Unlocking the Antibiofilm Potential of Natural Compounds by Targeting the NADH:quinone Oxidoreductase WrbA.

Biofilm-dwelling cells endure adverse conditions, including oxidative imbalances. The NADH:quinone oxidoreductase enzyme WrbA has a crucial role in the mechanism of action of antibiofilm molecules such as ellagic and salicylic acids. This study aimed to exploit the potential of the WrbA scaffold as a valuable target for identifying antibiofilm compounds at non-lethal concentrations. A three-dimensional computational model, based on the published WrbA structure, was used to screen natural compounds from a virtual library of 800,000 compounds. Fisetin, morin, purpurogallin, NZ028, and NZ034, along with the reference compound ellagic acid, were selected. The antibiofilm effect of the molecules was tested at non-lethal concentrations evaluating the cell-adhesion of wild-type and WrbA-deprived Escherichia coli strains through fluorochrome-based microplate assays. It was shown that, except for NZ028, all of the selected molecules exhibited notable antibiofilm effects. Purpurogallin and NZ034 showed excellent antibiofilm performances at the lowest concentration of 0.5 µM, in line with ellagic acid. The observed loss of activity and the level of reactive oxygen species in the mutant strain, along with the correlation with terms contributing to the ligand-binding free energy on WrbA, strongly indicates the WrbA-dependency of purpurogallin and NZ034. Overall, the molecular target WrbA was successfully employed to identify active compounds at non-lethal concentrations, thus revealing, for the first time, the antibiofilm efficacy of purpurogallin and NZ034.

Mechanistic Insights into the Antibiofilm Mode of Action of Ellagic Acid.

Bacterial biofilm is a major contributor to the persistence of infection and the limited efficacy of antibiotics. Antibiofilm molecules that interfere with the biofilm lifestyle offer a valuable tool in fighting bacterial pathogens. Ellagic acid (EA) is a natural polyphenol that has shown attractive antibiofilm properties. However, its precise antibiofilm mode of action remains unknown. Experimental evidence links the NADH:quinone oxidoreductase enzyme WrbA to biofilm formation, stress response, and pathogen virulence. Moreover, WrbA has demonstrated interactions with antibiofilm molecules, suggesting its role in redox and biofilm modulation. This work aims to provide mechanistic insights into the antibiofilm mode of action of EA utilizing computational studies, biophysical measurements, enzyme inhibition studies on WrbA, and biofilm and reactive oxygen species assays exploiting a WrbA-deprived mutant strain of Escherichia coli. Our research efforts led us to propose that the antibiofilm mode of action of EA stems from its ability to perturb the bacterial redox homeostasis driven by WrbA. These findings shed new light on the antibiofilm properties of EA and could lead to the development of more effective treatments for biofilm-related infections.

Biotechnologies in Perfume Manufacturing: Metabolic Engineering of Terpenoid Biosynthesis.

The fragrance industry is increasingly turning to biotechnology to produce sustainable and high-quality fragrance ingredients. Microbial-based approaches have been found to be particularly promising, as they offer a more practical, economical and sustainable alternative to plant-based biotechnological methods for producing terpene derivatives of perfumery interest. Among the evaluated works, the heterologous expression of both terpene synthase and mevalonate pathway into Escherichia coli has shown the highest yields. Biotechnology solutions have the potential to help address the growing demand for sustainable and high-quality fragrance ingredients in an economically viable and responsible manner. These approaches can help compensate for supply issues of rare or impermanent raw materials, while also meeting the increasing demand for sustainable ingredients and processes. Although scaling up biotransformation processes can present challenges, they also offer advantages in terms of safety and energy savings. Exploring microbial cell factories for the production of natural fragrance compounds is a promising solution to both supply difficulties and the demand for sustainable ingredients and processes in the fragrance industry.

Exploration of Novel Scaffolds Targeting Cytochrome b of Pyricularia oryzae.

The fulfilment of the European "Farm to Fork" strategy requires a drastic reduction in the use of "at risk" synthetic pesticides; this exposes vulnerable agricultural sectors-among which is the European risiculture-to the lack of efficient means for the management of devastating diseases, thus endangering food security. Therefore, novel scaffolds need to be identified for the synthesis of new and more environmentally friendly fungicides. In the present work, we employed our previously developed 3D model of P. oryzae cytochrome bc1 (cyt bc1) complex to perform a high-throughput virtual screening of two commercially available compound libraries. Three chemotypes were selected, from which a small collection of differently substituted analogues was designed and synthesized. The compounds were tested as inhibitors of the cyt bc1 enzyme function and the mycelium growth of both strobilurin-sensitive (WT) and -resistant (RES) P. oryzae strains. This pipeline has permitted the identification of thirteen compounds active against the RES cyt bc1 and five compounds that inhibited the WT cyt bc1 function while inhibiting the fungal mycelia only minimally. Serendipitously, among the studied compounds we identified a new chemotype that is able to efficiently inhibit the mycelium growth of WT and RES strains by ca. 60%, without inhibiting the cyt bc1 enzymatic function, suggesting a different mechanism of action.

Evaluation of Enzyme Agarose Gels for Cleaning Complex Substrates in Cultural Heritage.

This study starts from the need to remove a mix of proteins, oils and natural resin, called beverone in the Italian literature, from the back of canvas paintings. The aim of this study is to develop and evaluate the effectiveness of two different agarose/enzyme gels containing, respectively, a trypsin derived from porcine pancreas and a lipase from Candida rugosa, both in an aqueous solution of deoxycholic acid-triethanolamine soap. Enzymes were selected because of their action on peptide and ester bonds, effectiveness at maintaining a weak alkaline pH and low cost. Several series of model samples, resulting from a combination of rabbit skin glue, linseed oil and colophony, were prepared to test the enzyme gels with two different values for each of the following variables: agarose concentration, application modes and time of application. Measurements of weight loss after the gel application and Fourier transform infrared analysis were conducted to underline the hydrolysis occurring due to the enzyme gels and their effectiveness. Results confirmed what has been found in the literature and improved our knowledge about the action of agarose enzyme gels on complex substrates (hydrophilic/hydrophobic). The gels applied fluidly, with a longer contact time and a lower agarose concentration, are more effective. Furthermore, trypsin gels provided better results on substrates with oil and glue, while lipase gels turned out to be more effective on substrates made of a mix of oil, glue and colophony.

Effects of the Quinone Oxidoreductase WrbA on Escherichia coli Biofilm Formation and Oxidative Stress.

The effects of natural compounds on biofilm formation have been extensively studied, with the goal of identifying biofilm formation antagonists at sub-lethal concentrations. Salicylic and cinnamic acids are some examples of these compounds that interact with the quinone oxidoreductase WrbA, a potential biofilm modulator and an antibiofilm compound biomarker. However, WrbA's role in biofilm development is still poorly understood. To investigate the key roles of WrbA in biofilm maturation and oxidative stress, Escherichia coli wild-type and ∆wrbA mutant strains were used. Furthermore, we reported the functional validation of WrbA as a molecular target of salicylic and cinnamic acids. The lack of WrbA did not impair planktonic growth, but rather affected the biofilm formation through a mechanism that depends on reactive oxygen species (ROS). The loss of WrbA function resulted in an ROS-sensitive phenotype that showed reductions in biofilm-dwelling cells, biofilm thickness, matrix polysaccharide content, and H2O2 tolerance. Endogenous oxidative events in the mutant strain generated a stressful condition to which the bacterium responded by increasing the catalase activity to compensate for the lack of WrbA. Cinnamic and salicylic acids inhibited the quinone oxidoreductase activity of purified recombinant WrbA. The effects of these antibiofilm molecules on WrbA function was proven for the first time.

Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae.

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.

Non-Lethal Effects of N-Acetylcysteine on Xylella fastidiosa Strain De Donno Biofilm Formation and Detachment.

This study investigated in-vitro the non-lethal effects of N-acetylcysteine (NAC) on Xylella fastidiosa subspecies pauca strain De Donno (Xf-DD) biofilm. This strain was isolated from the olive trees affected by the olive quick decline syndrome in southern Italy. Xf-DD was first exposed to non-lethal concentrations of NAC from 0.05 to 1000 µM. Cell surface adhesion was dramatically reduced at 500 µM NAC (-47%), hence, this concentration was selected for investigating the effects of pre-, post- and co-treatments on biofilm physiology and structural development, oxidative homeostasis, and biofilm detachment. Even though 500 µM NAC reduced bacterial attachment to surfaces, compared to the control samples, it promoted Xf-DD biofilm formation by increasing: (i) biofilm biomass by up to 78% in the co-treatment, (ii) matrix polysaccharides production by up to 72% in the pre-treatment, and (iii) reactive oxygen species levels by 3.5-fold in the co-treatment. Xf-DD biofilm detachment without and with NAC was also investigated. The NAC treatment did not increase biofilm detachment, compared to the control samples. All these findings suggested that, at 500 µM, NAC diversified the phenotypes in Xf-DD biofilm, promoting biofilm formation (hyper-biofilm-forming phenotype) and discouraging biofilm detachment (hyper-attachment phenotype), while increasing oxidative stress level in the biofilm.

The Advent of the Internet of Things in Airfield Lightning Systems: Paving the Way from a Legacy Environment to an Open World.

This paper discusses the design and prototype implementation of a software solution facilitating the interaction of third-party developers with a legacy monitoring and control system in the airfield environment. By following the Internet of Things (IoT) approach and adopting open standards and paradigms such as REpresentational State Transfer (REST) and Advanced Message Queuing Protocol (AMQP) for message dispatching, the work aims at paving the way towards a more open world in the airfield industrial sector. The paper also presents performance results achieved by extending legacy components to support IoT standards. Quantitative results not only demonstrate the feasibility of the proposed solution, but also its suitability in terms of prompt message dispatching and increased fault tolerance.

Dual-active antifungal agents containing strobilurin and SDHI-based pharmacophores.

Crop disease management often implies repeated application of fungicides. However, the increasing emergence of fungicide-resistant pathogens requires their rotation or combined use. Tank-mix combinations using fungicides with different modes of action are often hard to manage by farmers. An alternative and unexploited strategy are bifunctional fungicides, i.e. compounds resulting from conjugation of the pharmacophores of fungicides with different mechanisms of action. In this paper we describe a new approach to antifungal treatments based on the synthesis of dual agents, obtained by merging the strobilurin and succinate dehydrogenase inhibitor pharmacophores into a new entity. The compounds were tested against important fungal plant pathogens and showed good inhibition of Pyricularia oryzae and Sclerotinia sclerotiorum with activity comparable to commercial fungicides. The inhibition of the cytochrome bc1 and the succinate dehydrogenase enzyme activity confirmed that the new molecules are endowed with a dual mechanism of action. These results were further supported by molecular modelling which showed that selected compounds form stable complexes with both cytochrome b subunit and succinate dehydrogenase enzyme. This work can be considered an important first step towards the development of novel dual-action agents with optimized structure and improved interaction with the targets.

Effect of Moringa oleifera Leaf Powder on Postprandial Blood Glucose Response: In Vivo Study on Saharawi People Living in Refugee Camps.

The hypoglycemic effect in humans of Moringa oleifera (MO) leaf powder has, to date, been poorly investigated. We assessed the chemical composition of MO leaf powder produced at Saharawi refugee camps, its in vitro ability to inhibit α-amylase activity, and its sensory acceptability in food. We then evaluated its effect on postprandial glucose response by randomly administering, on 2 different days, a traditional meal supplemented with 20 g of MO leaf powder (MOR20), or not (control meal, CNT), to 17 Saharawi diabetics and 10 healthy subjects. Capillary glycaemia was measured immediately before the meal and then at 30 min intervals for 3 h. In the diabetic subjects the postprandial glucose response peaked earlier with MOR20 compared to CNT and with lower increments at 90, 120, and 150 min. The mean glycemic meal response with MOR20 was lower than with CNT. The healthy subjects showed no differences. Thus, MO leaf powder could be a hypoglycemic herbal drug. However, given the poor taste acceptability of the 20 g MO meal, lower doses should be evaluated. Moreover, the hypoglycemic effects of MO leaf powder should also be demonstrated by trials evaluating its long-term effects on glycaemia.

Low density polyethylene functionalized with antibiofilm compounds inhibits Escherichia coli cell adhesion.

The present work concerns an efficient strategy to obtain novel medical devices materials able to inhibit biofilm formation. The new materials were achieved by covalent grafting of p-aminocinnamic or p-aminosalicylic acids on low density polyethylene coupons. The polyethylene surface, previously activated by oxygen plasma treatment, was functionalized using 2-hydroxymethylmetacrylate as linker. The latter was reacted with succinic anhydride affording the carboxylic end useful for the immobilization of the antibiofilm molecules. The modified surface was characterized by scanning electron microscope, X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared and fluorescence analyses. The antibiofilm activity of the modified materials were tested against Escherichia coli biofilm grown in the Center of Disease Control biofilm reactor. The results revealed that the grafted cinnamic and salicylic acid derivatives reduced biofilm biomass, in comparison with the control, by 73.7 ± 10.7% and 63.4 ± 7.1%, respectively. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3251-3261, 2017.

The response of Escherichia coli biofilm to salicylic acid.

In this research, salicylic acid is proposed as an alternative biocide-free agent suitable for a preventive or integrative anti-biofilm approach. Salicylic acid has been proved to: (1) reduce bacterial adhesion up to 68.1 ± 5.6%; (2) affect biofilm structural development, reducing viable biomass by 97.0 ± 0.7% and extracellular proteins and polysaccharides by 83.9 ± 2.5% and 49.5 ± 5.5% respectively; and (3) promote biofilm detachment 3.4 ± 0.6-fold. Moreover, salicylic acid treated biofilm showed an increased amount of intracellular (2.3 ± 0.2-fold) and extracellular (2.1 ± 0.3-fold) reactive oxygen species, and resulted in increased production of the quorum sensing signal indole (7.6 ± 1.4-fold). For the first time, experiments revealed that salicylic acid interacts with proteins that play a role in quorum sensing, reactive oxygen species accumulation, motility, extracellular polymeric matrix components, transport and metabolism.

Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests.

Sulfurtransferases (Strs) and thioredoxins (Trxs) are members of large protein families. Trxs are disulfide reductases and play an important role in redox-related cellular processes. They interact with a broad range of proteins. Strs catalyze the transfer of a sulfur atom from a suitable sulfur donor to nucleophilic sulfur acceptors in vitro, but the physiological roles of these enzymes are not well defined. Several studies in different organisms demonstrate protein-protein interactions of Strs with members of the Trx family. We are interested in investigating the specificity of the interaction between Str and Trx isoforms. In order to use the bimolecular fluorescence complementation (BiFC), several Str and Trx sequences from Arabidopsis thaliana were cloned into the pUC-SPYNE and pUC-SPYCE split-YFP vectors, respectively. Each couple of plasmids containing the sequences for the putative interaction partners were transformed into Arabidopsis protoplasts and screened using a confocal laser scanning microscope. Compartment- and partner-specific interactions could be observed in transformed protoplasts. Replacement of cysteine residues in the redox-active site of Trxs abolished the interaction signal. Therefore, the redox site is not only involved in the redox reaction but also responsible for the interaction with partner proteins. Biochemical assays support a specific interaction among Strs and certain Trxs. Based on the results obtained, the interaction of Strs and Trxs indicates a role of Strs in the maintenance of the cellular redox homeostasis.

Unravelling the Structural and Molecular Basis Responsible for the Anti-Biofilm Activity of Zosteric Acid.

The natural compound zosteric acid, or p-(sulfoxy)cinnamic acid (ZA), is proposed as an alternative biocide-free agent suitable for preventive or integrative anti-biofilm approaches. Despite its potential, the lack of information concerning the structural and molecular mechanism of action involved in its anti-biofilm activity has limited efforts to generate more potent anti-biofilm strategies. In this study a 43-member library of small molecules based on ZA scaffold diversity was designed and screened against Escherichia coli to understand the structural requirements necessary for biofilm inhibition at sub-lethal concentrations. Considerations concerning the relationship between structure and anti-biofilm activity revealed that i) the para-sulfoxy ester group is not needed to exploit the anti-biofilm activity of the molecule, it is the cinnamic acid scaffold that is responsible for anti-biofilm performance; ii) the anti-biofilm activity of ZA derivatives depends on the presence of a carboxylate anion and, consequently, on its hydrogen-donating ability; iii) the conjugated aromatic system is instrumental to the anti-biofilm activities of ZA and its analogues. Using a protein pull-down approach, combined with mass spectrometry, the herein-defined active structure of ZA was matrix-immobilized, and was proved to interact with the E. coli NADH:quinone reductase, WrbA, suggesting a possible role of this protein in the biofilm formation process.

A simple and reliable methodology to detect egg white in art samples.

A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th-16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides.

Desulfo-glucosinolate sulfotransferases isolated from several Arabidopsis thaliana ecotypes differ in their sequence and enzyme kinetics.

The goal was to investigate whether the diverse glucosinolate (Gl) profiles described for different Arabidopsis thaliana (L.) Heynh. ecotypes are at least partially shaped by the kinetic properties of sulfotransferases (SOTs) (EC 2.8.2.-) catalyzing the final step in Gl core structure biosynthesis. This study focuses on only one of the three SOTs that contribute to Gl biosynthesis. Homologues of AtSOT18 proteins were characterized, which was inspired by earlier findings on SOTs from ecotypes Col-0 and C24 differing in two amino acids (aa) and specific enzyme activities. Could there be a correlation of AtSOT18 enzyme activities and differences in Gl profiles between the ecotypes? SOT18 sequences from eight Arabidopsis ecotypes with highly diverse Gl patterns differed in two aa at various positions in the protein sequence. The SOT18 sequence from Col-0 showed the highest similarity to the largest number of other sequences in the alignment. The small differences in the primary sequence lead to important structural changes in secondary and tertiary structure that might be the key of different kinetic activities towards a broad range of substrates. All recombinant AtSOT18 proteins showed low substrate specificity with an indolic Gl, while the specificity for aliphatic substrates varied. There is no correlation in the kinetic behavior with the major ds-Gl contents or with the ratio of C(3)/C(4) ds-Gl in the respective ecotype. Therefore, is it unlikely that ds-Gl AtSOT18 proteins play a major role in shaping the Gl profile in Arabidopsis.

Involvement of the Azotobacter vinelandii rhodanese-like protein RhdA in the glutathione regeneration pathway.

The phenotypic features of the Azotobacter vinelandii RhdA mutant MV474 (in which the rhdA gene was deleted) indicated that defects in antioxidant systems in this organism were related to the expression of the tandem-domain rhodanese RhdA. In this work, further insights on the effects of the oxidative imbalance generated by the absence of RhdA (e.g. increased levels of lipid hydroperoxides) are provided. Starting from the evidence that glutathione was depleted in MV474, and using both in silico and in vitro approaches, here we studied the interaction of wild-type RhdA and Cys(230)Ala site-directed RhdA mutant with glutathione species. We found that RhdA was able to bind in vitro reduced glutathione (GSH) and that RhdA-Cys(230) residue was mandatory for the complex formation. RhdA catalyzed glutathione-disulfide formation in the presence of a system generating the glutathione thiyl radical (GS, an oxidized form of GSH), thereby facilitating GSH regeneration. This reaction was negligible when the Cys(230)Ala RhdA mutant was used. The efficiency of RhdA as catalyst in GS-scavenging activity is discussed on the basis of the measured parameters of both interaction with glutathione species and kinetic studies.

Effects of chronic sub-lethal oxidative stress on biofilm formation by Azotobacter vinelandii.

This work showed that perturbations of the physiological steady-state level of reactive oxygen species (ROS) affected biofilm genesis and the characteristics of the model bacterium Azotobacter vinelandii. To get a continuous endogenous source of ROS, a strain exposed to chronic sub-lethal oxidative stress was deprived of the gene coding for the antioxidant rhodanese-like protein RhdA (MV474). In this study MV474 biofilm showed (i) a seven-fold higher growth rate, (ii) induction of catalase and alkyl-hydroxyl-peroxidase enzymes, (iii) higher average thicknesses due to increased production of a polysaccharide-rich extracellular matrix and (iv) less susceptibility to hydrogen peroxide than the wild-type strain (UW136). MV474 showed increased swimming and swarming activity and the swarming colonies experienced a higher level of oxidative stress compared to UW136. A continuous exogenous source of ROS increased biofilm formation in UW136. Overall, chronic sub-lethal oxidative events promoted sessile behavior in A. vinelandii.

Altered expression level of Escherichia coli proteins in response to treatment with the antifouling agent zosteric acid sodium salt.

Zosteric acid sodium salt is a powerful antifouling agent. However, the mode of its antifouling action has not yet been fully elucidated. Whole cell proteome of Escherichia coli was analysed to study the different protein patterns expressed by the surface-exposed planktonic cells without and with sublethal concentrations of the zosteric acid sodium salt. Proteomic analysis revealed that at least 27 proteins showed a significant (19 upregulated and 8 downregulated, P < 0.001) altered expression level in response to the antifoulant. The proteomic signatures of zosteric acid sodium salt-treated cells are characterized by stress-associated (e.g. AhpC, OsmC, SodB, GroES, IscU, DnaK), motility-related (FliC), quorum-sensing-associated (LuxS) and metabolism/biosynthesis-related (e.g. PptA, AroA, FabD, FabB, GapA) proteins. Consistent with the overexpression of LuxS enzyme, the antifouling agent increased autoinducer-2 (AI-2) concentration by twofold. Moreover, treated cells experienced a statistically significant but modest increase of reactive oxygen species (+ 23%), tryptophanase (1.2-fold) and indole (1.2-fold) synthesis. Overall, our data suggest that zosteric acid sodium salt acts as environmental cue leading to global stress on E. coli cells, which favours the expression of various protective proteins, the AI-2 production and the synthesis of flagella, to escape from adverse conditions.