4-hydroxy-2-nonylquinoline: a novel iron chelator isolated from a bacterial cell membrane

The membrane associated iron chelator of Pseudomonas aeruginosa has been extracted from membranes of iron-rich cells with ethanol and purified by reverse phase HPLC. Using 13C NMR and FAB mass spectroscopy, the structure of the chelator has been determined to be 4-hydroxy-2-nonylquinoline. This compound has been previously isolated and named pseudan IX, a name which we use here. We synthesized pseudan IX and show that the spectral properties of the synthesized compound and the purified compound are nearly identical. Also purified from the ethanol extract of membranes is 4-hydroxy-2-heptylquinoline, i.e., pseudan VII. Bacterially purified pseudan IX binds iron as indicated by the incorporation of radiolabeled iron into the chelator and by the formation of pink micelles in a concentrated ethanol extract. The formation of pink micelles upon addition of iron to the synthesized compound indicates that it binds iron.     

喹诺酮信号系统研究进展

喹诺酮信号系统是铜绿假单胞菌群体感应调控网络中一个重要组成部分,对于绿脓菌素和弹性蛋白酶等毒力因子的表达及细菌生物被膜形成和细菌运动具有重要的调控作用,因此与临床细菌感染密切相关。3,4-二羟基-2-庚基-喹诺酮(Pseudomonas quinolone signal,PQS)及2-庚基-4喹诺酮(4-hydroxy-2-heptylquinoline,HHQ)是pqs调控系统中重要的信号分子。PQS对于细菌在压力下群体密度及细菌物质运输具有调控作用,从而增强细菌对于环境的适应能力。同时PQS等分子在一定程度上抑制了人体的免疫系统,帮助细菌在宿主体内生存。HHQ在其他革兰氏阴性细菌及革兰氏阳性细菌中也有合成并发挥调控作用,所以喹诺酮信号分子不仅是种内也是种间交流媒介。将喹诺酮系统作为靶点降低细菌的信号交流是抑制细菌感染的一个新思路。本文对喹诺酮信号系统进行概述。     

Inhibitors of pathogen intercellular signals as selective anti-infective compounds

Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens.     

The Stringent Response Modulates 4-Hydroxy-2-Alkylquinoline Biosynthesis and Quorum-Sensing Hierarchy in Pseudomonas aeruginosa

As a ubiquitous environmental organism and an important human pathogen, Pseudomonas aeruginosa readily adapts and responds to a wide range of conditions and habitats. The intricate regulatory networks that link quorum sensing and other global regulators allow P. aeruginosa to coordinate its gene expression and cell signaling in response to different growth conditions and stressors. Upon nutrient transitions and starvation, as well as other environmental stresses, the stringent response is activated, mediated by the signal (p)ppGpp. P. aeruginosa produces a family of molecules called HAQ (4-hydroxy-2-alkylquinolines), some of which exhibit antibacterial and quorum-sensing signaling functions and regulate virulence genes. In this study, we report that (p)ppGpp negatively regulates HAQ biosynthesis: in a (p)ppGpp-null (ΔSR) mutant, HHQ (4-hydroxyl-2-heptylquinoline) and PQS (3,4-dihydroxy-2-heptylquinoline) levels are increased due to upregulated pqsA and pqsR expression and reduced repression by the rhl system. We also found that (p)ppGpp is required for full expression of both rhl and las AHL (acyl-homoserine lactone) quorum-sensing systems, since the ΔSR mutant has reduced rhlI, rhlR, lasI, and lasR expression, butanoyl-homoserine lactone (C₄-HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C₁₂-HSL) levels, and rhamnolipid and elastase production. Furthermore, (p)ppGpp significantly modulates the AHL and PQS quorum-sensing hierarchy, as the las system no longer has a dominant effect on HAQ biosynthesis when the stringent response is inactivated.     

Influence of the Pseudomonas quinolone signal on denitrification in Pseudomonas aeruginosa

Denitrification is a well-studied respiratory system that is also important in the biogeochemical nitrogen cycle. Environmental signals such as oxygen and N-oxides have been demonstrated to regulate denitrification, though how denitrification is regulated in a bacterial community remains obscure. Pseudomonas aeruginosa is a ubiquitous bacterium that controls numerous genes through cell-to-cell signals. The bacterium possesses at least two N-acyl-L-homoserine lactone (AHL) signals. In our previous study, these quorum-sensing signals controlled denitrification in P. aeruginosa. In addition to the AHL signals, a third cell-to-cell communication signal, 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS), has been characterized. In this study, we examined the effect of PQS on denitrification to obtain more insight into the respiratory regulation in a bacterial community. Denitrification in P. aeruginosa was repressed by PQS, which was partially mediated by PqsR and PqsE. Measuring the denitrifying enzyme activities indicated that nitrite reductase activity was increased by PQS, whereas PQS inhibited nitric oxide reductase and the nitrate-respiratory chain activities. This is the first report to demonstrate that PQS influences enzyme activities, suggesting this effect is not specific to P. aeruginosa. Furthermore, when iron was supplied to the PQS-added medium, denitrifying activity was almost restored, indicating that the iron chelating property of PQS affected denitrification. Thus, our data indicate that PQS regulates denitrification primarily through iron chelation. The PQS effect on denitrification was relevant in a condition where oxygen was limited and denitrification was induced, suggesting its role in controlling denitrification where oxygen is present.     

Polyhydroxybutyrate accumulation by a Serratia sp.

A strain of Serratia sp. showed intracellular electron-transparent inclusion bodies when incubated in the presence of citrate and glycerol 2-phosphate without nitrogen source following pre-growth under carbon-limitation in continuous culture. About 1.3 mmol citrate were consumed per 450 mg biomass, giving a calculated yield of maximally 55% of stored material per g of biomass dry wt. The inclusion bodies were stained with Sudan Black and Nile Red (NR), suggesting a lipid material, which was confirmed as polyhydroxybutyrate (PHB) by analysis of molecular fragments by GC and by FTIR spectroscopy of isolated bio-PHB in comparison with reference material. Multi-parameter flow cytometry in conjunction with NR fluorescence, and electron microscopy, showed that not all cells contained heavy PHB bodies, suggesting the potential for increasing the overall yield. The economic attractiveness is enhanced by the co-production of nanoscale hydroxyapatite (HA), a possible high-value precursor for bone replacement materials.     

Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43

A bacterial strain, which based on the sequences of its 16S rRNA, gyrB, catA, and qsdA genes, was identified as a Rhodococcus sp. closely related to Rhodococcus erythropolis, was isolated from soil by enrichment on the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogen Pseudomonas aeruginosa. The isolate, termed Rhodococcus sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of P. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably, Rhodococcus sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite of P. aeruginosa to a quorum sensing signal molecule.     

Quorum sensing by 2-alkyl-4-quinolones in Pseudomonas aeruginosa and other bacterial species

Pseudomonas aeruginosa produces the cell-to-cell signal molecule 2-heptyl-3-hydroxy-4-quinolone (The Pseudomonas quinolone signal; PQS), which is integrated within a complicated quorum sensing signaling system. PQS belongs to the family of 2-alkyl-4-quinolones (AQs), which have been previously described for their antimicrobial activities. PQS is synthesized via the pqsABCDE operon which is responsible for generating multiple AQs including 2-heptyl-4-quinolone (HHQ), the immediate PQS precursor. In addition, PQS signaling plays an important role in P. aeruginosa pathogenesis because it regulates the production of diverse virulence factors including elastase, pyocyanin and LecA lectin in addition to affecting biofilm formation. Here, we summarize the most recent findings on the biosynthesis and regulation of PQS and other AQs including the discovery of AQs in other bacterial species.     

Three-dimensional analysis of the association of viral particles with mitochondria during the replication of Rice gall dwarf virus

Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication.     

铜绿假单胞菌群体感应信号分子PQS的功能多样性研究进展

铜绿假单胞菌(Pseudomonas aeruginosa)是一种革兰氏阴性条件致病菌,可对免疫功能低下或损伤的患者造成持续性感染。铜绿假单胞菌能成功感染离不开其自身产生的毒力因子,而这些毒力因子大多数都受群体感应系统(quorum sensing,QS)调控。铜绿假单胞菌有4个QS系统,分别为las系统、rhl系统、pqs系统和iqs系统。2-庚基-3-羟基-4-喹诺酮(Pseudomonas quinolone signal,PQS)作为铜绿假单胞菌pqs系统的信号分子,不仅能够调控许多毒力因子的表达,也能够影响一些微生物和宿主的多种生理过程。本文总结了PQS多种生物学功能,如介导QS系统、调控生物被膜形成、介导外膜囊泡产生及铁摄取、调节宿主免疫活性、介导细胞毒性作用,以及提供种群保护等。本文旨在突出铜绿假单胞菌PQS的功能多样性,并为PQS新功能研究和抗菌药物的研发提供指导。     

Isolation of a membrane associated iron chelator from Pseudomonas aeruginosa

A membrane associated iron chelator (MAIC) has been extracted with ethanol from the membranes of Pseudomonas aeruginosa, and isolated on thin-layer chromatograms. Also extracted from the membranes is the ferrated form of MAIC, FeMAIC. When cell-bound or in the complete ethanol extract of membranes, MAIC binds iron from exogenous iron sources forming FeMAIC. Methanol solutions of each compound exhibit similar absorption spectra with strong absorption in the ultraviolet, indicating the aromatic structure of the compounds. Colorimetric reactions reveal the presence of a phenolic moiety in these compounds. MAIC and FeMAIC are extracted from the membranes of cells grown in media supplemented with iron or in media containing significant trace levels of iron. Transport studies revealed that neither iron-fed nor iron-starved cells transport detectable levels of radiolabeled iron from exogenous iron sources, yet low amounts of 55FeMAIC are extracted from the membranes of cells incubated with [55Fe]ferric chelators. The MAIC may serve as an iron transporter in these cells, or may serve to bind iron following its transport into the cell via another mechanism.     

Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation

Bacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4'-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.     

Unusual chromenes from Peperomia blanda

From the methanol extract of the aerial parts of Peperomia blanda (Piperaceae), two chromenes were isolated and characterized mainly through application of 2D-NMR spectroscopy. The structures were 2S-(4-methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7-dimethyl-2H-chromene and 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-dimethyl-2H-chromene named as blandachromenes I and II, respectively.     

Growth inhibition of bloodstream forms of Trypanosoma brucei by the iron chelator deferoxamine

Treatment of bloodstream forms of Trypanosoma brucei with the iron chelator deferoxamine inhibits the proliferation of the parasites. Compared with mammalian cells, bloodstream forms of Trypanosoma brucei are 10 times more sensitive to iron depletion. The primary target of the chelator is obviously the intracellular iron as the toxicity of deferoxamine is abolished by addition of holotransferrin, the exogenous source of iron for the parasite. To identify probable target sites, the effect of deferoxamine on ribonucleotide reductase, alternative oxidase and superoxide dismutase, three iron-dependent enzymes in bloodstream-form trypanosomes, was studied. Incubation of the parasites with the chelator leads to inhibition of DNA synthesis and lowers oxygen consumption indicating that deferoxamine may affect ribonucleotide reductase and alternative oxidase. The compound does not inhibit the holoenzymes directly but probably acts by chelating cellular iron thus preventing its incorporation into the newly synthesised apoproteins. Treatment of the parasites with deferoxamine for 24 h has no effect on the activity of superoxide dismutase. The results have implications for antitrypanosomal drug development based on specific intervention with the parasite's iron metabolism.     

Burkholderia pseudomallei, B. thailandensis, and B. ambifaria produce 4-hydroxy-2-alkylquinoline analogues with a methyl group at the 3 position that is required for quorum-sensing regulation

4-Hydroxy-2-alkylquinolines (HAQs), especially 3,4-dihydroxy-2-heptylquinoline (Pseudomonas quinolone signal) and its precursor, 4-hydroxy-2-heptylquinoline, are attracting much attention, mainly because of their role as signaling molecules in Pseudomonas aeruginosa. The pqsABCDE operon is centrally involved in their biosynthesis. The presence of a homologous operon in Burkholderia pseudomallei and B. thailandensis was recently reported. Thus, we have investigated the abilities of 11 Burkholderia species to produce HAQ-like molecules by liquid chromatography/mass spectrometry. We have identified 29 different HAQ derivatives produced by the only three Burkholderia species where a pqsABCDE homologue was found among available sequenced Burkholderia species genomes, including B. ambifaria, a member of the Burkholderia cepacia complex. In contrast with those of P. aeruginosa, Burkholderia HAQs typically bear a methyl group, hence their designation as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). We identified three families of HMAQs with a saturated or unsaturated alkyl chain at the 2' position, in contrast with the 1' position of P. aeruginosa, including one with an N-oxide group. Furthermore, the operon in these species contains two more genes downstream of the pqsE homologue, resulting in the hmqABCDEFG operon. While the inactivation of hmqA inhibits the production of HMAQs, the methylation of the quinoline ring requires a putative methyltransferase encoded by hmqG. Interestingly, hmqA or hmqG mutations increase the production of acyl homoserine lactones and, consequently, phenotypes under the control of quorum sensing in B. ambifaria: antifungal activity, siderophore production, and proteolytic activity. These results indicate that only HAQs bearing a methyl group (HMAQs) are involved in quorum-sensing regulation.     

Isolation and Characterization of Antibacterial Compound from a Mangrove-Endophytic Fungus, Penicillium chrysogenum MTCC 5108

Microorganisms, especially endophytic fungi that reside in the tissue of living mangrove plants, seem to play a major role in meeting the general demand for new biologically active substances. During the course of screening for biologically active secondary metabolites from marine microorganisms, an antibiotic compound containing an indole and a diketopiperazine moiety was isolated from the culture medium of Penicilliumchrysogenum, (MTCC 5108), an endophytic fungus on the mangrove plant Porteresiacoarctata (Roxb.). The cell free culture medium of P. chrysogenum showed significant activity against Vibriocholerae, (MCM B-322), a pathogen causing cholera in humans. Bioassay guided chemical characterization of the crude extract led to the isolation of a secondary metabolite possessing a molecular formula C₁₉H₂₁O₂N₃. Its antibacterial activity was comparable with standard antibiotic, streptomycin. This compound (1) was found to be (3,1′-didehydro-3[2″(3′″,3′″-dimethyl-prop-2-enyl)-3″-indolylmethylene]-6-methyl pipera-zine-2,5-dione) on the basis of mass spectrometry, infrared spectroscopy and one and two-dimensional nuclear magnetic resonance analysis.     

Ascosonchine, the enol tautomer of 4-pyridylpyruvic acid with herbicidal activity produced by Ascochyta sonchi

A new phytotoxic enol tautomer of 4-pyridylpyruvic acid, named ascosonchine, was isolated from the culture filtrate of Ascochyta sonchi. Such a leaf pathogen is a potential biocontrol agent of Sonchus arvensis, a perennial herbaceous weed occurring throughout the temperate regions of the world. Ascosonchine, characterised as (Z)-2-hydroxy-3-(4-pyridyl)-2-propenoic acid by spectroscopic methods, showed selective herbicidal properties, that are not associated with antibacterial, antifungal or zootoxic activities.     

Antibacterial and antioxidant cassane diterpenoids from Caesalpinia benthamiana

Bioactivity-guided fractionation of the light petroleum extract of Caesalpinia benthamiana (=Mezoneuron benthamianum) root bark has led to the isolation of two cassane diterpenoids, designated as benthaminin 1 and 2. A third compound, a deoxy form of caesaldekarin C (also referred to as methyl vouacapenate) which has previously been isolated from Caesalpinia major, C. bonducella, Vouacapoua americana and V. macropetala, was also isolated, together with beta-sitosterol and stigmastenone. The antibacterial and antioxidant activities of these cassane diterpenoids have been assessed using the microdilution assay method and DPPH spectrophotometric and TBA lipid peroxidation assays. Benthaminin 1 was the more active antibacterial compound with MIC values of 47.8 microM for both Staphylococcus aureus and Micrococcus flavus. Benthaminin 2 was the more active antioxidant compound and showed IC50 values of 42.7 microM and 74.2 microM for the DPPH and TBA assays, respectively. Deoxycaesaldekarin C possessed both antibacterial and antioxidant activities. The presence of methyl ester and methyl functional groups as well as an unsaturated furan ring appears to confer antibacterial activity. On the other hand, the relatively stronger antioxidant activity of benthaminin 2 may be associated with the presence of an exocyclic methylene function.