Peroxynitrite stress is exacerbated by flavohaemoglobin-derived oxidative stress in Salmonella Typhimurium and is relieved by nitric oxide

Abstract

2 ) and peroxynitrite play key roles in determining the outcome of bacterial infections. In order to survive within the host and allow proliferation within immune cells such as macrophages, Salmonella isolates have a number of inducible proteins that are able to detoxify these highly reactive species, notably the anoxically functioning NO reductase NorVW, and the aerobically functioning flavohaemoglobin, Hmp, which catalyses the reaction between oxygen and NO to produce relatively inert nitrate. However, in the absence of NO but in the presence of reducing substrates and oxygen, O { :2 is generated from Hmp-mediated electron transfer to bound oxygen and may form a variety of further oxidative species. Hence, Hmp expression is under tight negative regulation by the transcription factor NsrR, abolition of which causes an increase in the production of Hmp. In a previous study, this increase in Hmp levels conferred resistance to the nitrosating agent S-nitrosoglutathione but, perhaps surprisingly, the organism became more sensitive to killing by macrophages. Here, we report that an nsrR mutant that constitutively overexpresses Hmp is also hypersensitive to peroxynitrite in vitro. This sensitivity is alleviated by deletion of the hmp gene or pre-incubation of growing bacteria with NO-releasing agents. We hypothesize that Hmp-expressing cells, in the absence of NO, generate reactive oxygen species, the toxicity of which is exacerbated by peroxynitrite in vitro and in macrophages. RT-PCR confirmed that peroxynitrite causes oxidative stress and upregulation of katG and ahpC, whilst hmp and norV expression are affected very little. The katG gene upregulated by peroxynitrite encodes a catalase peroxidase enzyme with wellestablished roles in detoxifying peroxides. Here, we report that KatG is also able to enhance the breakdown of peroxynitrite, suggesting that the protective role of this enzyme may be wider than previously thought. These data suggest that spatial and temporal fluctuations in the levels of NO and reactive oxygen species will have important consequences for bacterial survival in the macrophage. INTRODUCTIONIn parallel with the development of biological nitric oxide (NO) chemistry, peroxynitrite has become a focus for intense research. Peroxynitrite is formed in cells and tissues from the reaction of NO and superoxide (O { :2 ) at near diffusion-controlled rates (Huie & Padmaja, 1993)2 is a product of the immune system but also a by-product of incomplete oxygen respiration and, under normal physiological conditions, is removed by the potent activities of superoxide dismutases (SODs) (Beckman & Koppenol, 1996;Fridovich, 1995;Miao & St Clair, 2009) The peroxynitrite anion (ONOO 2 ) and its conjugate acid (ONOOH) are powerful nitrating and oxidizing agents, resulting in protein and lipid nitration and extensive damage to critical macromolecules. Previous studies have shown that peroxynitrite, generated within a macrophage from NO synthase-derived NO and O { : 2 generated in the respiratory burst, is able to reac...

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

“…For instance, it is well known that lipids are targets for peroxynitrite oxidation and could release secondary products that might mount the adaptive response of P. aeruginosa (57). The involvement of catalase in the response of bacteria to peroxynitrite was shown previously in F. tularensis and Salmonella enterica serovar Typhimurium, two microorganisms devoid of Ohr (47,58), although the latter does contain an osmC gene. Our data indicate that especially Ohr and Fig.…”

DksA-Dependent Resistance of Salmonella enterica Serovar Typhimurium against the Antimicrobial Activity of Inducible Nitric Oxide Synthase

“…The expression of DksA enhances the antioxidant defenses of Salmonella (19). Due to overlaps in cellular toxicity between ROS and RNS, many defense mechanisms serve a dual role in defending against oxidative and nitrosative stresses (5,29). Therefore, we evaluated whether DksA contributes to the antinitrosative defenses of Salmonella.…”

Oxidative and Nitrosative Stress on Phagocytes’ Function: from Effective Defense to Immunity Evasion Mechanisms

“…But hindering the oxidative-burst of macrophages is an active evasion mechanism of C. albicans (Wellington et al 2009). In the same manner, some NO reductase enzymes (NorvW) from S. typhimurium, and NorB from Neisseria meningitidis, and flavohemoglobins (Hmp) from Salmonella enterica, S. typhimurium, and E. coli are able to catalyze the reaction of NO and oxygen converting the first in the inert nitrate avoiding and escaping from peroxynitrite cytotoxicity (Laver et al 2010;McLean et al 2010). …”