Pseudomonas putida biofilm dynamics following a single pulse of silver nanoparticles.

Pseudomonas putida mono-species biofilms were exposed to silver nanoparticles (Ag NPs) in artificial wastewater (AW) under hydrodynamic conditions. Specifically, 48 h old biofilms received a single pulse of Ag NPs at 0, 0.01, 0.1, 1, 10 and 100 mg L(-1) for 24 h in confocal laser scanning microscopy (CLSM) compatible flow-cells. The biofilm dynamics (in terms of morphology, viability and activity) were characterised at 48, 72 and 96 h. Consistent patterns were found across flow-cells and experiments at 48 h. Dose dependent impacts of NPs were then shown at 72 h on biofilm morphology (e.g. biomass, surface area and roughness) from 0.01 mg L(-1). The microbial viability was not altered below 10 mg L(-1) Ag NPs. The activity (based on the d-glucose utilisation) was impacted by concentrations of Ag NPs equal and superior to 10 mg L(-1). Partial recovery of morphology, viability and activity were finally observed at 96 h. Comparatively, exposure to Ag salt resulted in ca. one order of magnitude higher toxicity when compared to Ag NPs. Consequently, the use of a continuous culture system and incorporation of a recovery stage extends the value of biofilm assays beyond the standard acute toxicity assessment.

Toxicity Testing of Pristine and Aged Silver Nanoparticles in Real Wastewaters Using Bioluminescent Pseudomonas putida.

Impact of aging on nanoparticle toxicity in real matrices is scarcely investigated due to a lack of suitable methodologies. Herein, the toxicity of pristine and aged silver nanoparticles (Ag NPs) to a bioluminescent Pseudomonas putida bioreporter was measured in spiked crude and final wastewater samples (CWs and FWs, respectively) collected from four wastewater treatment plants (WWTPs). Results showed lower toxicity of pristine Ag NPs in CWs than in FWs. The effect of the matrix on the eventual Ag NP toxicity was related to multiple physico-chemical parameters (biological oxygen demand (BOD), chemical oxygen demand (COD), total suspended solids (TSS) pH, ammonia, sulfide and chloride) based on a multivariate analysis. However, no collection site effect was concluded. Aged Ag NPs (up to eight weeks) were found less toxic than pristine Ag NPs in CWs; evident increased aggregation and decreased dissolution were associated with aging. However, Ag NPs exhibited consistent toxicity in FWs despite aging; comparable results were obtained in artificial wastewater (AW) simulating effluent. The study demonstrates the potency of performing nanoparticle acute toxicity testing in real and complex matrices such as wastewaters using relevant bacterial bioreporters.

Silver, zinc oxide and titanium dioxide nanoparticle ecotoxicity to bioluminescent Pseudomonas putida in laboratory medium and artificial wastewater.

Bacteria based ecotoxicology assessment of manufactured nanoparticles is largely restricted to Escherichia coli bioreporters in laboratory media. Here, toxicity effects of model OECD nanoparticles (Ag NM-300K, ZnO NM-110 and TiO2 NM-104) were assessed using the switch-off luminescent Pseudomonas putida BS566::luxCDABE bioreporter in Luria Bertani (LB) medium and artificial wastewater (AW). IC50 values ∼4 mg L(-1), 100 mg L(-1) and >200 mg L(-1) at 1 h were observed in LB for Ag NM-300K, ZnO NM-110 and TiO2 NM-104, respectively. Similar results were obtained in AW for Ag NM-300K (IC50∼5 mg L(-1)) and TiO2 NM-104 (IC50>200 mg L(-1)) whereas ZnO NM-110 was significantly higher (IC50>200 mg L(-1)). Lower ZnO NM-110 toxicity in AW compared to LB was associated with differences in agglomeration status and dissolution rate. This work demonstrates the importance of nanoecotoxicological studies in environmentally relevant matrices.

Binding of chondroitin 4-sulfate to cathepsin S regulates its enzymatic activity.

Human cysteine cathepsin S (catS) participates in distinct physiological and pathophysiological cellular processes and is considered as a valuable therapeutic target in autoimmune diseases, cancer, atherosclerosis, and asthma. We evaluated the capacity of negatively charged glycosaminoglycans (heparin, heparan sulfate, chondroitin 4/6-sulfates, dermatan sulfate, and hyaluronic acid) to modulate the activity of catS. Chondroitin 4-sulfate (C4-S) impaired the collagenolytic activity (type IV collagen) and inhibited the peptidase activity (Z-Phe-Arg-AMC) of catS at pH 5.5, obeying a mixed-type mechanism (estimated Ki = 16.5 ± 6 µM). Addition of NaCl restored catS activity, supporting the idea that electrostatic interactions are primarly involved. Furthermore, C4-S delayed in a dose-dependent manner the maturation of procatS at pH 4.0 by interfering with the intermolecular processing pathway. Binding of C4-S to catS was demonstrated by gel-filtration chromatography, and its affinity was measured by surface plasmon resonance (equilibrium dissociation constant Kd = 210 ± 40 nM). Moreover, C4-S induced subtle conformational changes in mature catS as observed by intrinsic fluorescence spectroscopy analysis. Molecular docking predicted three specific binding sites on catS for C4-S that are different from those found in the crystal structure of the cathepsin K-C4-S complex. Overall, these results describe a novel glycosaminoglycan-mediated mechanism of catS inhibition and suggest that C4-S may modulate the collagenase activity of catS in vivo.