Disruption of N-Αcyl Homoserine Lactone-Mediated Cell Signaling and Iron Acquisition in Epiphytic Bacteria by Leaf Surface Compounds

Since N-acyl homoserine lactones (AHLs) are key mediators of cell density-dependent regulation of traits involved in virulence and epiphytic fitness in gram-negative bacteria such as Pseudomonas syringae, a variety of plant species were examined to determine their production of leaf surface compounds that could interact with these signaling systems. Leaf washings of 17 of 52 plant species tested stimulated or inhibited AHL-dependent traits in at least one of the bacterial reporter strains used. The active compounds from most plants could be distinguished from known AHLs due to different patterns of mobility during C₈ and C₁₈ reverse-phase thin-layer chromatography (TLC) and normal-phase TLC compared to the patterns for authentic bacterial AHLs. All plant extracts were also tested to determine their abilities to sequester iron and trigger bacterial siderophore synthesis on a medium containing abundant iron. Leaf washings from 16 of the 52 plant species, as well as tannic acid solutions, stimulated pyoverdine synthesis in P. syringae in a high-iron medium. These preparations also inhibited the growth of a P. syringae mutant unable to produce pyoverdine siderophores but not the growth of the wild-type bacterium. The stimulation of siderophore production and the growth inhibition by plant extracts and purified tannins were both reversed by addition of ferric chloride to culture media, indicating that iron was made unavailable by the compounds released onto the leaf surface.     

Indole and 7‐hydroxyindole diminish Pseudomonas aeruginosa virulence

Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa.     

A convenient ‘one-pot’ synthesis and in vitro microbiological evaluation of novel 2,7-diaryl-[1,4]-diazepan-5-ones

A convenient method for the ‘one-pot’ synthesis of novel target molecule 2,7-diaryl-[1,4]-diazepan-5-ones from the respective 2,6-diaryl-piperidin-4-ones was catalyzed by NaHSO₄.Al₂O₃ heterogeneous catalyst in dry media under microwave irradiation in solvent-free conditions. Moreover, the catalyst could be recovered and re-used up to 4 times after washing with ethyl acetate. They were evaluated for potential antibacterial activity against Staphylococcus aureus, β-Haemolytic streptococcus, Vibreo cholerae, Salmonella typhii, Escherichia coli, Klebsiella pneumonia, Pseudomonas and antifungal activity against Aspergillus flavus, Aspergillus fumigatus, Mucor, Candida albicans and Rhizopus. Structure-Activity Relationship (SAR) led to the conclusion that, of all the compounds 25–32 tested, compound 30 exerted strong in vitro antibacterial activity against S. aureus, S. typhii, and Pseudomonas and all the compounds 25–32 were less active against E. coli, whereas all the compounds 25–32 displayed potent in vitro antifungal activity against all the fungal strains used, except compound 30, which was more effectual against Mucor.     

The structure of a pyoverdine from Pseudomonas sp. CFML 96.188 and its relation to other pyoverdines with a cyclic C-terminus

From Pseudomonas sp. CFML 96.188 a pyoverdine was isolated and its primary structure was elucidated by spectroscopic methods and degradation reactions. This strain is of interest as it accepts the structurally different pyoverdines from several other Pseudomonas strains. They all have in common as a specific structural feature a C-terminal cyclic substructure, the importance of which for the recognition of a pyoverdine at the cell surface of a given strain will be discussed.     

Selectively Increased Production of Acidic Actinomycin Congener,B-1625 FA2β, on Combined Use of Sarcosine and Ferrous Sulfate

In addition to actinomycins D, X₂ and X_0β, Streptomyces antibioticus No. B-1625 produces minor acidic actinomycin congeners (FA-components). To increase the production of the FA-components, improvement of medium constituents was attempted for both chemically defined and complex media. Addition of trace metals, especially FeSO₄, increased FA-components production and, moreover, the addition of sarcosine was found to increase the production of a selected component, B-1625 FA_2β. Finally, a complex medium, consisting of starch 3.0, Polypepton 0.1, meat extract 0.1, corn steep liquor 3.0, NaCl 0.3, CaCO₃ 0.3, sarcosine 0.1 and FeSO₄ · 7H₂O 0.05%, was developed for the increased production of FA-components, in particular, the selected component of B-1625 FA_2β.     

Recovery of soil streptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> pathogens

A total of 161 different Streptomyces isolates were recovered from 5 soil samples representing the driest habitats of Jordan. These were then characterized and assessed for their antagonistic activity against four clinical multi-drug resistant Pseudomonas aeruginosa test pathogens. Results indicated that only 3 strains out of 139 and 6 out of 22 isolated at 27°C and 45°C, respectively, were active against at least three strains of pathogenic Pseudomonas. However, three Streptomyces strains (J_2b, J₄, and J₁₂) that were isolated at 45°C inhibited all of the tested pathogens with an inhibition zone ranging between 5 and 16 mm in diameter. Data obtained from comparing the inhibition activity of these unique Streptomyces strains toward multi-resistant Pseudomonas pathogens with standard used antibiotics revealed that these isolates produce possible different inhibitory bioactive compounds other than the standard antibiotics.     

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.     

Synthesis,inhibition of NO production and antiproliferative activities of some indole derivatives

The synthesis and the biological evaluation of pyrano[3,2-e]indoles and their reaction intermediates are described. The compounds prepared were evaluated for their inhibition of NO production, antioxidant activity and also for their ability to inhibit in vitro the growth of four human tumor cell lines: large lung carcinoma (COR-L23), alveolar basal epithelial carcinoma (A549), amelanotic melanoma (C32) and melanoma (A375). The two reaction intermediates, 5a and 5b, showed the highest inhibition of NO production in murine monocytic macrophage (IC₅₀?=?1.1?μM and IC₅₀?=?2.3 μM respectively). Compound 5a was the most active against melanotic melanoma (IC₅₀?=?11.8?μM) while the other compounds exhibited weak cytotoxicity with IC₅₀ values >50?μM on all cell lines.     

Selection of antagonistic bacteria isolated from the Physalis peruviana rhizosphere against Fusarium oxysporum

Aims: Cape gooseberries (Physalis peruviana) have become increasingly important in Colombia for both domestic consumption and the international export market. Vascular wilting caused by Fusarium oxysporum is the most damaging disease to P. peruviana crops in Colombia. The control of this pathogen is mainly carried out by chemical and cultural practices, increasing production costs and generating resistance. Therefore, the objectives of this study were to test rhizobacteria isolates from P. peruviana rhizosphere against F. oxysporum under in vitro and in vivo conditions. Methods and Results: Over 120 strains were isolated, and five were selected for their high inhibition of F. oxysporum growth and conidia production under in vitro conditions. These strains inhibited growth by 41–58% and reduced three‐ to fivefold conidia production. In the in vivo assays, all the tested isolates significantly reduced fungal pathogenicity in terms of virulence. Isolate B‐3·4 was the most efficient in delaying the onset of the first symptoms. All isolates were identified as belonging to the genus Pseudomonas except for A‐19 (Bacillus sp.). Conclusions: Our results confirmed that there are prospective rhizobacteria strains that can be used as biological control agents; some of them being able to inhibit in vitro F. oxysporum growth and sporulation. Significance and Impact of the Study: Incorporating these bacteria into biological control strategies for the disease that causes high economical losses in the second most exported fruit from Colombia would result in a reduced impact on environment and economy.     

Porphyromonas gingivalis‐nucleoside‐diphosphate‐kinase inhibits ATP‐induced reactive‐oxygen‐species via P2X7 receptor/NADPH‐oxidase signalling and contributes to persistence

Ligation of P2X₇ receptors with a ‘danger signal’, extracellular ATP (eATP), has recently been shown to result in production of intracellular reactive‐oxygen‐species (ROS) in macrophages. We show that primary gingival epithelial cells (GECs) produce sustained, robust cellular ROS upon stimulation by eATP. The induction of ROS was mediated by P2X₇ receptor signalling coupled with NADPH‐oxidase activation, as determined by pharmacological inhibition and RNA interference. Furthermore, Porphyromonas gingivalis, an oral opportunistic pathogen, upregulated the antioxidant glutathione response, modulated eATP‐induced cytosolic and mitochondrial ROS generated through P2X₇/NADPH‐oxidase interactome, and subsequently blocked oxidative stress in GECs via temporal secretion of a P. gingivalis effector, nucleoside‐diphosphate‐kinase (Ndk). An ndk‐deficient P. gingivalis mutant lacked the ability to inhibit ROS production and persist intracellularly following eATP stimulation. Treatment with recombinant Ndk significantly diminished eATP‐evoked ROS production. P. gingivalis infection elicited a strong, time‐dependent increase in anti‐oxidativemitochondrial UCP₂ levels, whereas ndk‐deficient mutant did not cause any change. The results reveal a novel signalling cascade that is tightly coupled with eATP signalling and ROS regulation. Ndk by P. gingivalis counteracts these antimicrobial signalling activities by secreting Ndk, thus contributing to successful persistence of the pathogen.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Biological exploration of a novel 1,2,4-triazole-indole hybrid molecule as antifungal agent

Abstract

(2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1-(1,2,4-1H-triazol-1-yl)propan-2-ol (8?g), a new 1,2,4-triazole-indole hybrid molecule, showed a broad-spectrum activity against Candida, particularly against low fluconazole-susceptible species. Its activity was higher than fluconazole and similar to voriconazole on C. glabrata (MIC₉₀ = 0.25, 64 and 1?µg/mL, respectively), C. krusei (MIC₉₀ = 0.125, 64 and 0.125?µg/mL, respectively) and C. albicans (MIC₉₀ = 0.5, 8 and 0.25?µg/mL, respectively). The action mechanisms of 8?g were also identified as inhibition of ergosterol biosynthesis and phospholipase A2-like activity. At concentration as low as 4 ng/mL, 8g inhibited ergosterol production by 82% and induced production of 14a-methyl sterols, that is comparable to the results obtained with fluconazole at higher concentration. 8?g demonstrated moderate inhibitory effect on phospholipase A2-like activity being a putative virulence factor. Due to a low MRC5 cytotoxicity, this compound presents a high therapeutic index. These results pointed out that 8?g is a new lead antifungal candidate with potent ergosterol biosynthesis inhibition.     

PCR detection of novel non-ribosomal peptide synthetase genes in lipopeptide-producing Pseudomonas

Bacterial lipopeptides (LPs) are a diverse group of secondary metabolites synthesized through one or more non-ribosomal peptide synthetases (NRPSs). In certain genera, such as Pseudomonas and Bacillus, these enzyme systems are often involved in synthesizing biosurfactants or antimicrobial compounds. Several different types of LPs have been reported for non-pathogenic plant-associated Pseudomonas. Focusing on this group of bacteria, we devised and validated a PCR method to detect novel LP-synthesizing NRPS genes by targeting their lipoinitiation and tandem thioesterase domains, thus avoiding amplification of genes for non-LP metabolites, such as the pyoverdine siderophores present in all fluorescent Pseudomonas. This approach enabled detection of as yet unknown NRPS genes in strains producing viscosin, viscosinamide, WLIP, or lokisin. Furthermore, it proved valuable to identify novel candidate LP producers among Pseudomonas rhizosphere isolates. By phylogenetic analysis of these amplicons, several of the corresponding NRPS genes can be tentatively assigned to the viscosin, amphisin, or entolysin biosynthetic groups, while some others may represent novel NRPS systems.     

Polyphenolic compounds on leaves limit iron availability and affect growth of epiphytic bacteria

Polyphenolic compounds produced by plants can chelate iron, reducing its bioavailability to plant‐associated bacteria. In response to limited iron levels, most bacteria produce siderophores to acquire needed iron quantities. The amount of phenolic compounds detected in methanolic washings of leaves of different plant species varied greatly, being nearly sevenfold higher in Viburnum tinus than in Phaseolus vulgaris. In species with high levels of total phenolics (e.g. Pelargonium hortorum), tannin concentration of leaf washings was also high and accounted for up to 85% of total phenolics. Both stimulation of production of the siderophore pyoverdine in Pseudomonas syringae strain B728a and inhibition of growth of an isogenic mutant I‐1, deficient in pyoverdine production were associated with plants harbouring high levels of leaf surface phenolics. Levels of tannic acid sufficient to inhibit growth of the pyoverdine mutant in culture in an iron‐reversible fashion were similar to tannin levels found on leaves of plants such as P. hortorum. Additionally, the amount of pyoverdines produced by P. syringae and quantified in leaf washings from a variety of plants was directly related to the concentration of tannins released from the leaf, indicating that tannins were responsible for sequestering iron. Phenolic compounds, principally tannins, may thus play an important role in plant–microbe interactions.     

Response of Pseudomonas tolaasii,the causal agent of mushroom brown blotch disease to the volatile compounds produced by endofungal bacteria

Volatile organic compounds (VOCs) produced by bacteria have significant potential to control phytopathogens. In this study, the VOCs produced by endofungal bacteria Pseudomonas sp. Bi1, Bacillus sp. De3, Pantoea sp. Ma3 and Pseudomonas sp. De1 isolated from wild growing mushrooms were evaluated in vitro for their antagonistic activity against Pseudomonas tolaasii Pt18, the causal agent of mushroom brown blotch disease. The gas chromatography–mass spectrometry (GC–MS) analysis revealed that strains Pseudomonas sp. Bi1, Pseudomonas sp. De1, Bacillus sp. De3 and Pantoea sp. Ma3 produced eight, sixteen, nine, and twelve VOCs, respectively. All antagonistic endofungal bacteria produced VOCs which significantly reduced brown blotch symptoms on mushroom caps and inhibited the growth of P. tolaasii Pt18 at the varying levels. Scanning electron microscopy revealed severe morphological changes in cells of P. tolaasii Pt18 following exposure to the VOCs of Pseudomonas sp. Bi1 and De1. Furthermore, The VOCs produced by endofungal bacteria significantly reduced swarming, swimming, twitching, chemotaxis motility and biofilm formation by P. tolaasii Pt18 cells, which are essential contributors to pathogenicity. This is to first report about the inhibition effects of VOCs produced by antagonistic bacteria on virulence traits of P. tolaasii. Our findings provide new insights regarding the potential of antibacterial VOCs as a safe fumigant to control mushroom brown blotch disease.

     

Development of phenylthiourea derivatives as allosteric inhibitors of pyoverdine maturation enzyme PvdP tyrosinase

Infections caused by Pseudomonas aeruginosa become increasingly difficult to treat because these bacteria have acquired various mechanisms for antibiotic resistance, which creates the need for mechanistically novel antibiotics. Such antibiotics might be developed by targeting enzymes involved in the iron uptake mechanism because iron is essential for bacterial survival. For P. aeruginosa, pyoverdine has been described as an important virulence factor that plays a key role in iron uptake. Therefore, inhibition of enzymes involved in the pyoverdine synthesis, such as PvdP tyrosinase, can open a new window for the treatment of P. aeruginosa infections. Previously, we reported phenylthiourea as the first allosteric inhibitor of PvdP tyrosinase with high micromolar potency. In this report, we explored structure-activity relationships (SAR) for PvdP tyrosinase inhibition by phenylthiourea derivatives. This enables identification of a phenylthiourea derivative (3c) with a potency in the submicromolar range (IC₅₀ = 0.57 + 0.05 µM). Binding could be rationalized by molecular docking simulation and 3c was proved to inhibit the bacterial pyoverdine production and bacterial growth in P. aeruginosa PA01 cultures.     

N-glycosyl-N-hydroxysulfamides as potent inhibitors of Brucella suis carbonic anhydrases

Abstract

We investigated a series of N-hydroxysulfamides obtained by Ferrier sulfamidoglycosylation for the inhibition of two bacterial carbonic anhydrases (CAs, EC 4.2.1.1) present in the pathogen Brucella suis. bsCA I was moderately inhibited by these compounds with inhibition constants ranging between 522 and 958?nM and no notable differences of activity between the acetylated or the corresponding deacetylated derivatives. The compounds incorporating two trans-acetates and the corresponding deprotected ones were the most effective inhibitors in the series. bsCA II was better inhibited, with inhibition constants ranging between 59.8 and 799?nM. The acetylated derivatives were generally better bsCA II inhibitors compared to the corresponding deacetylated compounds. Although these compounds were not highly isoform-selective CA inhibitors (CAIs) for the bacterial over the human CA isoforms, some of them possess inhibition profiles that make them interesting leads for obtaining better and more isoform-selective CAIs targeting bacterial enzymes.