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Interplay between enterohaemorrhagic Escherichia coli and nitric oxide during the infectious process.

ABSTRACT: Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans such as bloody diarrhoea and the hemolytic and uremic syndrome. To date, no specific therapy is available and treatments remain essentially symptomatic. In recent years, we demonstrated in vitro that nitric oxide (NO), a major mediator of the intestinal immune response, strongly represses the synthesis of the two cardinal virulence factors in EHEC, namely Shiga toxins (Stx) and the type III secretion system, suggesting NO has a great potential to protect against EHEC infection. In this study, we investigated the interplay between NO and EHEC in vivo using mouse models of infection. Using a NO-sensing reporter strain, we determined that EHEC sense NO in the gut of infected mice. Treatment of infected mice with a specific NOS inhibitor increased EHEC adhesion to the colonic mucosa but unexpectedly decreased Stx activity in the gastrointestinal tract, protecting mice from renal failure. Taken together, our data indicate that NO can have both beneficial and detrimental consequences on the outcome of an EHEC infection, and underline the importance of in vivo studies to increase our knowledge in host-pathogen interactions.

SUBMITTER: Naili I

PROVIDER: S-EPMC7336997 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nitrite has been used as a bacteriostatic agent for centuries in food preservation. It is widely accepted that this biologically inert molecule functions indirectly, serving as a stable reservoir of bioactive nitric oxide (NO) and other reactive nitrogen species to impact physiology. As a result, to date, we know surprisingly little about in vivo targets of nitrite. Here, we carry out comparative analyses of nitrite and NO physiology in Escherichia coli and in Shewanella oneidensis, a Gram-negative environmental bacterium renowned for respiratory versatility. These two bacteria differ from each other in many aspects of nitrite and NO physiology, including NO generation, NO degradation, and unexpectedly, their contrary susceptibility to nitrite and NO. In cell extracts of both bacteria, most of the NO targets are also susceptible to nitrite, and vice versa. However, with respect to growth inhibition caused by NO, the targets are impacted distinctly; NO targets are responsible for the inhibition of growth of E. coli but not of S. oneidensis More surprisingly, all proteins identified to be implicated in NO tolerance in other bacteria appear to play a dispensable role in protecting S. oneidensis against NO. These data suggest that S. oneidensis is equipped with a robust but yet unknown NO protecting system. In the case of nitrite, it is clear that the target of physiological significance in both bacteria is cytochrome heme-copper oxidase.IMPORTANCE Nitrite is toxic to living organisms at high levels, but such antibacterial effects of nitrite are attributable to the formation of nitric oxide (NO), a highly reactive radical gas molecule. Here, we report that Shewanella oneidensis is highly resistant to NO but sensitive to nitrite compared to Escherichia coli by approximately 4-fold. In both bacteria, nitrite inhibits bacterial growth by targeting cytochrome heme-copper oxidase. In contrast, the targets of NO are diverse. Although these targets are similar in E. coli and S. oneidensis, they are responsible for growth inhibition caused by NO in the former but not in the latter. Overall, the presented data, along with the previous data, solidify a proposal that the in vivo targets of NO and nitrite in bacteria are largely different.

Project description:BACKGROUND:Plasmodium falciparum haemozoin, a detoxification product of digested haemoglobin from infected erythrocytes, is released into the bloodstream upon schizont rupture and accumulates in leukocytes. High levels of haemozoin correlate with disease severity. Some studies have shown that concentrations of the substrate of inducible nitric oxide synthase (iNOS), L-arginine, as well as nitric oxide are low in patients infected with P. falciparum malaria. The present study investigates, in vitro, the role of P. falciparum haemozoin on nitric oxide production, iNOS expression in macrophages, and the possible interaction between L-arginine and haemozoin. METHODS:Plasmodium falciparum haemozoin was obtained from in vitro cultures through magnetic isolation. Phagocytosis of haemozoin by immortalized bone marrow derived macrophages was detected by confocal reflection combined with fluorescence microscopy. Nitrite concentrations in the supernatants was evaluated by Griess assay as a standard indication of nitric oxide production, while iNOS expression was detected on cell extracts by western blotting. Detection of L-arginine in haemozoin-treated or untreated media was achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS:Haemozoin synergizes in vitro with interferon-gamma to produce nitric oxide. However, when mouse macrophages were stimulated with haemozoin, a proportional increase of nitric oxide was observed up to 25 ?M of haemozoin, followed by a decrease with doses up to 100 ?M, when nitric oxide release was completely abrogated. This was not due to reactive oxygen species production, nor to an effect on iNOS activity. Interestingly, when at 24 h, haemozoin-treated macrophages were washed and incubated in fresh medium for further 24 h, the nitric oxide production was restored in a dose-response manner. Similar results were seen when L-arginine-enriched media was used in the stimulation. Moreover, muramyldipeptide, a strong nitric oxide inducer, was unable to activate macrophages to release nitric oxide in the presence of haemozoin-treated medium. By LC-MS/MS a complete depletion of L-arginine was observed in this haemozoin-treated, conditioned medium. CONCLUSIONS:It is proposed that haemozoin interacts with L-arginine reducing its availability for iNOS, and thus decreasing nitric oxide production. The clinical (or pathological) implications of these results are discussed.

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Interplay between enterohaemorrhagic Escherichia coli and nitric oxide during the infectious process.

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