Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli

Abstract

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages. Key words Escherichia coli, hemolytic uremic syndrome, Shiga toxin 2.Shiga toxin-producing Escherichia coli infections are responsible for widespread disease, including HUS, which is characterized by thrombocytopenia, microangiopathic hemolytic anemia and renal failure (1-3). During intestinal infection, E. coli secretes different products into the intestinal lumen, including LPS and its main virulence factor, Stx (2). The latter is a holotoxin composed of an enzymatic A subunit (StxA) in association with five B subunits (StxB) (4). This toxin translocates across the intestinal epithelial cell layer into the circulation, allowing it to act on different cell populations at distant sites, such as the kidney and the brain (5). Numerous effects on different cellular populations have been attributed to Stx, including cytokine production, secretion of ROI such as List of Abbreviations: AG, aminoguanidine; CHOP, C/EBP homologous protein; E coli, Escherichia coli; ER, endoplasmic reticulum; HUS, hemolytic uremic syndrome; iNOS, inducible nitric oxide synthase; JNK, Jun N-terminal kinase; LPS, lipopolysaccharide; NO, nitric oxide; PI, propidium iodide; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates;rpm, revolutions per minute; SEM, standard error of the means; Stx2, Shiga toxin 2; TSB, tryptic soy broth the superoxide anion (O 2 − ), liberation of RNI such as NO, and apoptosis (6-9).Apoptosis (programmed cell death) is a phenomenon that has been demonstrated under both physiological and pathological conditions. This process is characterized by changes in cell morphology, chromatin condensation, nuclear DNA fragmentation, and apoptotic body formation (10). Induction of apoptosis occurs through two main apoptotic pathways: the extrinsic, or death receptor, pathway and the...

Relevance of Biofilms in the Pathogenesis of Shiga-Toxin-ProducingEscherichia coliInfection

“…Clinical isolates were kindly provided by the Microbiology Laboratory of the Pediatric Hospital of Córdoba, Provincia de Córdoba, Argentina [14, 17]. Stock cultures were preserved at −80°C using glycerol 15% (v/v) as the cryoprotectant, and the E. coli strains were grown in tryptic soy broth (TSB) at 37°C for 18 h. …”

“…A volume of 200  μ L of Griess reagent was then mixed with 100  μ L of sample aliquots, and this mixture incubated for 15 min in the dark. The OD at 540 nm was measured with an automated microplate reader (Bio-Rad, Hercules, CA, USA), and the concentration of nitrite calculated from a NaNO 2 standard curve [14, 22]. …”

“…A disturbance in the prooxidant-antioxidant balance in favor of the overproduction of ROS can result in damage to the cellular components. Another form of stress is termed nitrosative stress, where reactive nitrogen intermediates (RNI), such as nitrate (NO 3 − ) and nitrite (NO 2 − ), are used as terminal electron acceptors under anaerobic conditions, whereas nitric oxide (NO) is a short-lived free radical produced enzymatically by nitric oxide synthesis in various types of cells, and it has a great diffusion across membranes [14]. In the antioxidant defense system, some major enzymes involved in the detoxification of ROS are superoxide dismutase (SOD) and catalase (CAT), among others [15].…”

The antioxidant activity of a prenyl flavonoid alters its antifungal toxicity on Candida albicans biofilms

“…Their accumulation can lead to damage, such as protein cross-linking leading to protein inactivation, protein and lipid oxidation, breakage of DNA strands and cell death (Baronetti et al, 2011;Dickinson and Chang, 2011). The protective mechanisms either scavenge or detoxify ROS and include enzymatic and non-enzymatic antioxidant defenses.…”

“…) and mixed with 200 µl of 1.5% sulfanilamide in 1 N HCl and 50 µL of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride aqueous solution were added and was measured 15 min later by spectrophotometry at 540 nm. Results were expressed respect to BBU (RNI/BBU) (Angel Villegas et al, 2013; Arce Miranda et al, 2011;Baronetti et al, 2011). ArceMiranda et al, 2011;Marioni et al, 2017;Peralta et al, 2015;Peralta et al, 2017).Intracellular ROS production was determined by Confocal scanning laser microscopy (CSLM) using DCFH-DA.…”