The nitric oxide reductase of enterohaemorrhagic Escherichia coli plays an important role for the survival within macrophages

Abstract Background: For successful colonization, enterohaemorrhagic Escherichia coli (EHEC) injects virulence factors, called effectors, into target cells through the type three secretion system (T3SS), which is composed of a needle and basal body. Under anaerobic conditions, the T3SS machinery remains immature and does not have a needle structure. However, activation of nitrate respiration enhances the completion of the T3SS machinery. Because nitric oxide released by the host inflammatory response increases nitrate concentration, we sought to determine the effect of the inflammatory response on initiation of EHEC microcolony-formation. Results: The colony-forming capacity was increased in accordance with the increase of nitrate in the medium. The addition of the nitric oxide-producing agent NOR-4 also enhanced the adherence capacity, which was dependent on nitrate reductase encoded by the narGHJI genes. Culture supernatant of epithelial cells, which was stimulated by a cytokine mixture, enhanced the colony-forming capacity of wild-type EHEC but not of the narGHJI mutant. Finally, colony formation by wild-type EHEC on epithelial cells, which were preincubated with heat-killed bacteria, was higher than the narGHJI mutant, and this effect was abolished by aminoguanidine hydrochloride, which is an iNOS (inducible nitric oxide synthase) inhibitor. Conclusions: These results indicate that the inflammatory response enhances EHEC adherence by increasing nitrate concentration.

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Kinetic and thermodynamic resolution of the interactions between sulfite and the pentahaem cytochrome NrfA from Escherichia coli

Biochemical Journal ◽

10.1042/bj20100866 ◽

2010 ◽

Vol 431 (1) ◽

pp. 73-80 ◽

Cited By ~ 28

Author(s):

Gemma L. Kemp ◽

Thomas A. Clarke ◽

Sophie J. Marritt ◽

Colin Lockwood ◽

Susannah R. Poock ◽

...

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Active Site ◽

Dissociation Constants ◽

Cellular Activity ◽

Nitric Oxide Reductase ◽

Wide Range ◽

Nitric Oxide Reduction ◽

Nitric Oxide Detoxification ◽

Kinetic And Thermodynamic Studies

NrfA is a pentahaem cytochrome present in a wide-range of γ-, δ- and ε-proteobacteria. Its nitrite and nitric oxide reductase activities have been studied extensively and contribute to respiratory nitrite ammonification and nitric oxide detoxification respectively. Sulfite is a third substrate for NrfA that may be encountered in the micro-oxic environments where nrfA is expressed. Consequently, we have performed quantitative kinetic and thermodynamic studies of the interactions between sulfite and Escherichia coli NrfA to provide a biochemical framework from which to consider their possible cellular consequences. A combination of voltammetric, spectroscopic and crystallographic analyses define dissociation constants for sulfite binding to NrfA in oxidized (~54 μM), semi-reduced (~145 μM) and reduced (~180 μM) states that are comparable with each other, and the Km (~70 μM) for sulfite reduction at pH 7. Under comparable conditions Km values of ~22 and ~300 μM describe nitrite and nitric oxide reduction respectively, whereas the affinities of nitrate and thiocyanate for NrfA fall more than 50-fold on enzyme reduction. These results are discussed in terms of the nature of sulfite co-ordination within the active site of NrfA and their implications for the cellular activity of NrfA.

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Role of the Nitric Oxide Reductase NorVW in the Survival and Virulence of Enterohaemorrhagic Escherichia coli during Infection

Pathogens ◽

10.3390/pathogens9090683 ◽

2020 ◽

Vol 9 (9) ◽

pp. 683 ◽

Cited By ~ 1

Author(s):

Marion Gardette ◽

Julien Daniel ◽

Estelle Loukiadis ◽

Grégory Jubelin

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Nitrosative Stress ◽

Innate Response ◽

Enterohaemorrhagic Escherichia Coli ◽

Life Threatening ◽

Uremic Syndrome ◽

Synthase Inhibitor ◽

Strain Dependent

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans, such as hemolytic and uremic syndrome. It has been previously demonstrated that the interplay between EHEC and nitric oxide (NO), a mediator of the host immune innate response, is critical for infection outcome, since NO affects both Shiga toxin (Stx) production and adhesion to enterocytes. In this study, we investigated the role of the NO reductase NorVW in the virulence and fitness of two EHEC strains in a murine model of infection. We determined that the deletion of norVW in the strain O91:H21 B2F1 has no impact on its virulence, whereas it reduces the ability of the strain O157:H7 620 to persist in the mouse gut and to produce Stx. We also revealed that the fitness defect of strain 620 ΔnorVW is strongly attenuated when mice are treated with an NO synthase inhibitor. Altogether, these results demonstrate that the NO reductase NorVW participates in EHEC resistance against NO produced by the host and promotes virulence through the modulation of Stx synthesis. The contribution of NorVW in the EHEC infectious process is, however, strain-dependent and suggests that the EHEC response to nitrosative stress is complex and multifactorial.

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Binding of NorR to three DNA sites is essential for promoter activation of the flavorubredoxin gene, the nitric oxide reductase of Escherichia coli

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2005.01.008 ◽

2005 ◽

Vol 328 (2) ◽

pp. 540-544 ◽

Cited By ~ 11

Author(s):

Marta C. Justino ◽

Vera M.M. Gonçalves ◽

Lígia M. Saraiva

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Nitric Oxide Reductase ◽

Promoter Activation

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Nitric Oxide Induced stx2 Expression Is Inhibited by the Nitric Oxide Reductase, NorV, in a Clade 8 Escherichia coli O157:H7 Outbreak Strain

Microorganisms ◽

10.3390/microorganisms10010106 ◽

2022 ◽

Vol 10 (1) ◽

pp. 106

Author(s):

Rim Al Safadi ◽

Michelle L. Korir ◽

Shannon D. Manning

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Shiga Toxin ◽

Toxin Production ◽

Anaerobic Growth ◽

Escherichia Coli O157 ◽

Similar Increase ◽

Nitric Oxide Reductase ◽

Outbreak Strain ◽

Uremic Syndrome

Escherichia coli O157:H7 pathogenesis is due to Shiga toxin (Stx) production, though variation in virulence has been observed. Clade 8 strains, for instance, were shown to overproduce Stx and were more common among hemolytic uremic syndrome cases. One candidate gene, norV, which encodes a nitric oxide (NO) reductase found in a clade 8 O157:H7 outbreak strain (TW14359), was thought to impact virulence. Hence, we screened for norV in 303 O157 isolates representing multiple clades, examined stx2 expression following NO exposure in TW14359 for comparison to an isogenic mutant (ΔnorV), and evaluated survival in THP-1 derived macrophages. norV was intact in strains representing clades 6–9, whereas a 204 bp deletion was found in clades 2 and 3. During anaerobic growth, NO induced stx2 expression in TW14359. A similar increase in stx2 expression was observed for the ΔnorV mutant in anaerobiosis, though it was not impaired in its ability to survive within macrophages relative to TW14359. Altogether, these data suggest that NO enhances virulence by inducing Stx2 production in TW14359, and that toxin production is inhibited by NorV encoded by a gene found in most clade 8 strains. The mechanism linked to these responses, however, remains unclear and likely varies across genotypes.

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Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans

Microbiology ◽

10.1099/mic.0.26546-0 ◽

2003 ◽

Vol 149 (12) ◽

pp. 3405-3412 ◽

Cited By ~ 3

Author(s):

Jiří Mazoch ◽

Michal Kuňák ◽

Igor Kučera ◽

Rob J. M. van Spanning

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Nitrate Reductase ◽

Electron Acceptor ◽

Paracoccus Denitrificans ◽

Maximal Activity ◽

Galactosidase Activity ◽

Nitric Oxide Reductase ◽

Low Levels ◽

Insight Into

In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, β-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of β-galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic β-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N2O. Additions of nitrate or nitroprusside lowered β-galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.

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