A quorum-sensing regulatory cascade for siderophore-mediated iron homeostasis in Chromobacterium violaceum.

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

In Situ Spatiotemporal SERS Profiling of Bacterial Quorum Sensing by Hierarchical Hydrophobic Plasmonic Arrays in Agar Medium.

A feedback inhibition effect of high autoinducer levels on metabolite secretion in Chromobacterium subtsugae (C. subtsugae) was evidenced by in situ spatiotemporal surface-enhanced Raman spectroscopy (SERS) profiling. The hierarchical hydrophobic plasmonic array in agar medium is structured by oil/water/oil (OL/W/OH) triphasic interfacial self-assembly. The hydrophobic layer acts as a "door curtain" to selectively permit adsorption of a quorum sensing (QS)-regulated fat-soluble metabolite, i.e., violacein (Vio), and significantly blocks nonspecific adsorption of water-soluble proteins, etc. The SERS profiling clearly evidences that the diffusion of N-hexanoyl-l-homoserine lactone (C6-HSL) in agar medium quickly triggers the initial synthesis of Vio in C. subtsugae CV026 but surprisingly inhibits the intrinsic synthesis of Vio in C. subtsugae ATCC31532. The latter negative response might be related to the VioS repressor of ATCC31532, which negatively controls violacein production without influencing the expression of the CviI/R QS system. Moreover, two sender-receiver systems are constructed by separately coculturing CV026 or ATCC31532 with Hafnia alvei H4 that secretes large amounts of C6-HSL. Expectedly, the cocultivation similarly triggers the initial synthesis of Vio in CV026 but seems to have a quite weak negative effect on the intrinsic synthesis in ATCC31532. In fact, the negative regulation in ATCC31532 might be affected by a diffusion-dependent concentration effect. The H4 growth and its secretion of C6-HSL are a slow and continuous process, thereby avoiding the gathering of local high concentrations. Overall, our study put forward an in situ SERS strategy as an alternative to traditional bioluminescent tools for highly sensitively analyzing the spatiotemporal communication and cooperation in live microbial colonies.

Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion.

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.

Efflux Pump Inhibition-Based Screening and Anti-Infective Evaluation of Punica granatum Against Bacterial Pathogens.

Drug efflux pumps contribute to bacterial multidrug resistance (MDR), reducing antibiotic effectiveness and causing treatment failures. Besides their role in MDR, efflux pumps also assist in the transportation of quorum sensing (QS) signal molecules and increased the tolerance of biofilms. Recently, the search for efflux pump inhibitors from natural sources, including anti-infective plants, has gained attention as a potential therapy against drug-resistant bacteria. In this study, 19 traditional Indian medicinal plants were screened for their efflux pump inhibitory activity against Escherichia coli TGI. The promising extract, i.e., Punica granatum was subsequently fractioned in the solvents of increasing polarity. Among them, at sub-MIC active EPI fraction was PGEF (P. granatum ethyl acetate fraction), further investigated for anti-infective potential against Chromobacterium violaceum 12,472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MTCC 97. PGEF was also evaluated for in vivo efficacy in Caenorhabditis elegans model. Major phytocompounds were analyzed by mass spectroscopic techniques. At respective Sub-MIC, PGEF reduced violacein production by 71.14% in C. violaceum 12,472. Moreover, PGEF inhibited pyocyanin (64.72%), pyoverdine (48.17%), protease (51.35%), and swarming motility (44.82%) of P. aeruginosa PAO1. Furthermore, PGEF reduced the production of prodigiosin and exoprotease by 64.73% and 61.80%, respectively. Similarly, at sub-MIC, PGEF inhibited (≥ 50%) biofilm development in all test pathogens. The key phytocompounds detected in active fraction include 5-hydroxymethylfurfural, trans-p-coumaric acid 4- glucoside, (-)-Epicatechin 3'-O-glucuronide, and ellagic acid. Interestingly, PGEF also demonstrated anti-infective efficacy against the PAO1-infected C. elegans test model and highlighting its therapeutic potential as an anti-infective agent to combat drug-resistant problems.

Inhibition of Pseudomonas aeruginosa quorum sensing by methyl gallate from Mangifera indica.

Antipathogenic drugs are a potential source of therapeutics, particularly following the emergence of multiple drug-resistant pathogenic microorganisms in the last decade. The inhibition of quorum sensing (QS) is an advanced antipathogenic approach for suppression of bacterial virulence and dissemination. This study aimed to investigate the inhibitory effect of some Egyptian medicinal plants on the QS signaling system of Pseudomonas aeruginosa. Among the tested plants, Mangifera indica exhibited the highest quorum sensing inhibition (QSI) activity against Chromobacterium violaceum ATCC 12472. Four pure compounds were extracted and identified; of these, methyl gallate (MG) showed the most potent QSI. MG had a minimum inhibitory concentration (MIC) of 512 g/mL against P. aeruginosa strains PAO1, PA14, Pa21, Pa22, Pa23, Pa24, and PAO-JP2. The virulence factors of PAO1, PA14, Pa21, Pa22, Pa23, and Pa24 were significantly inhibited by MG at 1/4 and 1/2 sub-MICs without affecting bacterial viability. Computational insights were performed by docking the MG compound on the LasR receptor, and the QSI behavior of MG was found to be mediated by three hydrogen bonds: Trp60, Arg61, and Thr75. This study indicates the importance of M. indica and MG in the inhibition and modulation of QS and QS-related virulence factors in P. aeruginosa.

Synthesis and Biological Evaluation of New Quinoline and Anthranilic Acid Derivatives as Potential Quorum Sensing Inhibitors.

Inhibiting quorum sensing (QS), a central communication system, is a promising strategy to combat bacterial pathogens without antibiotics. Here, we designed novel hybrid compounds targeting the PQS (Pseudomonas quinolone signal)-dependent quorum sensing (QS) of Pseudomonas aeruginosa that is one of the multidrug-resistant and highly virulent pathogens with urgent need of new antibacterial strategies. We synthesized 12 compounds using standard procedures to combine halogen-substituted anthranilic acids with 4-(2-aminoethyl/4-aminobuthyl)amino-7-chloroquinoline, linked via 1,3,4-oxadiazole. Their antibiofilm activities were first pre-screened using Gram-negative Chromobacterium violaceum-based reporter, which identified compounds 15-19 and 23 with the highest anti-QS and minimal bactericidal effects in a single experiment. These five compounds were then evaluated against P. aeruginosa PAO1 to assess their ability to prevent biofilm formation, eradicate pre-formed biofilms, and inhibit virulence using pyocyanin as a representative marker. Compound 15 displayed the most potent antibiofilm effect, reducing biofilm formation by nearly 50% and pre-formed biofilm masses by 25%. On the other hand, compound 23 exhibited the most significant antivirulence effect, reducing pyocyanin synthesis by over 70%. Thus, our study highlights the potential of 1,3,4-oxadiazoles 15 and 23 as promising scaffolds to combat P. aeruginosa. Additionally, interactive QS systems should be considered to achieve maximal anti-QS activity against this clinically relevant species.

Identification of 2,4-diacetylphloroglucinol production in the genus Chromobacterium.

The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the biocontrol disease suppressing activity of Pseudomonas spp. In the current study, we report the discovery of the DAPG biosynthetic cluster in strains of Chromobacterium vaccinii isolated from Brazilian aquatic environments and the distribution of the biosynthetic cluster in the Chromobacterium genus. Phylogenetic analysis of the phlD protein suggests the biosynthetic cluster probably entered the genus of Chromobacterium after a horizontal gene transfer event with a member of the Pseudomonas fluorescens group. We were able to detect trace amounts of DAPG in wild type cultures and confirm the function of the cluster with heterologous expression in Escherichia coli. In addition, we identified and verified the presence of other secondary metabolites in these strains. We also confirmed the ability of C. vaccinii strains to produce bioactive pigment violacein and bioactive cyclic depsipeptide FR900359. Both compounds have been reported to have antimicrobial and insecticidal activities. These compounds suggest strains of C. vaccinii should be further explored for their potential as biocontrol agents.

Chromobacterium haemolyticum infection from hot springs near Yellowstone National Park: a case report.

BACKGROUND: Chromobacterium haemolyticum is a gram-negative anaerobic sporulated rod and was only first identified in 2008. It is very rare in people with only a handful of cases having been diagnosed around the world. CASE PRESENTATION: After suffering a fall near Yellowstone National Park, a white male patient in his 50 s presented to a hospital in Eastern Idaho. With many unexplained symptoms, several changes in patient stability and recovery, over a course of 18 days in the hospital, the infecting organism could not be easily identified. Labs in the hospital, state, and eventually outside of the state were consulted for pathogen identification, which was only accomplished after the patient was discharged. CONCLUSIONS: To our knowledge, this is only the seven reported human infection with Chromobacterium haemolyticum. This bacterium is difficult to identify and may be occur in rural areas without the proper testing facilities to quickly identify the pathogen, which is essential to timely treatment.

Chromobacterium Biopesticide Exposure Does Not Select for Resistance in Aedes Mosquitoes.

Developing effective tools to control mosquito populations is essential for reducing the incidence of diseases like malaria and dengue. Biopesticides of microbial origin are a rich, underexplored source of mosquitocidal compounds. We previously developed a biopesticide from the bacterium Chromobacterium sp. Panama that rapidly kills vector mosquito larvae, including Aedes aegypti and Anopheles gambiae. Here, we demonstrate that two independent Ae. aegypti colonies exposed to a sublethal dose of that biopesticide over consecutive generations persistently exhibited high mortality and developmental delays, indicating that resistance did not develop during the study period. Critically, the descendants of biopesticide-exposed mosquitoes experienced decreased longevity and did not display increased susceptibility to dengue virus or decreased susceptibility to common chemical insecticides. Through RNA sequencing, we observed no link between biopesticide exposure and the increased activity of xenobiotic metabolism and detoxification genes typically associated with insecticide resistance. These findings indicate that the Chromobacterium biopesticide is an exciting, emerging mosquito control tool. IMPORTANCE Vector control is an essential part of mitigating diseases caused by pathogens that mosquitoes spread. Modern vector control is highly reliant on using synthetic insecticides to eliminate mosquito populations before they can cause disease. However, many of these populations have become resistant to commonly used insecticides. There is a strong need to explore alternative vector control strategies that aim to mitigate disease burden. Biopesticides, insecticides of biological origin, can have unique mosquitocidal activities, meaning they can effectively kill mosquitoes that are already resistant to other insecticides. We previously developed a highly effective mosquito biopesticide from the bacterium Chromobacterium sp. Csp_P. Here, we investigate whether exposure to a sublethal dose of this Csp_P biopesticide over 9 to 10 generations causes resistance to arise in Aedes aegypti mosquitoes. We find no evidence of resistance at the physiological or molecular levels, confirming that the Csp_P biopesticide is a highly promising new tool for controlling mosquito populations.

Meningitis caused by Chromobacterium haemolyticum suspected to be derived from a canal in Japan: a case report.

BACKGROUND: The genus Chromobacterium, of which 12 species have been recognized, comprises bacteria that reside in tropical and subtropical environments. Of these species, Chromobacterium violaceum and Chromobacterium haemolyticum are known to cause infections in humans. There have been few reports of infections caused by Chromobacterium haemolyticum. CASE PRESENTATION: Chromobacterium haemolyticum was detected in spinal fluid and blood samples isolated from a 73-year-old Japanese male patient who fell into a canal in Kyoto City, Japan and developed bacteremia and meningitis. Although meropenem and vancomycin were administered, this patient died 9 days after admission. Although the infection was misidentified as being caused by Chromobacterium violaceum by conventional identification methods, average nucleotide identity analysis revealed that the causative pathogen was Chromobacterium haemolyticum. The same bacteria were also detected in the canal in which the accident occurred. Phylogenetic analysis of the strain isolated from the patient and the strain isolated from the canal suggested that the two strains were very closely related. CONCLUSIONS: Chromobacterium haemolyticum can be misidentified as Chromobacterium violaceum by conventional identification methods and tends to be more resistant to ß-lactams than Chromobacterium violaceum. Pigment production and ß-hemolysis on blood sheep agar can provide clues for the early identification of Chromobacterium haemolyticum.

Infection of the malaria vector Anopheles coluzzii with the entomopathogenic bacteria Chromobacterium anophelis sp. nov. IRSSSOUMB001 reduces larval survival and adult reproductive potential.

BACKGROUND: Vector control tools are urgently needed to control malaria transmission in Africa. A native strain of Chromobacterium sp. from Burkina Faso was recently isolated and preliminarily named Chromobacterium anophelis sp. nov. IRSSSOUMB001. In bioassays, this bacterium showed a promising virulence against adult mosquitoes and reduces their blood feeding propensity and fecundity. The current study assessed the entomopathogenic effects of C. anophelis IRSSSOUMB001 on larval stages of mosquitoes, as well as its impacts on infected mosquitoes reproductive capacity and trans-generational effects. METHODS: Virulence on larvae and interference with insemination were assayed by co-incubation with C. anophelis IRSSSOUMB001 at a range of 104 to 108 cfu/ml. Trans-generational effects were determined by measuring body size differences of progeny from infected vs. uninfected parent mosquitoes using wing size as a proxy. RESULTS: Chromobacterium anophelis IRSSSOUMB001 killed larvae of the pyrethroid-resistant Anopheles coluzzii with LT80 of ~ 1.75 ± 0.14 days at 108 cfu/ml in larval breeding trays. Reproductive success was reduced as a measure of insemination rate from 95 ± 1.99% to 21 ± 3.76% for the infected females. There was a difference in wing sizes between control and infected mosquito offsprings from 2.55 ± 0.17 mm to 2.1 ± 0.21 mm in infected females, and from 2.43 ± 0.13 mm to 1.99 ± 0.15 mm in infected males. CONCLUSIONS: This study showed that C. anophelis IRSSSOUMB001 was highly virulent against larvae of insecticide-resistant Anopheles coluzzii, and reduced both mosquito reproduction capacity and offspring fitness. Additional laboratory, field, safety and social acceptance studies are needed to draw firm conclusions about the practical utility of this bacterial strain for malaria vector control.

Biocatalytic synthesis of chiral five-membered carbasugars intermediates utilizing CV2025 ω-transaminase from Chromobacterium violaceum.

(S)-4-(Hydroxymethyl)cyclopent-2-enone is a key intermediate in the synthesis of chiral five-membered carbasugars, which can be used to synthesize a large number of pharmacologically relevant carbocyclic nucleosides. Herein, CV2025 ω-transaminase from Chromobacterium violaceum was selected based on substrate similarity to convert ((1S,4R)-4-aminocyclopent-2-enyl)methanol to (S)-4-(hydroxymethyl)cyclopent-2-enone. The enzyme was successfully cloned, expressed in Escherichia coli, purified and characterized. We show that it has R configuration preference in contrast with the conventional S preference. The highest activity was obtained below 60 °C and at pH 7.5. Cations Ca2+ and K+ enhanced activity by 21% and 13%, respectively. The conversion rate reached 72.4% within 60 min at 50 °C, pH 7.5, using 0.5 mM pyridoxal-5'-phosphate, 0.6 µM CV2025, and 10 mM substrate. The present study provides a promising strategy for preparing five-membered carbasugars economically and efficiently.

Modulation of quorum sensing activity by copper sulfate, potassium dichromate, and cadmium chloride in biosensor strains.

Beyond their biological roles, metals have a strong impact on the environment. It has been reported that metals are also inhibitory of Quorum Sensing (QS) mechanisms, ones of the best characterized signaling systems in bacteria and fungi. We analyzed the effect of CuSO4, CdCl2, and K2Cr2O7, on QS systems sharing or differing in the bacterial host or the QS signal. The results in this study show that CuSO4 can not only be inhibitory, but also stimulatory of QS activity: at 0.2 mM increased six fold the activity in Chromobacterium subtsugae CV026. This behavior is related to the concentration of the metal and the particular QS system: E. coli MT102 (pJBA132) was no affected, but CuSO4 decreased the QS activity of Pseudomonas putida F117 (pKR-C12) to half its control values. K2Cr2O7 increased four and three folds the QS activities of E. coli MT102 (pJBA132) and P. putida F117 (pAS-C8), respectively, but without effect when combined with CuSO4 or CdCl2. CdCl2 only showed a positive effect in CV026 when combined with CuSO4. Results suggest that factors related with the culture conditions impact on the influence of the metals, and reinforce the importance of the environment in the modulation of QS activity.

Chromobacterium violaceum: A rare opportunistic pathogen and clue for pediatric chronic granulomatous disease.

Chromobacterium violaceum is a rare bacterium found in water and soil in tropical regions and typically presents with a localized skin infection or lymphadenitis which can progress to fulminant septicemia and even death. We describe a case of a 2-year-old boy with C. violaceum septicemia from a suspected skin source which was confirmed by wound, tissue and blood cultures. The discovery of this rare organism, subsequently led to the evaluation and identification of underlying chronic granulomatous disease.

Effects of Rhapontigenin as a Novel Quorum-Sensing Inhibitor on Exoenzymes and Biofilm Formation of Pectobacterium carotovorum subsp. carotovorum and Its Application in Vegetables.

The aim of this study was to devise a method to protect Chinese cabbage (Brassica chinensis) and lettuce (Lactuca sativa) from bacterial-disease-induced damage during storage. Thus, the potential of rhapontigenin as a quorum sensing (QS) inhibitor against Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) was evaluated. The QS inhibitory effects of rhapontigenin were confirmed by significant inhibition of the production of violacein in Chromobacterium violaceum CV026 (C. violaceum, CV026). The inhibitory effects of rhapontigenin on the motility, exopolysaccharide (EPS) production, biofilm formation and virulence−exoenzyme synthesis of P. carotovorum were investigated. Acyl-homoserine lactones (AHLs) were quantified using liquid chromatography−mass spectrometry (LC−MS). The inhibitory effects of rhapontigenin on the development of biofilms were observed using fluorescence microscopy and scanning electron microscopy (SEM). A direct-inoculation assay was performed to investigate the QS inhibitory effects of rhapontigenin on P. carotovorum in Chinese cabbage and lettuce. Our results demonstrated that rhapontigenin exhibited significant inhibition (p < 0.05) of the motility, EPS production, biofilm formation, virulence−exoenzyme synthesis and AHL production of P. carotovorum. Additionally, the result of the direct-inoculation assay revealed that rhapontigenin might provide vegetables with significant shelf-life extension and prevent quality loss by controlling the spread of soft-rot symptoms. Consequently, the study provided a significant insight into the potential of rhapontigenin as a QS inhibitor against P. carotovorum.

Chromobacterium Csp_P biopesticide is toxic to larvae of three Diabrotica species including strains resistant to Bacillus thuringiensis.

The development of new biopesticides to control the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is urgent due to resistance evolution to various control methods. We tested an air-dried non-live preparation of Chromobacterium species Panama (Csp_P), against multiple corn rootworm species, including Bt-resistant and -susceptible WCR strains, northern (NCR, D. barberi Smith & Lawrence), and southern corn rootworm (SCR, D. undecimpunctata howardi Barber), in diet toxicity assays. Our results documented that Csp_P was toxic to all three corn rootworms species based on lethal (LC50), effective (EC50), and molt inhibition concentration (MIC50). In general, toxicity of Csp_P was similar among all WCR strains and ~ 3-fold less toxic to NCR and SCR strains. Effective concentration (EC50) was also similar among WCR and SCR strains, and 5-7-fold higher in NCR strains. Molt inhibition (MIC50) was similar among all corn rootworm strains except NCR diapause strain that was 2.5-6-fold higher when compared to all other strains. There was no apparent cross-resistance between Csp_P and any of the currently available Bt proteins. Our results indicate that Csp_P formulation was effective at killing multiple corn rootworm strains including Bt-resistant WCR and could be developed as a potential new management tool for WCR control.

Three New Xanthones from Hypericum scabrum and Their Quorum Sensing Inhibitory Activities against Chromobacterium violaceum.

Quorum sensing (QS) plays an important role in the production of virulence factors and pathogenicity in pathogenic bacteria and is, therefore, a hopeful target to fight against bacterial infections. During our search for natural QS inhibitors, two new xanthonolignoids (1 and 2), each existing as a racemic mixture, one new simple oxygenated xanthone (7), and eight known analogs (3-6, 8-11) were isolated from Hypericum scabrum Linn. Chiral separation of 1 yielded a pair of enantiomers 1a and 1b. The structures of these compounds were elucidated by spectroscopic analysis and ECD (electrostatic circular dichroism) calculations. All isolates were evaluated for their QS inhibitory activity against Chromobacterium violaceum. Both 9 and 10 exhibited the most potent QS inhibitory effects with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 31.25 and 62.5 µM, respectively. Crystal violet staining was used to further evaluate the biofilm inhibition potential of compounds 7, 9 and 10, and the formation of biofilms increased with decreasing drug concentration in a classic dose-dependent manner. The results of a cytotoxicity assay revealed that compounds 7, 9 and 10 exhibited no cytotoxic activity on PC-12 cells at the tested concentration.

A comparative study of antimicrobial, anti-quorum sensing, anti-biofilm, anti-swarming, and antioxidant activities in flower extracts of pecan (Carya illinoinensis) and chestnut (Castanea sativa).

Antibiotic resistance, which has increased rapidly in recent years because of uncontrolled and unconscious antibiotic consumption, poses a major threat to public health. The inadequacy of existing antibiotics has increased the need for new, effective, and less toxic antibiotic raw materials or antibiotic derivatives. Pecan (Carya illinoinensis) and Chestnut (Castanea sativa) flowers possess abundant pollen contents and exhibit similar morphological features. The purpose of this study was to compare these two flower extracts in terms of their antimicrobial and antioxidant activities. Total phenolic content, total flavonoid contents, and phenolic components were also analyzed in aquatic and ethanolic extracts. Antioxidant activities were measured using ferric reducing/antioxidant capacity (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Antimicrobial and antifungal activities were compared by means of agar diffusion tests against bacteria including Staphylococcus aureus, Bacillus cereus, Mycobacterium smegmatis, Acinetobacter haemolyticus, and Chromobacterium violaceum, and the yeasts Candida albicans and Candida parapsilosis. Anti-quorum sensing (anti-QS), anti-biofilm, and anti-swarming (SW) activities were also studied against Chromobacterium violaceum ATCC 31532, Chromobacterium violaceum ATCC 12472, and Pseudomonas aeruginosa PA01, respectively. Both extracts were rich in ellagic acid and gallic acid and exhibited similar antioxidant properties. Both flower extracts exhibited high antimicrobial and antifungal activities as well as anti-biofilm, anti-QS, and anti-SW activities.

The Derived Components of Gnaphalium hypoleucum DC. Reduce Quorum Sensing of Chromobacterium violaceum.

Gnaphalium hypoleucum DC. was first recorded in the Chinese National Pharmacopoeia "Yi Plant Medicine". There is no detailed report on its main components' activity in suppressing the quorum sensing activity (QS) of bacteria. Our study aimed to screen the main components in extracts of G. hypoleucum DC. in order to measure their effects on bacterial QS activity and to explore specific quorum sensing mechanisms that are affected by G. hypoleucum DC. extracts. Crude extracts of G. hypoleucum DC. contained significant amounts of two compounds shown to inhibit bacterial QS activity, namely apigenin and luteolin. Apigenin and luteolin in crude extracts of G. hypoleucum DC. showed substantial inhibition of pigment formation, biofilm production, and motility in Chromobacterium violaceum ATCC 12472 compared to the effects of other phytochemicals from G. hypoleucum DC. Apigenin and luteolin exhibited a strong QS inhibitory effect on C. violaceum, interfering with the violacein pigment biosynthesis by downregulating the vioB, vioC, and vioD genes. In the presence of signal molecules, the QS effect is prevented, and the selected compounds can still inhibit the production of the characteristic purple pigment in C. violaceum. Based on qualitative and quantitative research using genomics and bioinformatics, we concluded that apigenin and luteolin in crude extracts of G. hypoleucum DC can interfere with the generation of QS in C. violaceum by downregulating the vioB, vioC, and vioD genes. Indeed, G. hypoleucum DC. is used for the treatment of bacterial infections, and this research provides new ideas and potential alternative uses for medicinal plants.

Thermodynamics of iron, tetrahydrobiopterin, and phenylalanine binding to phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase (PheH) is a pterin-dependent, mononuclear nonheme iron(II) oxygenase that uses the oxidative power of O2 to hydroxylate phenylalanine to form tyrosine. PheH is a member of a superfamily of O2-activating enzymes that utilizes a common metal binding motif: the 2-His-1-carboxylate facial triad. Like most members of this superfamily, binding of substrates to PheH results in a reorganization of its active site to allow O2 activation. Exploring the energetics of each step before O2 activation can provide mechanistic insight into the initial steps that support the highly specific O2 activation pathway carried out by this metalloenzyme. Here the thermal stability of PheH and its substrate complexes were investigated under an anaerobic environment by using differential scanning calorimetry. In context with known binding constants for PheH, a thermodynamic cycle associated with iron(II), tetrahydrobiopterin (BH4), and phenylalanine binding to the active site was generated, showing a distinctive cooperativity between the binding of BH4 and Phe. The addition of phenylalanine and BH4 to PheH·Fe increased the stability of this enzyme (ΔTm of 8.5 (±0.7) °C with an associated δΔH of 43.0 (±2.9) kcal/mol). The thermodynamic data presented here gives insight into the complicated interactions between metal center, cofactor, and substrate, and how this interplay sets the stage for highly specific, oxidative C-H activation in this enzyme.