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The Staphylococcus aureus nitric oxide synthase (saNOS) modulates respiratory metabolism and cell physiology

ABSTRACT: 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.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE77400 | GEO | 2017/07/10

SECONDARY ACCESSION(S): PRJNA310224

REPOSITORIES: GEO

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Project description:The nitric oxide synthase (NOS) co-factor, tetrahydrobiopterin (BH4), is a redox-active molecule that regulates nitric oxide (NO) and reactive oxygen species (ROS) production by NOS, and is an example of redox-dependent signalling in the endothelium that underlies both the maintenance of vascular homeostasis and the development of cardiovascular disease (CVD). Loss of endothelial BH4 is observed in CVD states and results in decreased NO production and increased ROS generation by endothelial NO synthase (uncoupling). Genetic mouse models of augmented endothelial BH4 synthesis have shown proof of concept that endothelial BH4 can alter CVD pathogenesis. However, clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation and limited endothelial uptake of BH4, highlighting the need to explore the wider roles of BH4 in order to find novel therapeutic targets. In this study we aim to elucidate the effects of BH4 depletion on mitochondrial function and bioenergetics using targeted knockdown of the BH4 synthetic enzyme GTPCH in vitro. Knockdown of GTPCH (and, therefore, intracellular BH4 levels) by >90% lead to a striking induction of ROS generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH4 depleted GCH fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH4 in mitochondrial redox signalling. Furthermore, this BH4-dependent, mitochondrial-derived ROS oxidized mitochondrial BH4 and is seen alongside distinct changes in the thioredoxin and glutathione antioxidant pathways, revealed by mass spectrometry using an unbiased proteomic approach. These changes are accompanied by a modest increase in mitochondrial size, attenuated respiratory function, and marked changes in the cellular metabolic profile; including succinate accumulation. Taken together, these data reveal a novel NOS-independent role for BH4 in the regulation of mitochondrial redox signalling and metabolism.

Project description:This a model from the article: Vascular and perivascular nitric oxide release and transport: biochemical pathways of neuronal nitric oxide synthase (NOS1) and endothelial nitric oxide synthase (NOS3). Chen K, Popel AS Free Radic Biol Med. 2007 Mar 15;42(6):811-22. 17320763 , Abstract: Nitric oxide (NO) derived from nitric oxide synthase (NOS) is an important paracrine effector that maintains vascular tone. The release of NO mediated by NOS isozymes under various O(2) conditions critically determines the NO bioavailability in tissues. Because of experimental difficulties, there has been no direct information on how enzymatic NO production and distribution change around arterioles under various oxygen conditions. In this study, we used computational models based on the analysis of biochemical pathways of enzymatic NO synthesis and the availability of NOS isozymes to quantify the NO production by neuronal NOS (NOS1) and endothelial NOS (NOS3). We compared the catalytic activities of NOS1 and NOS3 and their sensitivities to the concentration of substrate O(2). Based on the NO release rates predicted from kinetic models, the geometric distribution of NO sources, and mass balance analysis, we predicted the NO concentration profiles around an arteriole under various O(2) conditions. The results indicated that NOS1-catalyzed NO production was significantly more sensitive to ambient O(2) concentration than that catalyzed by NOS3. Also, the high sensitivity of NOS1 catalytic activity to O(2) was associated with significantly reduced NO production and therefore NO concentrations, upon hypoxia. Moreover, the major source determining the distribution of NO was NOS1, which was abundantly expressed in the nerve fibers and mast cells close to arterioles, rather than NOS3, which was expressed in the endothelium. Finally, the perivascular NO concentration predicted by the models under conditions of normoxia was paradoxically at least an order of magnitude lower than a number of experimental measurements, suggesting a higher abundance of NOS1 or NOS3 and/or the existence of other enzymatic or nonenzymatic sources of NO in the microvasculature. This model was taken from the CellML repository and automatically converted to SBML. The original model was: Chen K, Popel AS (2007) - version02 The original CellML model was created by: Lloyd, Catherine, May c.lloyd@aukland.ac.nz The University of Auckland The Bioengineering Institute This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2011 The BioModels.net Team. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information. In summary, you are entitled to use this encoded model in absolutely any manner you deem suitable, verbatim, or with modification, alone or embedded it in a larger context, redistribute it, commercially or not, in a restricted way or not.. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

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The Staphylococcus aureus nitric oxide synthase (saNOS) modulates respiratory metabolism and cell physiology

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