Xiuhong Wang

Contribution of Oxidative Damage to Antimicrobial Lethality

ABSTRACT A potential pathway linking hydroxyl radicals to antimicrobial lethality was examined by using mutational and chemical perturbations of Escherichia coli. Deficiencies of sodA or sodB had no effect on norfloxacin lethality; however, the absence of both genes together reduced lethal activity, consistent with rapid conversion of excessive superoxide to hydrogen peroxide contributing to quinolone lethality. Norfloxacin was more lethal with a mutant deficient in katG than with its isogenic parent, suggesting that detoxification of peroxide to water normally reduces quinolone lethality. An iron chelator (bipyridyl) and a hydroxyl radical scavenger (thiourea) reduced the lethal activity of norfloxacin, indicating that norfloxacin-stimulated accumulation of peroxide affects lethal activity via hydroxyl radicals generated through the Fenton reaction. Ampicillin and kanamycin, antibacterials unrelated to fluoroquinolones, displayed behavior similar to that of norfloxacin except that these two agents showed hyperlethality with an ahpC (alkyl hydroperoxide reductase) mutant rather than with a katG mutant. Collectively, these data are consistent with antimicrobial stress increasing the production of superoxide, which then undergoes dismutation to peroxide, from which a highly toxic hydroxyl radical is generated. Hydroxyl radicals then enhance antimicrobial lethality, as suggested by earlier work. Such findings indicate that oxidative stress networks may provide targets for antimicrobial potentiation.

YihE Kinase Is a Central Regulator of Programmed Cell Death in Bacteria

Stress-mediated programmed cell death (PCD) in bacteria has recently attracted attention, largely because it raises novel possibilities for controlling pathogens. How PCD in bacteria is regulated to avoid population extinction due to transient, moderate stress remains a central question. Here, we report that the YihE protein kinase is a key regulator that protects Escherichia coli from antimicrobial and environmental stressors by antagonizing the MazEF toxin-antitoxin module. YihE was linked to a reactive oxygen species (ROS) cascade, and a deficiency of yihE stimulated stress-induced PCD even after stress dissipated. YihE was partially regulated by the Cpx envelope stress-response system, which, along with MazF toxin and superoxide, has both protective and destructive roles that help bacteria make a live-or-die decision in response to stress. YihE probably acts early in the stress response to limit self-sustaining ROS production and PCD. Inhibition of YihE may provide a way of enhancing antimicrobial lethality and attenuating virulence.

Inhibitors of Reactive Oxygen Species Accumulation Delay and/or Reduce the Lethality of Several Antistaphylococcal Agents

ABSTRACT Perturbation of hydroxyl radical accumulation by subinhibitory concentrations of 2,2′-bipyridyl plus thiourea protects Escherichia coli from being killed by 3 lethal antimicrobial classes. Here, we show that 2,2′-bipyridyl plus thiourea delays and/or reduces antimicrobial killing of Staphylococcus aureus by daptomycin, moxifloxacin, and oxacillin. While the protective effect of 2,2′-bipyridyl plus thiourea varied among strains and compounds, the data support the hypothesis that hydroxyl radical enhances antimicrobial lethality.

A Toxin-Antitoxin Module in Bacillus subtilis Can Both Mitigate and Amplify Effects of Lethal Stress

Background Bacterial type-2 (protein-protein) toxin-antitoxin (TA) modules are two-gene operons that are thought to participate in the response to stress. Previous work with Escherichia coli has led to a debate in which some investigators conclude that the modules protect from stress, while others argue that they amplify lethal stress and lead to programmed cell death. To avoid ambiguity arising from the presence of multiple TA modules in E. coli, the effect of the sole type-2 toxin-antitoxin module of Bacillus subtilis was examined for several types of lethal stress. Methodology/Principal Findings Genetic knockout of the toxin gene, ndoA (ydcE), conferred protection to lethal stressors that included kanamycin, moxifloxacin, hydrogen peroxide, and UV irradiation. However, at low doses of UV irradiation the ndoA deficiency increased lethality. Indeed, gradually increasing UV dose with the ndoA mutant revealed a crossover response – from the mutant being more sensitive than wild-type cells to being less sensitive. For high temperature and nutrient starvation, the toxin deficiency rendered cells hypersensitive. The ndoA deficiency also reduced sporulation frequency, indicating a role for toxin-antitoxin modules in this developmental process. In the case of lethal antimicrobial treatment, deletion of the toxin eliminated a surge in hydrogen peroxide accumulation observed in wild-type cells. Conclusions A single toxin-antitoxin module can mediate two opposing effects of stress, one that lowers lethality and another that raises it. Protective effects are thought to arise from toxin-mediated inhibition of translation based on published work. The enhanced, stress-mediated killing probably involves toxin-dependent accumulation of reactive oxygen species, since a deficiency in the NdoA toxin suppressed peroxide accumulation following antimicrobial treatment. The type and perhaps the level of stress appear to be important for determining whether this toxin will have a protective or detrimental effect.

Resveratrol Antagonizes Antimicrobial Lethality and Stimulates Recovery of Bacterial Mutants

Reactive oxygen species (ROS; superoxide, peroxide, and hydroxyl radical) are thought to contribute to the rapid bactericidal activity of diverse antimicrobial agents. The possibility has been raised that consumption of antioxidants in food may interfere with the lethal action of antimicrobials. Whether nutritional supplements containing antioxidant activity are also likely to interfere with antimicrobial lethality is unknown. To examine this possibility, resveratrol, a popular antioxidant dietary supplement, was added to cultures of Escherichia coli and Staphylococcus aureus that were then treated with antimicrobial and assayed for bacterial survival and the recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Resveratrol, at concentrations likely to be present during human consumption, caused a 2- to 3-fold reduction in killing during a 2-hr treatment with moxifloxacin or kanamycin. At higher, but still subinhibitory concentrations, resveratrol reduced antimicrobial lethality by more than 3 orders of magnitude. Resveratrol also reduced the increase in reactive oxygen species (ROS) characteristic of treatment with quinolone (oxolinic acid). These data support the general idea that the lethal activity of some antimicrobials involves ROS. Surprisingly, subinhibitory concentrations of resveratrol promoted (2- to 6-fold) the recovery of rifampicin-resistant mutants arising from the action of ciprofloxacin, kanamycin, or daptomycin. This result is consistent with resveratrol reducing ROS to sublethal levels that are still mutagenic, while the absence of resveratrol allows ROS levels to high enough to kill mutagenized cells. Suppression of antimicrobial lethality and promotion of mutant recovery by resveratrol suggests that the antioxidant may contribute to the emergence of resistance to several antimicrobials, especially if new derivatives and/or formulations of resveratrol markedly increase bioavailability.

Effect of Bile Pigments on the Compromised Gut Barrier Function in a Rat Model of Bile Duct Ligation

Background Studies have shown that the absence of bile in the gut lumen, either by bile duct ligation or bile diversion, induces mucosal injury. However, the mechanism remains elusive. In this study, the role of bile pigments in gut barrier function was investigated in a rat model of bile duct ligation. Methods Male Sprague Dawley (SD) rats were used in this study. After ligation of bile duct, the animals were administrated with free bilirubin, bilirubin ditaurate, or biliverdin by intragastric gavage. 1, 2, or 3 days later, the animals were sacrificed and the damage of mucosa was assessed by histological staining as well as biochemical parameters such as changes of diamine oxidase (DAO) and D-lactate (D-Lac) in the blood. Trypsin and chymotrypsin of the gut were also measured to determine how these digestive proteases may relate to the observed effects of bile pigments. Results Bile duct ligation (BDL) caused significant increases in gut trypsin and chymotrypsin along with damage of the mucosa as demonstrated by the histological findings under microscope, the reduced expression of tight junction molecules like occludin, and significant changes in DAO and D-lac in the blood. Free bilirubin but not bilirubin ditaurate or biliverdin showed significant inhibitions on trypsin and chymotrypsin as well as alleviated changes of histological and biochemical parameters related to gut barrier disruption. Conclusion Bile may protect the gut from damage through inhibiting digestive proteases like trypsin and chymotrypsin by free bilirubin.

Unconjugated bilirubin ameliorates the inflammation and digestive protease increase in TNBS-induced colitis

The authors previously demonstrated that unconjugated bilirubin (UCB) may inhibit the activities of various digestive proteases, including trypsin and chymotrypsin. The digestive proteases in the lower gut are important in the pathogenesis of inflammatory bowel diseases. The effects of UCB on the inflammation and levels of digestive proteases in feces of rats with colitis have not yet been revealed. The present study investigated the effect of UCB on the inflammatory status and levels of trypsin and chymotrypsin in the feces of rats with trinitrobenzenesulfonic acid (TNBS)-induced colitis. The data indicated that treatment with TNBS resulted in a marked reduction in weight gain, which was significantly alleviated in UCB-treated rats. Furthermore, UCB treatment alleviated the inflammation induced by TNBS, detected via macroscopic damage and microscopic inflammation scores, and pro-inflammatory markers including myeloperoxidase (MPO), tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Furthermore, rats with colitis demonstrated significant increases in fecal trypsin and chymotrypsin levels, whereas UCB treatment significantly alleviated these increases. A significant positive correlation was additionally revealed among the pro-inflammatory markers (MPO, TNF-α and IL-1β) and fecal digestive proteases (trypsin and chymotrypsin) in colitis. The results of the present study demonstrated that UCB ameliorated the inflammation and digestive protease increase in TNBS-induced colitis.

Increased Digestive Proteases and Decreased β-Glucuronidase in Feces of Rats Treated with Sucralose and Saccharin—Another Critical Evidence That These Dietary Chemicals May Be Important Causative Factors for Inflammatory Bowel Disease

To the Editor: We read with great interest the series of articles published recently in this journal. The article by Ganz et al highlighted the great economic and healthrelated impact of inflammatory bowel disease (IBD), with Crohn’s disease alone affecting up to 780,000 people and casting as high as

Contribution of reactive oxygen species to pathways of quinolone-mediated bacterial cell death

3.48 billion in the United States. IBD, including both ulcerative colitis and Crohn’s disease, emerged and dramatically increased since last century. There is urgent need to find out its cause. The recent large-scale prospective epidemiological study on dietary patterns and risk of IBD across Europe reflected the great effort, with the finding that high sugar and soft drinks are associated with increased risk of ulcerative colitis. Along with evidence collected during the last 15 years, Qin further proposed that dietary chemicals, such as sucralose and saccharin, may have played important causative role on IBD, as a result of inhibition of gut bacteria and impairment in inactivation of digestive proteases mediated by deconjugated bilirubin. It provided simple explanations for many puzzles in IBD. Despite that, coherent direct experimental evidence for the proposed mechanism is still lacking. In this regard, this study investigated the effect of sucralose and saccharin on fecal digestive proteases and b-glucuronidase, the enzyme needed for deconjugation of almost exclusively conjugated bilirubin released into the gut from the liver through the bile in animals. Sucralose was added in the drinking water of rats at 0.3, 1.0, and 1.5 mg/ mL, whereas saccharin was added to the drinking water at a single dose of 1.0 mg/ mL. The results showed a dose-dependent response for sucralose, i.e., a significant increases in both fecal chymotrypsin and trypsin in the highest dose group 4 weeks after sucralose treatment and significant increases in fecal chymotrypsin and trypsin and significant decrease in fecal b-glucuronidase in all groups 6 weeks after treatment with sucralose. Treatment with saccharin showed similar change, with significant increases in both fecal chymotrypsin and trypsin 4 weeks after the treatment and significant decrease in fecal b-glucuronidase 6 weeks after the treatment (Fig. 1). Our previous study showed that intragastric gavage of unconjugated bilirubin but not conjugated bilirubin alleviated increases in digestive proteases and gut damage in animals with bile duct ligation, suggesting the likely critical role of luminal b-glucuronidase in gut protection. Studies have showed that patients with IBD had significant increases in fecal digestive proteases and decrease in fecal bglucuronidase. This study suggested an intrinsic link between them. It added another piece of critical evidence that sucralose and saccharin may indeed be important causative factors for IBD through the proposed mechanism. Therefore, we advocate more studies on the possible link between these dietary chemicals and IBD.