John J. Rowe

Pseudomonas aeruginosa Anaerobic Respiration in Biofilms: Relationships to Cystic Fibrosis Pathogenesis

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells

Silver nanoparticles (Ag NPs) have recently received much attention for their possible applications in biotechnology and life sciences. Ag NPs are of interest to defense and engineering programs for new material applications as well as for commercial purposes as an antimicrobial. However, little is known about the genotoxicity of Ag NPs following exposure to mammalian cells. This study was undertaken to examine the DNA damage response to polysaccharide surface functionalized (coated) and non-functionalized (uncoated) Ag NPs in two types of mammalian cells; mouse embryonic stem (mES) cells and mouse embryonic fibroblasts (MEF). Both types of Ag NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and phosphorylated-H2AX expression. Furthermore both of them induced cell death as measured by the annexin V protein expression and MTT assay. Our observations also suggested that the different surface chemistry of Ag NPs induce different DNA damage response: coated Ag NPs exhibited more severe damage than uncoated Ag NPs. The results suggest that polysaccharide coated particles are more individually distributed while agglomeration of the uncoated particles limits the surface area availability and access to membrane bound organelles.

Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster

Due to the intensive commercial application of silver nanoparticles (Ag NPs), risk assessment of this nanoparticle is of great importance. Our previous in vitro study demonstrated that Ag NPs caused DNA damage and apoptosis in mouse embryonic stem cells and fibroblasts. However, toxicity of Ag NPs in vivo is largely lacking. This study was undertaken to examine the toxic effects of well-characterized polysaccharide coated 10 nm Ag NPs on heat shock stress, oxidative stress, DNA damage and apoptosis in Drosophila melanogaster. Third instar larvae of D. melanogaster were fed a diet of standard cornmeal media mixed with Ag NPs at the concentrations of 50 and 100 microg/ml for 24 and 48 h. Ag NPs up-regulated the expression of heat shock protein 70 and induced oxidative stress in D. melanogaster. Malondialdehyde level, an end product of lipid peroxidation was significantly higher while antioxidant glutathione content was significantly lower in Ag NPs exposed organisms. Activities of antioxidant enzyme superoxide dismutase and catalase were also significantly higher in the organisms exposed to Ag NPs. Furthermore, Ag NPs up-regulated the cell cycle checkpoint p53 and cell signaling protein p38 that are involved in the DNA damage repair pathway. Moreover, activities of caspase-3 and caspase-9, markers of apoptosis were significantly higher in Ag NPs exposed organisms. The results indicate that Ag NPs in D. melanogaster induce heat shock stress, oxidative stress, DNA damage and apoptosis. This study suggests that the organism is stressed and thus warrants more careful assessment of Ag NPs using in vivo models to determine if chronic exposure presents developmental and reproductive toxicity.

Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets

Recent evidence indicates that Pseudomonas aeruginosa residing as biofilms in airway mucus of cystic fibrosis (CF) patients is undergoing anaerobic metabolism, a form of growth requiring gene products that are not utilized during aerobic growth. The outer membrane protein, OprF, and the rhl quorum sensing circuit are two previously unrecognized cellular factors that are required for optimal anaerobic biofilm viability. Without OprF, bacteria grow extremely poorly because they lack nitrite reductase activity while lacking rhlR or rhlI forces bacteria to undergo metabolic suicide by overproduction of nitric oxide. Furthermore, anaerobic growth favors maintenance of the mucoid, alginate-overproducing phenotype. Thus, with increasing age of CF patients, mucoid populations predominate, indicating that anaerobic bacteria reside in the inspissated airway mucus. Because many frontline antibiotics used in the treatment of CF airway disease are either ineffective or show reduced efficacy during anaerobic conditions, we propose development of new drugs to combat anaerobic metabolism by P. aeruginosa for more effective treatment of chronic CF lung infections.

NarK is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia coli

Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest‐lived radioactive isotope of nitrogen, 13N‐nitrate (half‐life = 9.96 min) and the nitrite‐sensitive fluorophore N‐(ethoxycarbonylmethyl)‐6‐methoxyquinolinium bromide have now been used to define the function of NarK. At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange. This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase.

ZnO nanoparticles induce apoptosis in human dermal fibroblasts via p53 and p38 pathways.

The production of engineered nanoparticles is growing rapidly as the field of nanotechnology continues to expand. Zinc oxide nanoparticles (ZnO NPs) are used in various applications, including catalysis, electronics, biosensors, medicine, paints, sunscreens and cosmetics, thus it is important to understand the biological effects and risks of ZnO NPs. This study was designed to investigate the apoptosis induction by ZnO NPs via mitogen-activated protein kinase p38 and cell cycle checkpoint protein p53 pathways in human dermal fibroblasts. MTT-based cell viability assay showed a significant decrease in cell survivorship after ZnO NP exposure, and phase contrast images revealed that ZnO NP treated cells had lower density and a rounded morphology. Apoptosis induction was confirmed by the annexin V assay and Western blot analysis showed the up-regulation of p53 and phospho-p38 proteins. Furthermore, in ZnO NP exposed cells, p53 protein was phosphorylated at Ser33 and Ser46 sites known to be phosphorylated by p38. Our results suggest that ZnO NPs have the potential to induce apoptosis in human dermal fibroblasts via p53-p38 pathways.

Nitrite inhibition of aerobic bacteria

Nitrite was shown to inhibit active transport, oxygen uptake, and oxidative phosphorylation byPseudomonas aeruginosa. The evidence strongly suggested that nitrite exerted its inhibitory effect by oxidizing ferrous iron of an electron carrier(s), such as cytochrome oxidase, to ferric iron. The inhibitory effect of nitrite was readily reversible by washing the cells. Glucose transport byStreptococcus faecalis andS. lactis was not inhibited by nitrite, presumably because these species lack cytochromes and because glucose is transported by the phosphoenolpyruvate: phosphotransferase system rather than by active transport.

Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: Size, coatings and antioxidants matter

Silver and titanium dioxide nanoparticles are known to induce oxidative stress in vitro and in vivo. Here we test if they impact development, mating success, and survivorship in Drosophila melanogaster, and if so, if these effects are reversible by antioxidants. Ingestion of nanotitanium dioxide during the larval stage of the life cycle showed no effects on development or survivorship, up to doses of 200 μg mL(-1). Conversely, ingestion of nanosilver had major dose, size, and coating-dependent effects on each of these aspects of life history. Each of these effects was partially or fully reversible by vitamin C. Larvae growing on nanosilver supplemented with vitamin C showed a greater than twofold increase in survivorship compared to flies reared on nanosilver alone, and a threefold increase in mating success. Vitamin C also rescued cuticular and pigmentation defects in nanosilver fed flies. Biochemical assays of superoxide dismutase and glutathione show these markers respond to nanotitanium dioxide and nanosilver induced oxidative stress, and this response is reduced by vitamin C. These results indicate that life history effects of nanosilver ingestion result from oxidative stress, and suggest antioxidants as a potential remediation for nanosilver toxicity. Conversely, the lack of nanotitanium dioxide life history toxicity shows that oxidative stress does not necessarily result in whole organism effects, and argues that nanoparticle toxicity needs to be examined at different levels of biological organization.

Involvement of NarK1 and NarK2 Proteins in Transport of Nitrate and Nitrite in the Denitrifying Bacterium Pseudomonas aeruginosa PAO1

ABSTRACT Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the ΔnarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ΔnarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions.

Molecular mechanism of the antimicrobial action of pyocyanin

The mechanism by which pyocyanin inhibits bacterial growth was investigated. Several organisms possessing varying levels of superoxide dismutase were analyzed for their sensitivity to pyocyanin to test the possibility that reduced pyocyanin univalently reduces oxygen to superoxide, thus causing cell death. No correlation was found between the amount of superoxide dismutase possessed by an organism and resistance to pyocyanin. In addition, it was demonstrated that organisms growing anaerobically with nitrate as a terminal electron acceptor were as sensitive as, or more sensitive to the action of pyocyanin than organisms grown under aerobic conditions. We thus rule out the possibility that excess superoxide generation is the primary mechanism by which pyocyanin exerts its antibiotic effect. Oxygen electrode and radioisotope studies demonstrated that pyocyanin does inhibit bacterial respiration and active transport of solutes. Thus, it was concluded that the mechanism of action is the result of pyocyanin interacting with the cell membrane respiratory chain in such a way to render the cell unable to perform energy-requiring, membrane-bound metabolic processes such as active transport.