ABSTRACT
A talented endophytic Streptomyces sp. PH9030 is derived from the medicinal plant Kadsura coccinea (Lem.) A.C. Smith. The undescribed naphthoquinone naphthgeranine G (5) and seven previously identified compounds, 6-12, were obtained from Streptomyces sp. PH9030. The structure of 5 was identified by comprehensive examination of its HRESIMS, 1D NMR, 2D NMR and ECD data. The inhibitory activities of all the compounds toward α-glucosidase and their antibacterial properties were investigated. The α-glucosidase inhibitory activities of 5, 6, 7 and 9 were reported for the first time, with IC50 values ranging from 66.4 ± 6.7 to 185.9 ± 0.2 µM, as compared with acarbose (IC50 = 671.5 ± 0.2 µM). The molecular docking and molecular dynamics analysis of 5 with α-glucosidase further indicated that it may have a good binding ability with α-glucosidase. Both 9 and 12 exhibited moderate antibacterial activity against methicillin-resistant Staphylococcus aureus, with minimum inhibitory concentration (MIC) values of 16 µg/mL. These results indicate that 5, together with the naphthoquinone scaffold, has the potential to be further developed as a possible inhibitor of α-glucosidase.
Subject(s)
Anti-Bacterial Agents , Glycoside Hydrolase Inhibitors , Molecular Docking Simulation , Naphthoquinones , Phenazines , Streptomyces , alpha-Glucosidases , Streptomyces/chemistry , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Naphthoquinones/isolation & purification , Glycoside Hydrolase Inhibitors/chemistry , Glycoside Hydrolase Inhibitors/pharmacology , Glycoside Hydrolase Inhibitors/isolation & purification , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , alpha-Glucosidases/metabolism , alpha-Glucosidases/chemistry , Phenazines/chemistry , Phenazines/pharmacology , Phenazines/isolation & purification , Microbial Sensitivity Tests , Endophytes/chemistry , Molecular Structure , Molecular Dynamics Simulation , Methicillin-Resistant Staphylococcus aureus/drug effectsABSTRACT
1,4-Dimethylphenazine endoperoxide releases singlet oxygen with a half-life of 89 hours at 37 °C. The thermal cycloreversion reaction is accompanied by a strong increase in the emission intensity with a peak at 490 nm, due to the formation of the phenazine core. The endoperoxide is effective against cancer cells in culture medium and tumor spheroids, with singlet oxygen-mediated cytotoxicity.
Subject(s)
Phenazines , Singlet Oxygen , Singlet Oxygen/metabolism , Singlet Oxygen/chemistry , Humans , Phenazines/chemistry , Phenazines/pharmacology , Cell Survival/drug effects , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Molecular StructureABSTRACT
Antimicrobial resistance has become a global threat to human health, which is coupled with the lack of novel drugs. Metallocompounds have emerged as promising diverse scaffolds for the development of new antibiotics. Herein, we prepared some metal compounds mainly focusing on cis-[Ru(bpy)(dppz)(SO3)(NO)](PF6) (PR02, bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine), in which phenazinic and nitric oxide ligands along with sulfite conferred some key properties. This compound exhibited a redox potential for bound NO+/0 of -0.252 V (vs. Ag|AgCl) and a high pH for nitrosyl-nitro conversion of 9.16, making the nitrosyl ligand the major species. These compounds were still able to bind to DNA structures. Interestingly, reduced glutathione (GSH) was unable to promote significant NO/HNO release, an uncommon feature of many similar systems. However, this reducing agent was essential to generate superoxide radicals. Antimicrobial studies were carried out using six bacterial strains, where none or very low activity was observed for Gram-negative bacteria. However, PR02 and PR (cis-[Ru(bpy)(dppz)Cl2]) showed high antibacterial activity in some Gram-positive strains (MBC for S. aureus up to 4.9 µmol L-1), where the activity of PR02 was similar to or at least 4-fold better than that of PR. Besides, PR02 showed capacity to inhibit bacterial biofilm formation, a major health issue leading to bacterial tolerance to antibiotics. Interestingly, we also showed that PR02 can function in synergism with the known antibiotic ampicillin, improving their action up to 4-fold even against resistant strains. Altogether, these results showed that PR02 is a promising antimicrobial nitrosyl ruthenium compound combining features beyond its killing action, which deserves further biological studies.
Subject(s)
Anti-Bacterial Agents , Biofilms , Coordination Complexes , Microbial Sensitivity Tests , Phenazines , Ruthenium , Phenazines/chemistry , Phenazines/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/chemical synthesis , Ruthenium/chemistry , Ruthenium/pharmacology , Biofilms/drug effects , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Coordination Complexes/chemical synthesis , Drug Synergism , Staphylococcus aureus/drug effectsABSTRACT
In this study, we showed that phenazine-1 carboxylic acid (PCA) of Pseudomonas aeruginosa induced the expression of Tet38 efflux pump triggering Staphylococcus aureus resistance to tetracycline and phenazines. Exposure of S. aureus RN6390 to supernatants of P. aeruginosa PA14 and its pyocyanin (PYO)-deficient mutants showed that P. aeruginosa non-PYO phenazines could induce the expression of Tet38 efflux pump. Direct exposure of RN6390 to PCA compound at 0.25× MIC led to a five-fold increase in tet38 transcripts. Expression of Tet38 protein was identified through confocal microscopy using RN6390(pRN-tet38p-yfp) that expressed YFP under control of the tet38 promoter by PCA at 0.25× MIC. The MICs of PCA of a Tet38-overexpressor and a Δtet38 mutant showed a three-fold increase and a two-fold decrease, respectively, compared with that of wild-type. Pre-exposure of RN6390 to PCA (0.25× MIC) for 1 hour prior to addition of tetracycline (1× or 10× MIC) improved bacteria viability of 1.5-fold and 2.6-fold, respectively, but addition of NaCl 7% together with tetracycline at 10× MIC reduced the number of viable PCA-exposed RN6390 of a 2.0-log10 CFU/mL. The transcript levels of tetR21, a repressor of tet38, decreased and increased two-fold in the presence of PCA and NaCl, respectively, suggesting that the effects of PCA and NaCl on tet38 production occurred through TetR21 expression. These data suggest that PCA-induced Tet38 protects S. aureus against tetracycline during coinfection with P. aeruginosa; however, induced tet38-mediated S. aureus resistance to tetracycline is reversed by NaCl 7%, a nebulized treatment used to enhance sputum mobilization in CF patients.
Subject(s)
Anti-Bacterial Agents , Microbial Sensitivity Tests , Phenazines , Pseudomonas aeruginosa , Staphylococcus aureus , Phenazines/pharmacology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Anti-Bacterial Agents/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Tetracycline/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Gene Expression Regulation, Bacterial/drug effects , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolismABSTRACT
Fusarium head blight caused by Fusarium graminearum is a devastating disease in wheat that seriously endangers food security and human health. Previous studies have found that the secondary metabolite phenazine-1-carboxamide produced by biocontrol bacteria inhibited F. graminearum by binding to and inhibiting the activity of histone acetyltransferase Gcn5 (FgGcn5). However, the detailed mechanism of this inhibition remains unknown. Our structural and biochemical studies revealed that phenazine-1-carboxamide (PCN) binds to the histone acetyltransferase (HAT) domain of FgGcn5 at its cosubstrate acetyl-CoA binding site, thus competitively inhibiting the histone acetylation function of the enzyme. Alanine substitution of the residues in the binding site shared by PCN and acetyl-CoA not only decreased the histone acetylation level of the enzyme but also dramatically impacted the development, mycotoxin synthesis, and virulence of the strain. Taken together, our study elucidated a competitive inhibition mechanism of Fusarium fungus by PCN and provided a structural template for designing more potent phenazine-based fungicides.
Subject(s)
Fungal Proteins , Fungicides, Industrial , Fusarium , Histone Acetyltransferases , Phenazines , Plant Diseases , Triticum , Fusarium/metabolism , Fusarium/drug effects , Fusarium/genetics , Phenazines/metabolism , Phenazines/pharmacology , Phenazines/chemistry , Fungal Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/chemistry , Fungicides, Industrial/pharmacology , Fungicides, Industrial/chemistry , Fungicides, Industrial/metabolism , Plant Diseases/microbiology , Histone Acetyltransferases/metabolism , Histone Acetyltransferases/genetics , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/antagonists & inhibitors , Triticum/microbiology , Binding Sites , AcetylationABSTRACT
In this study, we propose an Ethanol Pretreatment Gram staining method that significantly enhances the color contrast of the stain, thereby improving the accuracy of judgement, and demonstrated the effectiveness of the modification by eliminating unaided-eye observational errors with unsupervised machine learning image analysis. By comparing the traditional Gram staining method with the improved method on various bacterial samples, results showed that the improved method offers distinct color contrast. Using multimodal assessment strategies, including unaided-eye observation, manual image segmentation, and advanced unsupervised machine learning automatic image segmentation, the practicality of ethanol pretreatment on Gram staining was comprehensively validated. In our quantitative analysis, the application of the CIEDE2000, and CMC color difference standards confirmed the significant effect of the method in enhancing the discrimination of Gram staining.This study not only improved the efficacy of Gram staining, but also provided a more accurate and standardized strategy for analyzing Gram staining results, which might provide an useful analytical tool in microbiological diagnostics.
Subject(s)
Ethanol , Image Processing, Computer-Assisted , Staining and Labeling , Unsupervised Machine Learning , Ethanol/pharmacology , Staining and Labeling/methods , Image Processing, Computer-Assisted/methods , Gentian Violet , Phenazines/pharmacology , Bacteria/drug effects , Bacteria/isolation & purificationABSTRACT
Halogenated phenazine meroterpenoids are a structurally unusual family of marine actinobacterial natural products that exhibit antibiotic, antibiofilm, and cytotoxic bioactivities. Despite a lack of established phenazine halogenation biochemistry, genomic analysis of Streptomyces sp. CNZ-289, a prolific lavanducyanin and C2-halogenated derivative producer, suggested the involvement of vanadium-dependent haloperoxidases. We subsequently discovered lavanducyanin halogenase (LvcH), characterized it in vitro as a regioselective vanadium-dependent chloroperoxidase, and applied it in late-stage chemoenzymatic synthesis.
Subject(s)
Chloride Peroxidase , Halogenation , Vanadium , Chloride Peroxidase/metabolism , Chloride Peroxidase/chemistry , Vanadium/chemistry , Molecular Structure , Streptomyces/chemistry , Stereoisomerism , Phenazines/chemistry , Phenazines/pharmacology , Phenazines/chemical synthesis , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/chemical synthesisABSTRACT
Rhenium complexes show great promise as anticancer drug candidates. Specifically, compounds with a Re(CO)3(NN)(py)+ core in their architecture have shown cytotoxicity equal to or greater than that of well-established anticancer drugs based on platinum or organic molecules. This study aimed to evaluate how the strength of the interaction between rhenium(I) tricarbonyl complexes fac-[Re(CO)3(NN)(py)]+, NN = 1,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq) or dipyrido[3,2-a:2'3'-c]phenazine (dppz) and biomolecules (protein, lipid and DNA) impacted the corresponding cytotoxic effect in cells. Results showed that fac-[Re(CO)3(dppz)(py)]+ has higher Log Po/w and binding constant (Kb) with biomolecules (protein, lipid and DNA) compared to complexes of fac-[Re(CO)3(phen)(py)]+ and fac-[Re(CO)3(dpq)(py)]+. As consequence, fac-[Re(CO)3(dppz)(py)]+ exhibited the highest cytotoxicity (IC50 = 8.5 µM for HeLa cells) for fac-[Re(CO)3(dppz)(py)]+ among the studied compounds (IC50 > 15 µM). This highest cytotoxicity of fac-[Re(CO)3(dppz)(py)]+ are probably related to its lipophilicity, higher permeation of the lipid bilayers of cells, and a more potent interaction of the dppz ligand with biomolecules (protein and DNA). Our findings open novel avenues for rational drug design and highlight the importance of considering the chemical structures of rhenium complexes that strongly interact with biomolecules (proteins, lipids, and DNA).
Subject(s)
Antineoplastic Agents , Coordination Complexes , DNA , Rhenium , Rhenium/chemistry , Humans , Coordination Complexes/pharmacology , Coordination Complexes/chemistry , Coordination Complexes/chemical synthesis , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , DNA/chemistry , DNA/metabolism , Phenanthrolines/chemistry , Phenanthrolines/pharmacology , Quinoxalines/chemistry , Quinoxalines/pharmacology , Phenazines/chemistry , Phenazines/pharmacology , Cell Line, Tumor , HeLa CellsABSTRACT
The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by 1H NMR, 13C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC50 value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1-F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 µmol/L, which was six times higher than Metalaxyl (3.56 µmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the "ion trap" effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds.
Subject(s)
Alanine , Alanine/analogs & derivatives , Phenazines , Phenazines/chemistry , Phenazines/pharmacology , Phenazines/chemical synthesis , Alanine/chemistry , Alanine/pharmacology , Phytophthora/drug effects , Antifungal Agents/pharmacology , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Phloem/metabolism , Phloem/drug effects , Ascomycota/drug effects , Ascomycota/metabolism , Fungicides, Industrial/pharmacology , Fungicides, Industrial/chemical synthesis , Fungicides, Industrial/chemistry , Drug Design , Esters/chemistry , Esters/pharmacology , Esters/chemical synthesisABSTRACT
The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.
Subject(s)
Antineoplastic Agents , Cations , Phenazines , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Cations/chemistry , Cations/pharmacology , Phenazines/chemistry , Phenazines/pharmacology , Lysosomes/metabolism , Lysosomes/drug effects , HEK293 Cells , Apoptosis/drug effects , Drug Screening Assays, Antitumor , Cell Line, Tumor , Animals , Theranostic Nanomedicine , Molecular StructureABSTRACT
AIMS: The current work aims to fully characterize a new antimicrobial agent against Acinetobacter baumannii, which continues to represent a growing threat to healthcare settings worldwide. With minimal treatment options due to the extensive spread of resistance to almost all the available antimicrobials, the hunt for new antimicrobial agents is a high priority. METHODS AND RESULTS: An Egyptian soil-derived bacterium strain NHM-077B proved to be a promising source for a new antimicrobial agent. Bio-guided fractionation of the culture supernatants of NHM-077B followed by chemical structure elucidation identified the active antimicrobial agent as 1-hydroxy phenazine. Chemical synthesis yielded more derivatives, including dihydrophenazine (DHP), which proved to be the most potent against A. baumannii, yet it exhibited a marginally safe cytotoxicity profile against human skin fibroblasts. Proteomics analysis of the cells treated with DHP revealed multiple proteins with altered expression that could be correlated to the observed phenotypes and potential mechanism of the antimicrobial action of DHP. DHP is a multipronged agent that affects membrane integrity, increases susceptibility to oxidative stress, interferes with amino acids/protein synthesis, and modulates virulence-related proteins. Interestingly, DHP in subinhibitory concentrations re-sensitizes the highly virulent carbapenem-resistant A. baumannii strain AB5075 to carbapenems providing great hope in regaining some of the benefits of this important class of antibiotics. CONCLUSIONS: This work underscores the potential of DHP as a promising new agent with multifunctional roles as both a classical and nonconventional antimicrobial agent that is urgently needed.
Subject(s)
Acinetobacter baumannii , Anti-Bacterial Agents , Carbapenems , Drug Resistance, Multiple, Bacterial , Microbial Sensitivity Tests , Oxidative Stress , Phenazines , Acinetobacter baumannii/drug effects , Phenazines/pharmacology , Phenazines/chemistry , Oxidative Stress/drug effects , Carbapenems/pharmacology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Humans , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Soil MicrobiologyABSTRACT
Quorum sensing is a type of cell-cell communication that modulates various biological activities of bacteria. Previous studies indicate that quorum sensing contributes to the evolution of bacterial resistance to antibiotics, but the underlying mechanisms are not fully understood. In this study, we grew Pseudomonas aeruginosa in the presence of sub-lethal concentrations of ciprofloxacin, resulting in a large increase in ciprofloxacin minimal inhibitory concentration. We discovered that quorum sensing-mediated phenazine biosynthesis was significantly enhanced in the resistant isolates, where the quinolone circuit was the predominant contributor to this phenomenon. We found that production of pyocyanin changed carbon flux and showed that the effect can be partially inhibited by the addition of pyruvate to cultures. This study illustrates the role of quorum sensing-mediated phenotypic resistance and suggests a strategy for its prevention.
Subject(s)
Anti-Bacterial Agents , Ciprofloxacin , Drug Resistance, Bacterial , Microbial Sensitivity Tests , Phenazines , Pseudomonas aeruginosa , Pyocyanine , Quorum Sensing , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Ciprofloxacin/pharmacology , Quorum Sensing/drug effects , Phenazines/pharmacology , Phenazines/metabolism , Anti-Bacterial Agents/pharmacology , Pyocyanine/biosynthesis , Drug Resistance, Bacterial/genetics , Gene Expression Regulation, Bacterial/drug effects , Quinolones/pharmacologyABSTRACT
Microbial symbionts of plants constitute promising sources of biocontrol organisms to fight plant pathogens. Bacillus sp. G2112 and Pseudomonas sp. G124 isolated from cucumber (Cucumis sativus) leaves inhibited the plant pathogens Erwinia and Fusarium. When Bacillus sp. G2112 and Pseudomonas sp. G124 were co-cultivated, a red halo appeared around Bacillus sp. G2112 colonies. Metabolite profiling using liquid chromatography coupled to UV and mass spectrometry revealed that the antibiotic phenazine-1-carboxylic acid (PCA) released by Pseudomonas sp. G124 was transformed by Bacillus sp. G2112 to red pigments. In the presence of PCA (>40 µg/mL), Bacillus sp. G2112 could not grow. However, already-grown Bacillus sp. G2112 (OD600 > 1.0) survived PCA treatment, converting it to red pigments. These pigments were purified by reverse-phase chromatography, and identified by high-resolution mass spectrometry, NMR, and chemical degradation as unprecedented 5N-glucosylated phenazine derivatives: 7-imino-5N-(1'ß-D-glucopyranosyl)-5,7-dihydrophenazine-1-carboxylic acid and 3-imino-5N-(1'ß-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid. 3-imino-5N-(1'ß-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid did not inhibit Bacillus sp. G2112, proving that the observed modification constitutes a resistance mechanism. The coexistence of microorganisms-especially under natural/field conditions-calls for such adaptations, such as PCA inactivation, but these can weaken the potential of the producing organism against pathogens and should be considered during the development of biocontrol strategies.
Subject(s)
Bacillus , Bacillus/metabolism , Pseudomonas/metabolism , Phenazines/pharmacology , Phenazines/chemistry , Carboxylic Acids/pharmacology , Carboxylic Acids/metabolismABSTRACT
Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. In this study, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity-an apparent beneficial effect-in hypoxic/anoxic subpopulations within biofilms. However, we also find that under specific conditions, biofilms lacking RpoS and/or Crc show increased sensitivity to phenazines indicating that the increased metabolic activity in these mutants comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.
Subject(s)
Phenazines , RNA , Methylation , Phenazines/pharmacology , RNA/metabolism , Biofilms , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/metabolismABSTRACT
Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.
Subject(s)
Bacterial Proteins , Oxides , Bacterial Proteins/metabolism , Molecular Docking Simulation , Anti-Bacterial Agents/pharmacology , Phenazines/pharmacologyABSTRACT
Pathogenic bacteria have devastating impacts on human health as a result of acquired antibiotic resistance and innate tolerance. Every class of our current antibiotic arsenal was initially discovered as growth-inhibiting agents that target actively replicating (individual, free-floating) planktonic bacteria. Bacteria are notorious for utilizing a diversity of resistance mechanisms to overcome the action of conventional antibiotic therapies and forming surface-attached biofilm communities enriched in (non-replicating) persister cells. To address problems associated with pathogenic bacteria, our group is developing halogenated phenazine (HP) molecules that demonstrate potent antibacterial and biofilm-eradicating activities through a unique iron starvation mode of action. In this study, we designed, synthesized, and investigated a focused collection of carbonate-linked HP prodrugs bearing a quinone trigger to target the reductive cytoplasm of bacteria for bioactivation and subsequent HP release. The quinone moiety also contains a polyethylene glycol group, which dramatically enhances the water-solubility properties of the HP-quinone prodrugs reported herein. We found carbonate-linked HP-quinone prodrugs 11, 21-23 to demonstrate good linker stability, rapid release of the active HP warhead following dithiothreitol (reductive) treatment, and potent antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, and Enterococcus faecalis. In addition, HP-quinone prodrug 21 induced rapid iron starvation in MRSA and S. epidermidis biofilms, illustrating prodrug action within these surface-attached communities. Overall, we are highly encouraged by these findings and believe that HP prodrugs have the potential to address antibiotic resistant and tolerant bacterial infections.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Prodrugs , Humans , Prodrugs/pharmacology , Solubility , Anti-Bacterial Agents/pharmacology , Staphylococcus epidermidis , Quinones , Phenazines/pharmacology , Iron , WaterABSTRACT
Phenazines are a large group of heterocyclic nitrogen-containing compounds with demonstrated insecticidal, antimicrobial, antiparasitic, and anticancer activities. These natural compounds are synthesized by several microorganisms originating from diverse habitats, including marine and terrestrial sources. The most well-studied producers belong to the Pseudomonas genus, which has been extensively investigated over the years for its ability to synthesize phenazines. This review is focused on the research performed on pseudomonads' phenazines in recent years. Their biosynthetic pathways, mechanism of regulation, production processes, bioactivities, and applications are revised in this manuscript.
Subject(s)
Phenazines , Pseudomonas , Pseudomonas/metabolism , Phenazines/pharmacology , Ecosystem , Biosynthetic PathwaysABSTRACT
Fourteen new 2,3-dialkoxyphenazine derivatives with two different alkoxy groups bearing R1 and R2 alkyl chains, defined as -CH2CH(CH3)2 and -(CH2)n-1CH3 for n = 1, 2, 4, 6, 8, and 10, were prepared via regioselective synthesis. The applied synthetic protocol is based on the following reactions: the Buchwald-Hartwig coupling of a nonsymmetrically substituted 4,5-dialkoxy-2-nitroaniline with a 1-bromo-2-nitrobenzene derivative featuring additional tert-butyl, trifluoromethyl or two methoxy groups; the reduction of bis(2-nitrophenyl)amine; and a final step of tandem-like oxidation that leads to the preparation of a heterocyclic phenazine system. The regioselectivity of these steps and the molecular structure of the compounds under investigation were confirmed by nuclear magnetic resonance and additionally by single-crystal X-ray diffraction performed for some examples of 5 and 6 phenazine series. For 7-(tert-butyl)-3-isobutoxy-2-(octyloxy)phenazine (5f), 3-(hexyloxy)-2-isobutoxy-7-(trifluoromethyl)phenazine (6e), and 2,3-bis(hexyloxy)-7,8-dimethoxyphenazine (7), viability and cytotoxicity assays were performed on the LoVo human colon adenocarcinoma cell line, with 5f confirmed to exhibit cytotoxicity.
Subject(s)
Adenocarcinoma , Colonic Neoplasms , Humans , Molecular Structure , Amines , Phenazines/pharmacologyABSTRACT
Endophytic microorganisms residing within the diverse parts of plants play a significant role in the plant growth and defense response. In the case of the vertically transmitted seed-borne endophytes, they form the promising initiator of the juvenile plant microbiome by supporting the growth and establishment of the seedlings. Hence, the current study emphasizes the isolation and screening of plant beneficial traits of seed endophytes from the cultivated rice variety Jyothi of Kerala, India. Among the 14 bacterial endophytes obtained in the study, the isolate S3 was found to have promising activity against the phytopathogens such as Fusarium oxysporum, Pythium aphanidermatum, Pythium myriotylum, Phytophthora infestans, Rhizoctonia solani, Colletotrichum acutatum, and Sclerotium rolfsii. The isolate S3 was further identified as Paenibacillus polymyxa by the 16S rRNA-based sequence analysis. Furthermore, the isolate was confirmed for its capability for hydrogen cyanide (HCN) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, biofilm formation, and nitrogen fixation. The P. polymyxa S3 was also found to have the potential to provide post-harvest protection to the rice kernels from Sclerotium rolfsii. By the LC-MS/MS analysis, the organism was confirmed for the production of phenazine 1-carboxylic acid which could be the prime chemical basis of its antifungal activity. The in vivo plant growth evaluation has also demonstrated the root length enhancement effect of P. polymyxa S3 in Vigna unguiculata. Here, the root length of P. polymyxa S3-treated plant was enhanced to 12.44 ± 0.58223 cm when compared with distilled water control (10.261 ± 0.38151 cm) and the observed change was statistically significant as per the analysis of variance at P value less than 0.05. Based on all these properties, the isolated P. polymyxa S3 could be considered as a promising agent to be used for the development of competent plant probiotic formulations.
Subject(s)
Oryza , RNA, Ribosomal, 16S/genetics , Chromatography, Liquid , Tandem Mass Spectrometry , Bacteria/genetics , Seeds , Phenazines/pharmacology , Carboxylic AcidsABSTRACT
Previously, it was reported that natural phenazines are able to support the anaerobic survival of Pseudomonas aeruginosa PA14 cells via electron shuttling, with electrodes poised as the terminal oxidants (Y. Wang, S. E. Kern, and D. K. Newman, J Bacteriol 192:365-369, 2010, https://doi.org/10.1128/JB.01188-09). The present study shows that both pyocyanin (PYO) and 1-hydroxyphenazine (1-OHPHZ) promoted the anaerobic killing of PA14 Δphz cells presumably via a single-electron transfer reaction with ferrous iron. However, phenazine-1-carboxylic acid (PCA) did not affect anaerobic survival in the presence of ferrous iron. Anaerobic cell death was alleviated by the addition of antioxidant compounds, which inhibit electron transfer via DNA damage. Neither superoxide dismutase (SOD) nor catalase was able to alleviate P. aeruginosa cell death, ruling out the possibility of reactive oxygen species (ROS)-induced killing. Further, the phenazine degradation profile and the redox state-associated color changes suggested that phenazine radical intermediates are likely generated by single-electron transfer. In this study, we showed that the phenazines 1-OHPHZ and PYO anaerobically killed the cell via single-electron transfer with ferrous iron and that the killing might have resulted from phenazine radicals. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen which infects patients with burns, immunocompromised individuals, and in particular, the mucus that accumulates on the surface of the lung in cystic fibrosis (CF) patients. Phenazines as redox-active small molecules have been reported as important compounds for the control of cellular functions and virulence as well as anaerobic survival via electron shuttles. We show that both pyocyanin (PYO) and 1-hydroxyphenazine (1-OHPHZ) generate phenazine radical intermediates via presumably single-electron transfer reaction with ferrous iron, leading to the anaerobic killing of Pseudomonas cells. The recA mutant defect in the DNA repair system was more sensitive to anaerobic conditions. Our results collectively suggest that both phenazines anaerobically kill cells via DNA damage during electron transfer with iron.