriad of presentations. The ability of S. aureus to cause infection is strongly linked with its capacity to overcome the effects of innate immunity, whether by directly killing immune cells or expressing factors that diminish the impact of immune effectors. One such scenario is the induction of lactic acid fermentation by S. aureus in response to host nitric oxide (NO·). This fermentative activity allows S. aureus to balance redox during NO·-induced respiration inhibition. However, little is known about the metabolic substrates and pathways that support this activity. Here, we identify glycolytic hexose catabolism as being essential for S. aureus growth in the presence of high levels of NO·. We determine that glycolysis supports S. aureus NO· resistance by allowing for ATP and precursor metabolite production in a redox-balanced and respiration-independent manner. We further demonstrate that glycolysis is required for NO· resistance during phagocytosis and that increased levels of extracellular glucose limit the effectiveness of phagocytic killing by enhancing NO· resistance. Finally, we demonstrate that S. aureus glycolysis is essential for virulence in both sepsis and skin/soft tissue models of infection in a time frame consistent with the induction of innate immunity and host NO· production. Staphylococcus aureus is a leading human bacterial pathogen capable of causing a wide variety of diseases that, as a result of antibiotic resistance, are very difficult to treat. The frequency of S. aureus tissue invasion suggests that this bacterium has evolved to resist innate immunity and grow using the nutrients present in otherwise sterile host tissue. We have identified glycolysis as an essential component of S. aureus virulence and attribute its importance to promoting nitric oxide resistance and growth under low oxygen conditions. Our data suggest that diabetics, a patient population characterized by excess serum glucose, may be more susceptible to S. aureus as a result of increased glucose availability. Furthermore, the essential nature of S. aureus glycolysis indicates that a newly developed glycolysis inhibitor may be a highly effective treatment for S. aureus infections.
| S-EPMC4453550 | biostudies-literature
Project description:UnlabelledThe ability of Staphylococcus aureus to resist host innate immunity augments the severity and pervasiveness of its pathogenesis. Nitric oxide (NO˙) is an innate immune radical that is critical for the efficient clearance of a wide range of microbial pathogens. Exposure of microbes to NO˙ typically results in growth inhibition and induction of stress regulons. S. aureus, however, induces a metabolic state in response to NO˙ that allows for continued replication and precludes stress regulon induction. The regulatory factors mediating this distinctive response remain largely undefined. Here, we employ a targeted transposon screen and transcriptomics to identify and characterize five regulons essential for NO˙ resistance in S. aureus: three virulence regulons not formerly associated with NO˙ resistance, SarA, CodY, and Rot, as well as two regulons with established roles, Fur and SrrAB. We provide new insights into the contributions of Fur and SrrAB during NO˙ stress and show that the S. aureus ΔsarA mutant, the most sensitive of the newly identified mutants, exhibits metabolic dysfunction and widespread transcriptional dysregulation following NO˙ exposure. Altogether, our results broadly characterize the regulatory requirements for NO˙ resistance in S. aureus and suggest an intriguing overlap between the regulation of NO˙ resistance and virulence in this well-adapted human pathogen.ImportanceThe prolific human pathogen Staphylococcus aureus is uniquely capable of resisting the antimicrobial radical nitric oxide (NO˙), a crucial component of the innate immune response. However, a complete understanding of how S. aureus regulates an effective response to NO˙ is lacking. Here, we implicate three central virulence regulators, SarA, CodY, and Rot, as major players in the S. aureus NO˙ response. Additionally, we elaborate on the contribution of two regulators, SrrAB and Fur, already known to play a crucial role in S. aureus NO˙ resistance. Our study sheds light on a unique facet of S. aureus pathogenicity and demonstrates that the transcriptional response of S. aureus to NO˙ is highly pleiotropic and intrinsically tied to metabolism and virulence regulation.
| S-EPMC4944221 | biostudies-literature
Project description:Staphylococcus aureus nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of nos expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring nos mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a nos srrAB mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The nos srrAB mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the nos srrAB double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits S. aureus to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The nos, srrAB, and nos srrAB mutants showed comparable defects in endothelial intracellular survival, whereas the srrAB and nos srrAB mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant in vivo.
| S-EPMC6346124 | biostudies-literature
Project description:Staphylococcus aureus is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. S. aureus presents a remarkable capacity to adapt to different niches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of S. aureus, focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. S. aureus presents two genes encoding NDH-2s (NDH-2A and NDH-2B) and lacks genes coding for Complex I, the canonical respiratory NADH:quinone oxidoreductase. This observation makes the action of NDH-2s crucial for the regeneration of NAD+ and, consequently, for the progression of metabolism. Our study involved the comprehensive biochemical characterization of NDH-2B and the exploration of the cellular roles of NDH-2A and NDH-2B, utilizing knockout mutants (Δndh-2a and Δndh-2b). We show that NDH-2B uses NADPH instead of NADH, does not establish a charge-transfer complex in the presence of NADPH, and its reduction by this substrate is the catalytic rate-limiting step. In the case of NDH-2B, the reduction of the flavin is inherently slow, and we suggest the establishment of a charge transfer complex between NADP+ and FADH2, as previously observed for NDH-2A, to slow down quinone reduction and, consequently, prevent the overproduction of reactive oxygen species, which is potentially unnecessary. Furthermore, we observed that the lack of NDH-2A or NDH-2B impacts cell growth, volume, and division differently. The absence of these enzymes results in distinct metabolic phenotypes, emphasizing the unique cellular roles of each NDH-2 in energy metabolism.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen, posing a global challenge in clinical medicine due to the increased incidence of its drug resistance. For this reason, it is essential to explore and understand the mechanisms behind its resistance, as well as the fundamental biological features such as energy metabolism and the respective players that allow S. aureus to live and survive. Despite its prominence as a pathogen, the energy metabolism of S. aureus remains underexplored, with its respiratory enzymes often escaping thorough investigation. S. aureus bioenergetic plasticity is illustrated by its ability to use different respiratory enzymes, two of which are investigated in the present study. Understanding the metabolic adaptation strategies of S. aureus to bioenergetic challenges may pave the way for the design of therapeutic approaches that interfere with the ability of the pathogen to successfully adapt when it invades different niches within its host.
| S-EPMC11302666 | biostudies-literature
Project description:Nitric oxide synthase (NOS) enzymes produce nitric oxide (NO), a highly reactive free radical capable of interacting with multiple cellular targets. Although saNOS contributes to Staphylococcus aureus virulence, as well as protection against exogenous oxidative stress and antimicrobials, the current mechanism behind these phenotypes is unknown. Here we report a previously-undescribed role for saNOS in modulating S. aureus physiology. When grown aerobically, endogenous reactive oxygen species (ROS) and superoxide levels were elevated in a S. aureus nos mutant. NO has been shown to slow respiration in other organisms, and likewise, comparison of respiratory dehydrogenase activity and membrane potential in wild-type, nos mutant, and complement strains suggested that saNOS-derived NO limits S. aureus aerobic respiration. Multiple transcriptional and metabolic changes were also observed in a S. aureus nos mutant, as assessed by RNAseq and targeted metabolomics analyses, respectively. Specifically, expression of genes associated with oxidative and nitrosative stress responses, anaerobic and lactate metabolism, and cytochrome biosynthesis and assembly were increased in the nos mutant relative to wild-type. Metabolites utilized to produce reducing equivalents by the right arm of the TCA cycle were depleted in a nos mutant (citrate and α-ketoglutarate), whereas fumarate and malate levels were increased relative to wild-type and complement strains. A significant reduction in lactate levels was also observed in the nos mutant. Collectively, these results support a model in which the absence of saNOS results in increased respiration and ROS accumulation, which may signal the cells to switch to an alternative lactate-based respiratory metabolism.
2017-07-10 | GSE77400 | GEO
Project description:Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme.
| S-EPMC4475277 | biostudies-literature
Project description:Staphylococcus aureus is a major human pathogen capable of causing a variety of diseases ranging from skin and soft tissue infections to systemic presentations such as sepsis, endocarditis, and osteomyelitis. For S. aureus to persist as a pathogen in these environments, it must be able to resist the host immune response, including the production of reactive oxygen and nitrogen species (e.g., nitric oxide, NO·). Extensive work from our lab has shown that S. aureus is highly resistant to NO·, especially in the presence of glucose. RNA-seq performed on S. aureus exposed to NO· in the presence and absence of glucose showed a new system important for NO· resistance-phosphate transport. The phosphate transport systems pstSCAB and nptA are both upregulated upon NO·-exposure, particularly in the presence of glucose. Both are key for phosphate transport at an alkaline pH, which the cytosol of S. aureus becomes under NO· stress. Accordingly, the ΔpstSΔnptA mutant is attenuated under NO stress in vitro as well as in macrophage and murine infection models. This work defines a new role in infection for two phosphate transporters in S. aureus and provides insight into the complex system that is NO· resistance in S. aureus.IMPORTANCEStaphylococcus aureus is a bacterial pathogen capable of causing a wide variety of disease in humans. S. aureus is unique in its ability to resist the host immune response, including the antibacterial compound known as nitric oxide (NO·). We used an RNA-sequencing approach to better understand the impact of NO· on S. aureus in different environments. We discovered that inorganic phosphate transport is induced by the presence of NO·. Phosphate is important for the generation of energy from glucose, a carbon source favored by S. aureus. We show that the absence of these phosphate transporters causes lowered energy levels in S. aureus. We find that these phosphate transporters are essential for S. aureus to grow in the presence of NO· and to cause infection. Our work here contributes significantly to our understanding of S. aureus NO· resistance and provides a new context in which S. aureus phosphate transporters are essential.
| S-EPMC10746193 | biostudies-literature
Project description:Nitric oxide (NO) is generated from arginine and oxygen via NO synthase (NOS). Staphylococcus aureus NOS (saNOS) has previously been shown to affect virulence and resistance to exogenous oxidative stress, yet the exact mechanism is unknown. Herein, a previously undescribed role of saNOS in S. aureus aerobic physiology was reported. Specifically, aerobic S. aureus nos mutant cultures presented with elevated endogenous reactive oxygen species (ROS) and superoxide levels, as well as increased membrane potential, increased respiratory dehydrogenase activity and slightly elevated oxygen consumption. Elevated ROS levels in the nos mutant likely resulted from altered respiratory function, as inhibition of NADH dehydrogenase brought ROS levels back to wild-type levels. These results indicate that, in addition to its recently reported role in regulating the switch to nitrate-based respiration during low-oxygen growth, saNOS also plays a modulatory role during aerobic respiration. Multiple transcriptional changes were also observed in the nos mutant, including elevated expression of genes associated with oxidative/nitrosative stress, anaerobic respiration and lactate metabolism. Targeted metabolomics revealed decreased cellular lactate levels, and altered levels of TCA cycle intermediates, the latter of which may be related to decreased aconitase activity. Collectively, these findings demonstrate a key contribution of saNOS to S. aureus aerobic respiratory metabolism.
| S-EPMC5641370 | biostudies-literature