Inhibition of AcrAB-TolC enhances antimicrobial activity of phytochemicals in Pectobacterium brasiliense.

Introduction: The eons-long co-evolvement of plants and bacteria led to a plethora of interactions between the two kingdoms, in which bacterial pathogenicity is counteracted by plant-derived antimicrobial defense molecules. In return, efflux pumps (EP) form part of the resistance mechanism employed by bacteria to permit their survival in this hostile chemical environment. In this work we study the effect of combinations of efflux pump inhibitors (EPIs) and plant-derived phytochemicals on bacterial activity using Pectobacteriun brasiliense 1692 (Pb1692) as a model system. Methods: We measured the minimal inhibitory concentration (MIC) of two phytochemicals, phloretin (Pht) and naringenin (Nar), and of one common antibiotic ciprofloxacin (Cip), either alone or in combinations with two known inhibitors of the AcrB EP of Escherichia coli, a close homolog of the AcrAB-TolC EP of Pb1692. In addition, we also measured the expression of genes encoding for the EP, under similar conditions. Results: Using the FICI equation, we observed synergism between the EPIs and the phytochemicals, but not between the EPIs and the antibiotic, suggesting that EP inhibition potentiated the antimicrobial activity of the plant derived compounds, but not of Cip. Docking simulations were successfully used to rationalize these experimental results. Discussion: Our findings suggest that AcrAB-TolC plays an important role in survival and fitness of Pb1692 in the plant environment and that its inhibition is a viable strategy for controlling bacterial pathogenicity.

Single-Fluorophore Indicator to Explore Cellular and Sub-cellular Lactate Dynamics.

Lactate is an energy substrate and an intercellular signal, which can be monitored in intact cells with the genetically encoded FRET indicator Laconic. However, the structural complexity, need for sophisticated equipment, and relatively small fluorescent change limit the use of FRET indicators for subcellular targeting and development of high-throughput screening methodologies. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed a single-fluorophore indicator for lactate, CanlonicSF. This indicator exhibits a maximal fluorescence change of 200% and a KD of ∼300 µM. The fluorescence is not affected by other monocarboxylates. The lactate indicator was not significantly affected by Ca2+ at the physiological concentrations prevailing in the cytosol, endoplasmic reticulum, and extracellular space, but was affected by Ca2+ in the low micromolar range. Targeting the indicator to the endoplasmic reticulum revealed for the first time sub-cellular lactate dynamics. Its improved lactate-induced fluorescence response permitted the development of a multiwell plate assay to screen for inhibitors of the monocarboxylate transporters MCTs, a pharmaceutical target for cancer and inflammation. The functionality of the indicator in living tissue was demonstrated in the brain of Drosophila melanogaster larvae. CanlonicSF is well suited to explore lactate dynamics with sub-cellular resolution in intact systems.

Phloretin, an Apple Phytoalexin, Affects the Virulence and Fitness of Pectobacterium brasiliense by Interfering With Quorum-Sensing.

The effects of phloretin a phytoalexin from apple, was tested on Pectobacterium brasiliense (Pb1692), an emerging soft-rot pathogen of potato. Exposure of Pb1692 to 0.2 mM phloretin a concentration that does not affect growth, or to 0.4 mM a 50% growth inhibiting concentration (50% MIC), reduced motility, biofilm formation, secretion of plant cell wall-degrading enzymes, production of acyl-homoserine lactone (AHL) signaling molecules and infection, phenotypes that are associated with bacterial population density-dependent system known as quorum sensing (QS). To analyze the effect of growth inhibition on QS, the activity of ciprofloxacin, an antibiotic that impairs cell division, was compared to that of phloretin at 50% MIC. Unlike phloretin, the antibiotic hardly affected the tested phenotypes. The use of DH5α, a QS-negative Escherichia coli strain, transformed with an AHL synthase (ExpI) from Pb1692, allowed to validate direct inhibition of AHL production by phloretin, as demonstrated by two biosensor strains, Chromobacterium violaceaum (CV026) and E. coli (pSB401). Expression analysis of virulence-related genes revealed downregulation of QS-regulated genes (expI, expR, luxS, rsmB), plant cell wall degrading enzymes genes (pel, peh and prt) and motility genes (motA, fim, fliA, flhC and flhD) following exposure to both phloretin concentrations. The results support the inhibition of ExpI activity by phloretin. Docking simulations were used to predict the molecular associations between phloretin and the active site of ExpI, to suggest a likely mode of action for the compound's inhibition of virulence.

Simultaneous extraction and separation of bioactive compounds from apple pomace using pressurized liquids coupled on-line with solid-phase extraction.

The objective of this work was the development of an on-line extraction/fractionation method based on the coupling of pressurized liquid extraction and solid-phase extraction for the separation of phenolic compounds from apple pomace. Several variables of the process were evaluated, including the amount of water of the first stage (0-120 mL), temperature (60-80 °C), solid-phase extraction adsorbent (Sepra, Isolute, Strata X and Oasis) and activation/elution solvent (methanol and ethanol). The best results were observed with the adsorbent Sepra. The temperature had a small effect on recovery, but significant differences were observed for phlorizin and a quercetin derivative. Results indicate that ethanol can be used to replace methanol as an activation, extraction/elution solvent. While using mostly green solvents (water, ethanol, and a small amount of methanol that could be reused), the developed method produced higher or similar yields of acids (2.85 ± 0.19 mg/g) and flavonoids (0.97 ± 0.11 mg/g) than conventional methods.

Probing the antioxidant potential of phloretin and phlorizin through a computational investigation.

The structures and energetics of two dihydrochalcones (phloretin and its glycoside phlorizin) were examined with density functional theory, using the B3LYP, M06-2X, and LC-ω PBE functionals with both the 6-311G(d,p) and 6-311 + G(d,p) basis sets. Properties connected to antioxidant activity, i.e., bond dissociation enthalpies (BDEs) for OH groups and ionization potentials (IPs), were computed in a variety of environments including the gas-phase, n-hexane, ethanol, methanol, and water. The smallest BDEs among the four OH groups for phloretin (three for phlorizin) were determined (using B3LYP/6-311 + G(d,p) in water) to be 79.36 kcal/mol for phloretin and 79.98 kcal/mol for phlorizin while the IPs (at the same level of theory) were obtained as 139.48 and 138.98 kcal/mol, respectively. By comparing with known antioxidants, these values for the BDEs indicate both phloretin and phlorizin show promise for antioxidant activity. In addition, the presence of the sugar moiety has a moderate (0-6 kcal/mol depending on functional) effect on the BDEs for all OH groups. Interestingly, the BDEs suggest that (depending on the functional chosen) the sugar moiety can lead to an increase, decrease, or no change in the antioxidant activity. Therefore, further experimental tests are encouraged to understand the substituent effect on the BDEs for phloretin and to help determine the most appropriate functional to probe BDEs for dihydrochalcones.

Black bean anthocyanin-rich extracts as food colorants: Physicochemical stability and antidiabetes potential.

Black beans contain anthocyanins that could be used as colorants in foods with associated health benefits. The objective was to optimize anthocyanins extraction from black bean coats and evaluate their physicochemical stability and antidiabetes potential. Optimal extraction conditions were 24% ethanol, 1:40 solid-to-liquid ratio and 29°C (P<0.0001). Three anthocyanins were identified by MS ions, delphinidin-3-O-glucoside (465.1m/z), petunidin-3-O-glucoside (479.1m/z) and malvidin-3-O-glucoside (493.1m/z). A total of 32mg of anthocyanins were quantified per gram of dry extract. Bean anthocyanins were stable at pH 2.5 and low-temperature 4°C (89.6%), with an extrapolated half-life of 277days. Anthocyanin-rich extracts inhibited α-glucosidase (37.8%), α-amylase (35.6%), dipeptidyl peptidase-IV (34.4%), reactive oxygen species (81.6%), and decreased glucose uptake. Black bean coats are a good source of anthocyanins and other phenolics with the potential to be used as natural-source food colorants with exceptional antidiabetes potential.

Phloretin-induced cytoprotective effects on mammalian cells: A mechanistic view and future directions.

Phloretin (C15 H14 O5 ), a dihydrochalcone flavonoid, is mainly found in fruit, leaves, and roots of apple tree. Phloretin exerts antioxidant, anti-inflammatory, and anti-tumor activities in mammalian cells through mechanisms that have been partially elucidated throughout the years. Phloretin bioavailability is well known in humans, but still remains to be better studied in experimental animals, such as mouse and rat. The focus of the present review is to gather information regarding the mechanisms involved in the phloretin-elicited effects in different in vitro and in vivo experimental models. Several manuscripts were analyzed and data raised by authors were described and discussed here in a mechanistic manner. Comparisons between the effects elicited by phloretin and phloridzin were made whenever possible, as well as with other polyphenols, clarifying questions about the use of phloretin as a potential therapeutic agent. Toxicological aspects associated to phloretin exposure were also discussed here. Furthermore, a special section containing future directions was created as a suggestive guide towards the elucidation of phloretin-related actions in mammalian cells and tissues.