A comprehensive proteomics analysis of the response of Pseudomonas aeruginosa to nanoceria cytotoxicity.

The increased commercial use and spread of nanoceria raises concerns about the risks associated with its effects on living organisms. Although Pseudomonas aeruginosa may be ubiquitous in nature, it is largely found in locations closely linked with human activity. P. aeruginosa san ai was used as a model organism for a deeper understanding of the interaction between biomolecules of the bacteria with this intriguing nanomaterial. A comprehensive proteomics approach along with analysis of altered respiration and production of targeted/specific secondary metabolites was conducted to study the response of P. aeruginosa san ai to nanoceria. Quantitative proteomics found that proteins associated with redox homeostasis, biosynthesis of amino acids, and lipid catabolism were upregulated. Proteins from outer cellular structures were downregulated, including transporters responsible for peptides, sugars, amino acids and polyamines, and the crucial TolB protein of the Tol-Pal system, required for the structural formation of the outer membrane layer. In accordance with the altered redox homeostasis proteins, an increased amount of pyocyanin, a key redox shuttle, and the upregulation of the siderophore, pyoverdine, responsible for iron homeostasis, were found. Production of extracellular molecules, e.g. pyocyanin, pyoverdine, exopolysaccharides, lipase, and alkaline protease, was significantly increased in P. aeruginosa san ai exposed to nanoceria. Overall, nanoceria at sublethal concentrations induces profound metabolic changes in P. aeruginosa san ai and provokes increased secretion of extracellular virulence factors, revealing the powerful influence this nanomaterial has on the vital functions of the microorganism.

Bioactivity and phenolics profile of aqueous and ethyl acetate extracts of Satureja kitaibelii Wierzb. ex Heuff. obtained by ultrasound-assisted extraction.

The aim of the study was to investigate the biological activity and chemical composition of Satureja kitaibelii Wierzb. ex Heuff. LC-PDA/MS analyses for the aqueous extracts (A1-stem, leaves and flowers, A2-leaves and flowers) and ethyl-acetate extracts (E1-stem, leaves and flowers, E2-leaves and flowers) obtained by ultrasound-assisted extraction enabled the identification of thirty-four compounds. Quantitative analysis revealed that the aqueous extract obtained from leaves and flowers was the richest in total phenolic acids (65.36 mg/g) and flavonoids (21.17 mg/g). The total polyphenol content was the highest in the aqueous extract obtained from leaves and flowers (274 ± 2.4 mg Gallic Acid equivalents/g). The best antioxidant activity was observed for the same extract using the DPPH (SC50 20 ± 10 µg/mL), ABTS (2.834 ± 0.02 mg Ascorbic Acid/g), FRAP (1.922 ± 0.03 mmol Fe2+/mg), and total reducing power tests (16.4 ± 1.0 mg Ascorbic Acid/g). Both ethyl acetate extracts were the most active against strains of Bacillus cereus and Micrococcus flavus (MIC 1.70-1.99 mg/mL and 1.99-3.41 mg/mL, respectively). They were more efficient against Aspergillus ochraceus (MFC 0.86 mg/mL) and towards HeLa cell lines. All the obtained results implied the good potential of the investigated extracts to be used as effective preservatives and functional ingredients in food products and dietary supplements.

Study of the venom proteome of Vipera ammodytes ammodytes (Linnaeus, 1758): A qualitative overview, biochemical and biological profiling.

Vipera ammodytes (Va), is the European venomous snake of the greatest medical importance. We analyzed whole venom proteome of the subspecies V. ammodytes ammodytes (Vaa) from Serbia for the first time using the shotgun proteomics approach and identified 99 proteins belonging to four enzymatic families: serine protease (SVSPs), L-amino acid oxidase (LAAOs), metalloproteinases (SVMPs), group II phospholipase (PLA2s), and five nonenzymatic families: cysteine-rich secretory proteins (CRISPs), C-type lectins (snaclecs), growth factors -nerve (NGFs) and vascular endothelium (VEGFs), and Kunitz-type protease inhibitors (SPIs). Considerable enzymatic activity of LAAO, SVSPs, and SVMPs and a high acidic PLA2 activity was measured implying potential of Vaa to produce haemotoxic, myotoxic, neuro and cardiotoxic effects. Moreover, significant antimicrobial activity of Vaa venom against Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) was found. The crude venom shows considerable potential cytotoxic activity on the C6 and HL60 and a moderate level of potency on B16 cell lines. HeLa cells showed the same sensitivity, while DU 145 and PC-3 are less sensitive than as normal cell line. Our data demonstrated a high complexity of Vaa and considerable enzymatic, antibacterial and cytotoxic activity, implying a great medical potential of Vaa venom as a promising source for new antibacterial and cytostatic agents.

High-quality draft genome sequence of Pseudomonas aeruginosa san ai, an environmental isolate resistant to heavy metals.

The strain Pseudomonas aeruginosa san ai, isolated from an extreme environment (industrial mineral cutting oil, pH 10), is able to survive and persist in the presence of a variety of pollutants such as heavy metals and organic chemicals. The genome of P. aeruginosa san ai is 6.98 Mbp long with a GC content of 66.08% and 6485 protein encoding genes. A large number of genes associated with proteins, responsible for microbial resistance to heavy metal ions and involved in catabolism of toxic aromatic organic compounds were identified. P. aeruginosa san ai is a highly cadmium-resistant strain. Proteome analysis of biomass after cadmium exposal confirmed a high tolerance to sublethal concentrations of cadmium (100 mg/L), based on: extracellular biosorption, bioaccumulation, biofilm formation, controlled siderophore production and a pronounced metalloprotein synthesis. Proteins responsible for survival in osmostress conditions during exposure to elevated concentrations of cadmium (200 mg/L) demonstrate a strong genetic potential of P. aeruginosa san ai for survival and adaptation. Sequencing of P. aeruginosa san ai genome provides valuable insights into the evolution and adaptation of this microbe to environmental extremes at the whole-genome level, as well as how to optimally use the strain in bioremediation of chemically polluted sites.

Phytochemical Properties of Satureja kitaibelii, Potential Natural Antioxidants: a New Insight.

Satureja kitaibelii Wierzb. ex Heuff. has a great importance in Serbian ethnopharmacology/herbal traditional medicine, as well as a flavoring food additive. Ethanol extract of aerial parts of Satureja kitaibelii analyzed by liquid chromatography-mass spectrometry revealed the presence of 18 compounds among which the most abundant were phenolic acids, flavonoids, jasmonic acid derivatives and rosmanol. The extracts were rich in total phenolics and flavonoid contents, while rosmarinic acid was the dominant compound (18.30-29.52 mg/g). As assessments of antioxidant properties of natural extracts are important because of their growing use in medicine and food industry, antioxidant activity of ethanol extracts of Satureja kitaibelii was analyzed by several assays. The half maximal scavenging capacity (SC50) of 2,2'-diphenyl-1-picrylhydrazyl ranging from 71.20 to 125.65 µg/mL, the total antioxidant capacity from 272.37 to 714.12 mg ascorbic acid/g, and ferric ion reducing antioxidant power ranging from 0.74 to 1.94 µmol Fe/mg, clearly imply a significant antioxidant potential of Satureja kitaibelii. The extracts inhibit growth of Micrococcus luteus and Pseudomonas aeruginosa with inhibition zones 20-30 and 16-26 mm, respectively. Antioxidant and antibacterial activity of compounds identified in extracts suggest a great potential for Satureja kitaibelii application as valuable food ingredient.

A comprehensive study of conditions of the biodegradation of a plastic additive 2,6-di-tert-butylphenol and proteomic changes in the degrader Pseudomonas aeruginosa san ai.

The Pseudomonas aeruginosa san ai strain was investigated for its capability to degrade the 2,6-di-tert-butylphenol (2,6-DTBP) plastic additive, a hazardous and toxic substance for aquatic life. This investigation was performed under different parameter values: 2,6-DTBP concentration, inoculum size, pH, and temperature. The GC-MS study showed that P. aeruginosa efficiently degraded 2,6-DTBP in the pH range of 5-8 at higher temperatures. Under exposure to 2,6-DTBP concentrations of 2, 10, and 100 mg L-1, the strain degraded by 100, 100, and 85%, respectively, for 7 days. Crude enzyme preparation from the biomass of P. aeruginosa san ai showed higher efficiency in 2,6-DTBP removal than that shown by whole microbial cells. Gene encoding for the enzymes involved in the degradation of aromatic compounds in P. aeruginosa san ai was identified. To complement the genomic data, a comparative proteomic study of P. aeruginosa san ai grown on 2,6-DTBP or sunflower oil was conducted by means of nanoLC-MS/MS. The presence of aromatic substances resulted in the upregulation of aromatic ring cleavage enzymes, whose activity was confirmed by enzymatic tests; therefore, it could be concluded that 2,6-DTBP might be degraded by ortho-ring cleavage. A comparative proteomics study of P. aeruginosa san ai indicated that the core molecular responses to aromatic substances can be summarized as the upregulation of proteins responsible for amino acid metabolism with emphasized glutamate metabolism and energy production with upregulated enzymes of glyoxylate bypass. P. aeruginosa san ai has a high capacity to efficiently degrade aromatic compounds, and therefore its whole cells or enzymes could be used in the treatment of contaminated areas.

Cadmium specific proteomic responses of a highly resistant Pseudomonas aeruginosa san ai.

Pseudomonas aeruginosa san ai is a promising candidate for bioremediation of cadmium pollution, as it resists a high concentration of up to 7.2 mM of cadmium. Leaving biomass of P. aeruginosa san ai exposed to cadmium has a large biosorption potential, implying its capacity to extract heavy metal from contaminated medium. In the present study, we investigated tolerance and accumulation of cadmium on protein level by shotgun proteomics approach based on liquid chromatography and tandem mass spectrometry coupled with bioinformatics to identify proteins. Size exclusion chromatography was used for protein prefractionation to preserve native forms of metalloproteins and protein complexes. Using this approach a total of 60 proteins were observed as up-regulated in cadmium-amended culture. Almost a third of the total numbers of up-regulated were metalloproteins. Particularly interesting are denitrification proteins which are over expressed but not active, suggesting their protective role in conditions of heavy metal exposure. P. aeruginosa san ai developed a complex mechanism to adapt to cadmium, based on: extracellular biosorption, bioaccumulation, the formation of biofilm, controlled siderophore production, enhanced respiration and modified protein profile. An increased abundance of proteins involved in: cell energy metabolism, including denitrification proteins; amino acid metabolism; cell motility and posttranslational modifications, primarily based on thiol-disulfide exchange, were observed. Enhanced oxygen consumption of biomass in cadmium-amended culture versus control was found. Our results signify that P. aeruginosa san ai is naturally well equipped to overcome and survive high doses of cadmium and, as such, has a great potential for application in bioremediation of cadmium polluted sites.

Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: compatibility study with detergent ingredients and washing performance.

An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol® XP80 and Triton® X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1h) while the protease activity was enhanced by the addition of Triton® WR1339 and Tween® 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%.

Enzymatic characterization of 30 kDa lipase from Pseudomonas aeruginosa ATCC 27853.

An extracellular lipase from Pseudomonas aeruginosa ATCC 27853 has been purified and its enzymatic characteristics were determined. According to SDS-PAGE and gel filtration molecular mass estimated to be 30 kDa, what classified the lipase in group I.1. Although 14 lipases from P. aeruginosa with similar molecular mass are referred to date, their basic enzymatic properties have not been reported yet. To address the gap we found: the optimal temperature and pH in water solution being 50 degrees C and 9.3, respectively; the lipase was inhibited with Hg2+ ions and sodium dodecylsulphate (SDS), while non-ionic detergent Triton X-100 activated the enzyme; the lipase hydrolyzed more rapidly middle chain triglycerides and it was not regiospecific; the lipase demonstrated naturally occurring stability in different organic solvents with concentrations ranging from 30 to 70%, including good thermal stability in 30% organic solvent solution. Even though strain P. aeruginosa ATCC 27853 was not isolated from extreme environment it showed activity in organic solvent suggesting that this lipase is suitable for variety of applications, including reactions in water restricted medium and bioremediation of contaminations by organic solvents.

Detection of gelatinase B activity in serum of gastric cancer patients.

AIM: To determine the proteolytic activity and expression of gelatinase B in serum of gastric cancer patients and their correlation with the stage of the tumor. METHODS: Sera from 23 patients who underwent surgery for primary gastric cancer as the experimental group and from 11 as the control group were used to determine the proteolytic activity and its inhibition by EDTA and 1,10-phenanthroline. Gelatinase B activity was detected by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE zymography. RESULTS: Proteolytic enzyme activity was increased in gastric cancer patients when compared to the control group (P < 0.05). The proteinases were determined to be metalloproteinases upon inhibition test with specific metalloproteinase inhibitors 1,10-phenanthroline (P < 0.05) and EDTA (P < 0.01). SDS-PAGE and SDS-PAGE zymography revealed gelatinase B (proMMP-9) activity and its molecular mass of 92 ku. CONCLUSION: Proteinase activity is overexpressed in serum of gastric cancer patients. Gelatinase B in serum plays an important role in the progression of gastric cancer. ProMMP-9 can be used as a marker for invasiveness of gastric cancer.