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Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement
in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III
(FabH) like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-a...
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We have previously shown that the eukaryotic C-type natriuretic peptide hormone (CNP) regulates Pseudomonas aeruginosa virulence and biofilm formation after binding on the AmiC sensor, triggering the amiE transcription. Herein, the involvement of the aliphatic amidase AmiE in P. aeruginosa virulence regulation has been investigated. The proteome an...
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... Furthermore, a broad specificity thioesterase, TesB, can partially offset the function of PqsE [42]. Fourth, 2-ABA condenses with octanoyl-coenzyme A to form 2-heptyl-4-quinolone (HHQ) in the presence of the dimer PqsBC [41][42][43]. Finally, HHQ is hydroxylated by monooxygenase PqsH under aerobic conditions to form PQS [16,44]. PQS is a unique cell-to-cell signal, but the potential mechanism of PQS transport by P. aeruginosa to the extracellular environment remains unclear. ...
Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to survive in the host; however, the host immune system limits the availability of iron. Pyochelin (PCH) is a major siderophore produced by P. aeruginosa during infection, which can help P. aeruginosa survive in an iron-restricted environment and cause infection. The infection activity of P. aeruginosa is regulated by the Pseudomonas quinolone signal (PQS) quorum-sensing system. The system uses 2-heptyl-3-hydroxy-4-quinolone (PQS) or its precursor, 2-heptyl-4-quinolone (HHQ), as the signal molecule. PQS can control specific life processes such as mediating quorum sensing, cytotoxicity, and iron acquisition. This review summarizes the biosynthesis of PCH and PQS, the shared transport system of PCH and PQS, and the regulatory relationship between PCH and PQS. The correlation between the PQS and PCH is emphasized to provide a new direction for future research.
... PqsBC is FabH-like β-ketoac yl-(ac yl carrier protein) synthase III, whic h catal yzes the final step in quinolone synthesis (Dulcey et al. 2013 ). It is structur all y similar to FabH and PqsD (Drees et al. 2016 al ytic activity wher eas PqsB has a r ole in structur al stability. The position of the active site overlaps with the one in the PqsD. ...
... The complex shows eac h pr otein contains αβ4 α type of two subdomains . T he active site of PqsC has an extended wider cavity with lar ge volume differ ent fr om other members of the famil y, to accommodate the large bicyclic ring of its product (Drees et al. 2016 ). The catalytic site is marked by the presence of a dyad of Cys129, and His269. ...
Pseudomonas aeruginosa is an opportunistic human pathogen responsible for acute and chronic, hard to treat infections. Persistence of P. aeruginosa is due to its ability to develop into biofilms which are sessile bacterial communities adhered to substratum and encapsulated in layers of self-produced exopolysaccharides. These biofilms provide enhanced protection from the host immune system and resilience towards antibiotics which poses a challenge for treatment. Various strategies have been expended for combating biofilms which involve inhibiting biofilm formation or promoting their dispersal. The current remediation approaches offer some hope for clinical usage however treatment and eradication of preformed biofilms is still a challenge. Thus, identifying novel targets and understanding the detailed mechanism of biofilm regulation becomes imperative. Structure-based drug discovery (SBDD) provides a powerful tool that exploits the knowledge of atomic resolution details of the targets to search for high affinity ligands. This review describes the available structural information on the putative target protein structures that can be utilised for high throughput in silico drug discovery against P. aeruginosa biofilms. Integrating available structural information on the target proteins in readily accessible format will accelerate the process of drug discovery.
... This variety is likely a result of the rather broad substrate specificity of the β-ketoacyl-(acyl-carrier-protein) synthase III (FabH)-like enzymes PqsB and PqsC. The heterodimeric PqsBC complex uses CoA-activated fatty acids to catalyze a decarboxylative Claisencondensation of acyl-ACP and 2-aminobenzoylacetate (2-ABA) or 2-hydroxy-aminobenzoylacetate (2-HABA) to form AQs or AQNOs, respectively 8,9 . The fatty acids used in the biosynthesis of AQs and AQNOs may be provided by the bacterial fatty acid synthesis, β-oxidation, or be utilized from the medium (Fig. 1). ...
The human pathogen Pseudomonas aeruginosa produces various 4(1H)-quinolones with diverse functions. Among these, 2-nonyl-4(1H)-quinolone (NQ) and its N-oxide (NQNO) belong to the main metabolites. Their biosynthesis involves substrates from the fatty acid metabolism and we hypothesized that oxidized fatty acids could be responsible for a so far undetected class of metabolites. We developed a divergent synthesis strategy for 2′-hydroxy (2′-OH) and 2′-oxo- substituted quinolones and N-oxides and demonstrated for the first time that 2′-OH-NQ and 2′-OH-NQNO but not the corresponding 2′-oxo compounds are naturally produced by PAO1 and PA14 strains of P. aeruginosa. The main metabolite 2′-OH-NQ is produced even in concentrations comparable to NQ. Exogenous availability of β-hydroxydecanoic acid can further increase the production of 2′-OH-NQ. In contrast to NQ, 2′-OH-NQ potently induced the cytokine IL-8 in a human cell line at 100 nм, suggesting a potential role in host immune modulation.
... Although both bacteria contain a LuxI/R-type QS, the PQS system is unique for P. aeruginosa (9)(10)(11)21). More specifically, the PqsBC complex is a FabH-like enzyme that catalyzes the condensation of an octanoyl-CoA with 2-ABA (38,39). This complex does not show similarity to the CviI synthase in C. violaceum. ...
Pseudomonas aeruginosa is an opportunistic pathogen that causes major health care concerns due to its virulence and high intrinsic resistance to antimicrobial agents. Therefore, new treatments are greatly needed. An interesting approach is to target quorum sensing (QS). QS regulates the production of a wide variety of virulence factors and biofilm formation in P. aeruginosa. This study describes the identification of paecilomycone as an inhibitor of QS in both Chromobacterium violaceum and P. aeruginosa. Paecilomycone strongly inhibited the production of virulence factors in P. aeruginosa, including various phenazines, and biofilm formation. In search of the working mechanism, we found that paecilomycone inhibited the production of 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), but not 2'-aminoacetophenone (2-AA). Therefore, we suggest that paecilomycone affects parts of QS in P. aeruginosa by targeting the PqsBC complex and alternative targets or alters processes that influence the enzymatic activity of the PqsBC complex. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research. IMPORTANCE Antibiotics are becoming less effective against bacterial infections due to the evolution of resistance among bacteria. Pseudomonas aeruginosa is a Gram-negative pathogen that causes major health care concerns and is difficult to treat due to its high intrinsic resistance to antimicrobial agents. Therefore, new targets are needed, and an interesting approach is to target quorum sensing (QS). QS is the communication system in bacteria that regulates multiple pathways, including the production of virulence factors and biofilm formation, which leads to high toxicity in the host and low sensitivity to antibiotics, respectively. We found a compound, named paecilomycone, that inhibited biofilm formation and the production of various virulence factors in P. aeruginosa. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research.
... AI-2 was a type II QS signal molecule in E. coli. It can affect phenotypes of E. coli, including biofilm formation, motility, virulence, bacterial adhesion, and biofilm matrix production [68][69][70][71][72]. Results showed that Gin reduces the transcription of luxS, lsrB, lsrK, and lsrR, which was consistent with the results obtained in the AI-2 bioluminescence assay (Figure 6). ...
As an opportunistic pathogen, Escherichia coli (E. coli) forms biofilm that increases the virulence of bacteria and antibiotic resistance, posing a serious threat to human and animal health. Recently, ginkgetin (Gin) has been discovered to have antiinflammatory, antioxidant, and antitumor properties. In the present study, we evaluated the antibiofilm and antibacterial synergist of Gin against E. coli. Additionally, Alamar Blue assay combined with confocal laser scanning microscope (CLSM) and crystal violet (CV) staining was used to evaluate the effect of antibiofilm and antibacterial synergist against E. coli. Results showed that Gin reduces biofilm formation, exopolysaccharide (EPS) production, and motility against E. coli without limiting its growth and metabolic activity. Furthermore, we identified the inhibitory effect of Gin on AI-2 signaling molecule production, which showed apparent anti-quorum sensing (QS) properties. The qRT-PCR also indicated that Gin reduced the transcription of curli-related genes (csgA, csgD), flagella-formation genes (flhC, flhD, fliC, fliM), and QS-related genes (luxS, lsrB, lsrK, lsrR). Moreover, Gin showed obvious antibacterial synergism to overcome antibiotic resistance in E. coli with marketed antibiotics, including gentamicin, colistin B, and colistin E. These results suggested the potent antibiofilm and novel antibacterial synergist effect of Gin for treating E. coli infections.
... In contrast to A. aurantiaca and H. rubripertinctus, the BGC of C. nematophagum moreover comprises putative anthranilate synthase genes, which may be involved in supplying the precursor for AQ synthesis (Fig. 1). Due to the highly specific reactions of PqsBC [36], PqsD [37] and PqsE [38], we hypothesized that key intermediates of the AQ biosynthetic pathway such as anthraniloyl-CoA, Fig. 2. Sequence-based phylogenetic tree of selected clade I flavoprotein monooxygenases. Clade I comprises GR2-fold FPMOs, namely group A, E and F [23]. ...
... Heterologous production and purification of monooxygenases was performed as described previously for PqsL [35]. PqsD Cn , HpaC and PqsBC were produced and purified according to established protocols [36,70,71]. ...
Many natural products comprise N‐O containing functional groups with crucial roles for biological activity. Their enzymatic formation is predominantly achieved by oxidation of an amine to form a hydroxylamine, which enables further functionalization. N‐hydroxylation by flavin‐dependent enzymes has so far been attributed to a distinct group of flavoprotein monooxygenases (FPMOs) containing two dinucleotide binding domains. Here, we present three flavoprotein N‐hydroxylases that exhibit a glutathione reductase 2 (GR2)‐type topology with only one nucleotide binding domain, which belong to a distinct phylogenetic branch within the GR2‐fold FPMOs. In addition to PqsL of Pseudomonas aeruginosa, which catalyses the N‐hydroxylation of a primary aromatic amine during biosynthesis of 2‐alkyl‐4‐hydroxyquinoline N‐oxide respiratory chain inhibitors, we analysed isofunctional orthologs from Burkholderia thailandensis (HmqL) and Chryseobacterium nematophagum (PqsLCn). Pre‐steady‐state kinetics revealed that the oxidative half‐reaction of all three enzymes is highly efficient despite the soft nucleophile substrate. Ligand binding studies indicated that HmqL and PqsLCn show displacement of the oxidized flavin cofactor from the active site by the organic substrate, which likely abolishes the substrate inhibition observed in PqsL. Despite mechanistic heterogeneity, the investigated monooxygenases in principle follow the catalytic mechanism of GR2‐fold FPMOs and thus differ from previously described N‐hydroxylating enzymes. The discovery of this yet unrecognized family of flavoprotein N‐hydroxylases expands the current knowledge on the catalytic repertoire of GR2‐type FPMOs and provides a basis for the discovery of other nitrogen functionalizing reactions.
... The latter is converted to 2-aminobenzoylacetate (2-ABA) via the thioesterase functionality of PqsE [21]. The PqsBC heterodimer condenses 2-ABA with octanoyl-CoA to generate HHQ [22,23]. PQS is formed through the oxidation of HHQ by PqsH [24] while formation of the AQ N-oxides such as 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) requires the alternative mono-oxygenase PqsL [25]. ...
Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.
... Biogenetically, compounds 1-10 were probably generated from anthranilic acid, malonyl-CoA, and proline as reported previously (Silva et al., 2019). The condensation reaction between anthranilic acid and malonyl-CoA provided 2-aminobenzoylacetyl-CoA (2-ABA-CoA), which then coupled with D/L-Pro to yield the key intermediate I (Drees et al., 2016). The intramolecular cyclization of the β-keto amide I might generate intermediate II, which was further cyclized to form the enantiomeric PQA (5) (Tatsuta et al., 2001). ...
Six undescribed 4-quinolone alkaloids, including four racemic mixtures, (±)-oxypenicinolines A−D, and two related ones, penicinolines F and G, together with seven known analogues, were isolated from the mangrove-derived fungus Penicillium steckii SCSIO 41025 (Trichocomaceae). The racemates were separated by HPLC using chiral columns. Their structures including absolute configurations were elucidated by extensive spectroscopic analysis, electronic circular dichroism (ECD) experiments, and single-crystal X-ray diffraction analysis. Structurally, (±)-oxypenicinolines A−D shared with an unusual 6/6/5/5 tetracyclic system incorporating a rare tetrahydro-pyrrolyl moiety. A plausible biosynthetic pathway for pyrrolyl 4-quinolone alkaloids is proposed. (±)-oxypenicinoline A and quinolactacide displayed α-glucosidase inhibitory activity with the IC50 values of 317.8 and 365.9 μΜ, respectively, which were more potent than that of acarbose (461.0 μM). Additionally, penicinoline and penicinoline E showed weak inhibitions toward acetylcholinesterase (AChE).
... A previous observation of HQNO 3-hydroxylation by R. erythropolis BG43 (21) raised In P. aeruginosa, biosynthesis of HHQ is mediated by PqsABCDE (51)(52)(53)(54). Subsequent hydroxylation of HHQ by PqsH PA yields PQS (red frame) (24). ...
The multiple biological activities of 2-alkylquinolones (AQs) are crucial for virulence of Pseudomonas aeruginosa , conferring advantages during infection and in polymicrobial communities. Whereas 2-heptyl-3-hydroxyquinolin-4(1 H )-one (the “ Pseudomonas quinolone signal”, PQS) is an important quorum sensing signal molecule, 2-heptyl-1-hydroxyquinolin-4(1 H )-ones (also known as 2-alkyl-4-hydroxyquinoline- N -oxides, AQNOs) are antibiotics inhibiting respiration. Hydroxylation of the PQS precursor 2-heptylquinolin-4(1 H )-one (HHQ) by the signal synthase PqsH boosts AQ quorum sensing. Remarkably, the same reaction, catalyzed by the ortholog AqdB, is used by Mycobacteroides abscessus to initiate degradation of AQs. The antibiotic 2-heptyl-1-hydroxyquinolin-4(1 H )-one (HQNO) is hydroxylated by Staphylococcus aureus to the less toxic derivative PQS- N -oxide (PQS-NO), a reaction probably also catalyzed by a PqsH/AqdB ortholog. In this study, we provide a comparative analysis of four AQ 3-monooxygenases of different organisms. Due to the major impact of AQ/AQNO 3-hydroxylation on the biological activities of the compounds, we surmised adaptations on enzymatic and/or physiological level to serve either the producer or target organisms. Our results indicate that all enzymes share similar features and are incapable of discriminating between AQs and AQNOs. PQS-NO hence occurs as native metabolite of P. aeruginosa although the unfavorable AQNO 3-hydroxylation is minimized by export as shown for HQNO, involving at least one multidrug efflux pump. Moreover, M. abscessus is capable of degrading the AQNO heterocycle by concerted action of AqdB and dioxygenase AqdC. However, S. aureus and M. abscessus orthologs disfavor AQNOs despite their higher toxicity, suggesting that catalytic constraints, rather than evolutionary adaptation, lead to the preference of non- N -oxide substrates by AQ 3-monooxygenases.
IMPORTANCE Pseudomonas aeruginosa , Staphylococcus aureus and Mycobacteroides abscessus are major players in bacterial chronic infections and particularly common colonizers of cystic fibrosis (CF) lung tissue. Whereas S. aureus is an early onset pathogen in CF, P. aeruginosa establishes at later stages. M. abscessus occurs at all stages but has a lower epidemiological incidence. The dynamics of how these pathogens interact can affect survival and therapeutic success.
2-Alkylquinolone (AQ) and 2-alkylhydroxyquinoline N -oxide (AQNO) production is a major factor of P. aeruginosa virulence. The 3-position of the AQ scaffold is critical, both for attenuation of AQ toxicity or degradation by competitors, as well as for full unfolding of quorum sensing. Despite lacking signaling functionality, AQNOs have the strongest impact on suppression of Gram-positives. Because evidence for 3-hydroxylation of AQNOs has been reported, it is desirable to understand the extent by which AQ 3-monooxygenases contribute to manipulation of the AQ/AQNO equilibrium, resistance, and degradation.
... The Mycobacterium tuberculosis FabH is also thought to undergo large conformation changes that allow it to accommodate acyl-CoAs having a wide spectrum of acyl chain lengths (C6 to C 20) 40 . In addition to PsqD the FabH scaffold seems able to accommodate a wide variety of primers since there are other FabH-like proteins that are not involved in fatty-acid synthesis 41,42 . Examples are the archaeal proteins often annotated as FabH homologs (e.g., microbesonline. ...
Pimelic acid, a seven carbon α,ω-dicarboxylic acid (heptanedioic acid), is known to provide seven of the ten biotin carbon atoms including all those of the valeryl side chain. Distinct pimelate synthesis pathways were recently elucidated in Escherichia coli and Bacillus subtilis where fatty acid synthesis plus dedicated biotin enzymes produce the pimelate moiety. In contrast, the α-proteobacteria which include important plant and mammalian pathogens plus plant symbionts, lack all of the known pimelate synthesis genes and instead encode bioZ genes. Here we report a pathway in which BioZ proteins catalyze a 3-ketoacyl-acyl carrier protein (ACP) synthase III-like reaction to produce pimeloyl-ACP with five of the seven pimelate carbon atoms being derived from glutaryl-CoA, an intermediate in lysine degradation. Agrobacterium tumefaciens strains either deleted for bioZ or which encode a BioZ active site mutant are biotin auxotrophs, as are strains defective in CaiB which catalyzes glutaryl-CoA synthesis from glutarate and succinyl-CoA.
