RESUMO
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(1H)-one (the "Pseudomonas quinolone signal" [PQS]) is an important quorum sensing signal molecule, 2-alkyl-1-hydroxyquinolin-4(1H)-ones (also known as 2-alkyl-4-hydroxyquinoline N-oxides [AQNOs]) are antibiotics inhibiting respiration. Hydroxylation of the PQS precursor 2-heptylquinolin-4(1H)-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(1H)-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 the 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 a 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 restrict evolutionary adaptation and lead to the preference of non-N-oxide substrates by AQ 3-monooxygenases.IMPORTANCEPseudomonas 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 AQ/AQNO equilibrium, resistance, and degradation.
Assuntos
Oxigenases de Função Mista/metabolismo , Óxidos/metabolismo , Pseudomonas aeruginosa/metabolismo , Quinolonas/metabolismo , Antibacterianos/metabolismo , Hidroxilação , Mycobacterium abscessus/metabolismo , Staphylococcus aureus/metabolismoRESUMO
The opportunistic pathogen Pseudomonas aeruginosa can utilize unusual carbon sources, like sodium dodecyl sulfate (SDS) and alkanes. Whereas the initiating enzymatic steps of the corresponding degradation pathways have been characterized in detail, the oxidation of the emerging long-chain alcohols has received little attention. Recently, the genes for the Lao (long-chain-alcohol/aldehyde oxidation) system were discovered to be involved in the oxidation of long-chain alcohols derived from SDS and alkane degradation. In the Lao system, LaoA is predicted to be an alcohol dehydrogenase/oxidase; however, according to genetic studies, efficient long-chain-alcohol oxidation additionally required the Tat-dependent protein LaoB. In the present study, the Lao system was further characterized. In vivo analysis revealed that the Lao system complements the substrate spectrum of the well-described Exa system, which is required for growth with ethanol and other short-chain alcohols. Mutational analysis revealed that the Tat site of LaoB was required for long-chain-alcohol oxidation activity, strongly suggesting a periplasmic localization of the complex. Purified LaoA was fully active only when copurified with LaoB. Interestingly, in vitro activity of the purified LaoAB complex also depended on the presence of the Tat site. The copurified LaoAB complex contained a flavin cofactor and preferentially oxidized a range of saturated, unbranched primary alcohols. Furthermore, the LaoAB complex could reduce cytochrome c550-type redox carriers like ExaB, a subunit of the Exa alcohol dehydrogenase system. LaoAB complex activity was stimulated by rhamnolipids in vitro. In summary, LaoAB constitutes an unprecedented protein complex with specific properties apparently required for oxidizing long-chain alcohols. IMPORTANCE Pseudomonas aeruginosa is a major threat to public health. Its ability to thrive in clinical settings, water distribution systems, or even jet fuel tanks is linked to detoxification and degradation of diverse hydrophobic substrates that are metabolized via alcohol intermediates. Our study illustrates a novel flavoprotein long-chain-alcohol dehydrogenase consisting of a facultative two-subunit complex, which is unique among related enzymes, while the homologs of the corresponding genes are found in numerous bacterial genomes. Understanding the catalytic and compartmentalization processes involved is of great interest for biotechnological and hygiene research, as it may be a potential starting point for rationally designing novel antibacterial substances with high specificity against this opportunistic pathogen.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Álcoois/química , Álcoois/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Cinética , Oxirredução , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMO
The nosocomial pathogen Pseudomonas aeruginosa regulates its virulence via a complex quorum sensing network, which, besides N-acylhomoserine lactones, includes the alkylquinolone signal molecules 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal [PQS]) and 2-heptyl-4(1H)-quinolone (HHQ). Mycobacteroides abscessus subsp. abscessus, an emerging pathogen, is capable of degrading the PQS and also HHQ. Here, we show that although M. abscessus subsp. abscessus reduced PQS levels in coculture with P. aeruginosa PAO1, this did not suffice for quenching the production of the virulence factors pyocyanin, pyoverdine, and rhamnolipids. However, the levels of these virulence factors were reduced in cocultures of P. aeruginosa PAO1 with recombinant M. abscessus subsp. massiliense overexpressing the PQS dioxygenase gene aqdC of M. abscessus subsp. abscessus, corroborating the potential of AqdC as a quorum quenching enzyme. When added extracellularly to P. aeruginosa cultures, AqdC quenched alkylquinolone and pyocyanin production but induced an increase in elastase levels. When supplementing P. aeruginosa cultures with QsdA, an enzyme from Rhodococcus erythropolis which inactivates N-acylhomoserine lactone signals, rhamnolipid and elastase levels were quenched, but HHQ and pyocyanin synthesis was promoted. Thus, single quorum quenching enzymes, targeting individual circuits within a complex quorum sensing network, may also elicit undesirable regulatory effects. Supernatants of P. aeruginosa cultures grown in the presence of AqdC, QsdA, or both enzymes were less cytotoxic to human epithelial lung cells than supernatants of untreated cultures. Furthermore, the combination of both aqdC and qsdA in P. aeruginosa resulted in a decline of Caenorhabditis elegans mortality under P. aeruginosa exposure.
Assuntos
Hidrolases de Éster Carboxílico/genética , Dioxigenases/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium abscessus/genética , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/genética , Células A549 , Animais , Antibiose/genética , Caenorhabditis elegans/microbiologia , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Dioxigenases/metabolismo , Dioxigenases/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Mycobacterium abscessus/enzimologia , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/genética , Piocianina/metabolismo , Quinolonas/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Alkyl quinolones (AQs) are multifunctional bacterial secondary metabolites generally known for their antibacterial and algicidal properties. Certain representatives are also employed as signalling molecules of Burkholderia strains and Pseudomonas aeruginosa. The marine Gammaproteobacterium Microbulbifer sp. HZ11 harbours an AQ biosynthetic gene cluster with unusual topology but does not produce any AQ-type metabolites under laboratory conditions. In this study, we demonstrate the potential of strain HZ11 for AQ production by analysing intermediates and key enzymes of the pathway. Moreover, we demonstrate that exogenously added AQs such as 2-heptyl-1(H)-quinolin-4-one (referred to as HHQ) or 2-heptyl-1-hydroxyquinolin-4-one (referred to as HQNO) are brominated by a vanadium-dependent haloperoxidase (V-HPOHZ11 ), which preferably is active towards AQs with C5-C9 alkyl side chains. Bromination was specific for the third position and led to 3-bromo-2-heptyl-1(H)-quinolin-4-one (BrHHQ) and 3-bromo-2-heptyl-1-hydroxyquinolin-4-one (BrHQNO), both of which were less toxic for strain HZ11 than the respective parental compounds. In contrast, BrHQNO showed increased antibiotic activity against Staphylococcus aureus and marine isolates. Therefore, bromination of AQs by V-HPOHZ11 can have divergent consequences, eliciting a detoxifying effect for strain HZ11 while simultaneously enhancing antibiotic activity against other bacteria.
Assuntos
Alteromonadaceae/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Quinolonas/metabolismo , Quinolonas/farmacologia , Alteromonadaceae/genética , Alteromonadaceae/isolamento & purificação , Antibacterianos/química , Halogenação , Quinolonas/química , Água do Mar/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Programmed ribosomal frameshifting (PRF) is a translational anomaly causing the ribosome to shift into an alternative reading frame. PRFs are common in viral genomes, using a single nucleotide sequence to code for two proteins in overlapping frames. In bacteria and eukaryota, PRFs are less frequent. We report on a PRF in the copper detoxification system of Escherichia coli where a metallochaperone is generated out of the first 69 amino acids and a C-terminal out-of-frame glycine of the gene copA. copA besides codes for the P1B -ATPase CopA, a membrane-integral protein and principal interaction target of the chaperone. To enhance the production of the frameshift-generated cytosolic copper binding protein a truncated transcript is produced from the monocistronic copA gene. This shorter transcript is essential for producing sufficient amounts of the chaperone to support the membrane pump. The findings close the gap in our understanding of the molecular physiology of cytoplasmic copper transport in E. coli, revealing that a chaperone-like entity is required for full functionality of the P1B -ATPase copper pump. We, moreover, demonstrate that the primary transcriptional response to copper results in formation of the small transcript and concurrently, the metallochaperone plays a key role in resistance against copper shock.
Assuntos
ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Ribossomos/metabolismoRESUMO
P1 B -ATPases are among the most common resistance factors to metal-induced stress. Belonging to the superfamily of P-type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P1 B -ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N-terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal-binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein-internal pathway of copper and demonstrate the distal N-terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane-integral ion-binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P1 B -ATPases, which is governed by the length of the inter-domain linker.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Escherichia coli/metabolismo , Estrutura Terciária de Proteína , Sítios de Ligação , ATPases Transportadoras de Cobre , Proteínas de Escherichia coli , Modelos Biológicos , Modelos MolecularesRESUMO
The extraction of disease specific information from Fourier transform infrared (FTIR) spectra of human body fluids demands the highest standards of accuracy and reproducibility of measurements because the expected spectral differences between healthy and diseased subjects are very small in relation to a large background absorbance of the whole sample. Here, we demonstrate that with the increased sensitivity of modern FTIR spectrometers, automatisation of sample preparation and modern bioinformatics, it is possible to identify and validate spectral biomarker candidates for distinguishing between urinary bladder cancer (UBC) and inflammation in suspected bladder cancer patients. The current dataset contains spectra of blood serum and plasma samples of 135 patients. All patients underwent cytology and pathological biopsy characterization to distinguish between patients without UBC (46) and confirmed UBC cases (89). A minimally invasive blood test could spare control patients a repeated cystoscopy including a transurethral biopsy, and three-day stationary hospitalisation. Blood serum, EDTA and citrate plasma were collected from each patient and processed following predefined strict standard operating procedures. Highly reproducible dry films were obtained by spotting sub-nanoliter biofluid droplets in defined patterns, which were compared and optimized. Particular attention was paid to the automatisation of sample preparation and spectral preprocessing to exclude errors by manual handling. Spectral biomarker candidates were identified from absorbance spectra and their 1(st) and 2(nd) derivative spectra using an advanced Random Forest (RF) approach. It turned out that the 2(nd) derivative spectra were most useful for classification. Repeat validation on 21% of the dataset not included in predictor training with Linear Discriminant Analysis (LDA) classifiers and Random Forests (RFs) yielded a sensitivity of 93 ± 10% and a specificity of 46 ± 18% for bladder cancer. The low specificity can be most likely attributed to the unbalanced and small number of control samples. Using this approach, spectral biomarker candidates in blood-derived biofluids were identified, which allow us to distinguish between cancer and inflammation, but the observed differences were tiny. Obviously, a much larger sample number has to be investigated to reliably validate such candidates.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Papilar/diagnóstico , Recidiva Local de Neoplasia/diagnóstico , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Idoso , Carcinoma Papilar/sangue , Estudos de Casos e Controles , Análise Discriminante , Feminino , Humanos , Masculino , Microscopia de Força Atômica , Pessoa de Meia-Idade , Gradação de Tumores , Recidiva Local de Neoplasia/sangue , Estadiamento de Neoplasias , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/sangueRESUMO
Certain heavy metal ions such as copper and zinc serve as essential cofactors of many enzymes, but are toxic at high concentrations. Thus, intracellular levels have to be subtly balanced. P-type ATPases of the P(IB)-subclass play a major role in metal homeostasis. The thermoacidophile Sulfolobus solfataricus possesses two P(IB)-ATPases named CopA and CopB. Both enzymes are present in cells grown in copper-depleted medium and are accumulated upon an increase in the external copper concentration. We studied the physiological roles of both ATPases by disrupting genes copA and copB. Neither of them affected the sensitivity of S. solfataricus to reactive oxygen species, nor were they a strict prerequisite to the biosynthesis of the copper protein cytochrome oxidase. Deletion mutant analysis demonstrated that CopA is an effective copper pump at low and high copper concentrations. CopB appeared to be a low-affinity copper export ATPase, which was only relevant if the media copper concentration was exceedingly high. CopA and CopB thus act as resistance factors to copper ions at overlapping concentrations. Moreover, growth tests on solid media indicated that both ATPases are involved in resistance to silver.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Arqueais/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Sulfolobus solfataricus/enzimologia , Adenosina Trifosfatases/genética , Proteínas Arqueais/genética , Proteínas de Transporte de Cátions/genética , ATPases Transportadoras de Cobre , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Espécies Reativas de Oxigênio/metabolismo , Sulfolobus solfataricus/classificação , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismoRESUMO
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.
Assuntos
Produtos Biológicos , Oxigenases de Função Mista , Aminas , Flavinas/metabolismo , Flavoproteínas/química , Glutationa Redutase/metabolismo , Hidroxilaminas , Cinética , Ligantes , Oxigenases de Função Mista/metabolismo , Nitrogênio , Nucleotídeos/metabolismo , Oxirredução , Óxidos , FilogeniaRESUMO
In contrast to many steroid hormones and cholesterol, mammalian bile salts are 5ß-steroids, which leads to a bent structure of the steroid core. Bile salts are surface-active steroids excreted into the environment in large amounts, where they are subject to bacterial degradation. Bacterial steroid degradation is initiated by the oxidation of the A-ring leading to canonical Δ4-3-keto steroids with a double bond in the A-ring. For 5ß-bile salts, this Δ4-double bond is introduced into 3-keto-bile salts by a 5ß-Δ4-ketosteroid dehydrogenase (5ß-Δ4-KSTD). With the Nov2c019 protein from bile-salt degrading Sphingobium sp. strain Chol11, a novel 5ß-Δ4-KSTD for bile-salt degradation belonging to the Old Yellow Enzyme family was identified and named 5ß-Δ4-KSTD1. By heterologous production in Escherichia coli, 5ß-Δ4-KSTD function could be shown for 5ß-Δ4-KSTD1 as well as the homolog CasH from bile-salt degrading Rhodococcus jostii RHA1. The deletion mutant of 5ß-Δ4-kstd1 had a prolonged lag-phase with cholate as sole carbon source and, in accordance with the function of 5ß-Δ4-KSTD1, showed delayed 3-ketocholate transformation. Purified 5ß-Δ4-KSTD1 was specific for 5ß-steroids in contrast to 5α-steroids and converted steroids with a variety of hydroxy groups regardless of the presence of a side chain. 5ß-Δ4-KSTD1 showed a relatively low K m for 3-ketocholate, a very high specific activity and pronounced substrate inhibition. With respect to the toxicity of bile salts, these kinetic properties indicate that 5ß-Δ4-KSTD1 can achieve fast detoxification of the detergent character as well as prevention of an overflow of the catabolic pathway in presence of increased bile-salt concentrations.
RESUMO
Light-dependent or light-stimulated catalysis provides a multitude of perspectives for implementation in technological or biomedical applications. Despite substantial progress made in the field of photobiocatalysis, the number of usable light-responsive enzymes is still very limited. Flavoproteins have exceptional potential for photocatalytic applications because the name-giving cofactor intrinsically features light-dependent reactivity, undergoing photoreduction with a variety of organic electron donors. However, in the vast majority of these enzymes, photoreactivity of the enzyme-bound flavin is limited or even suppressed. Here, we present a flavoprotein monooxygenase in which catalytic activity is controllable by blue light illumination. The reaction depends on the presence of nicotinamide nucleotide-type electron donors, which do not support the reaction in the absence of light. Employing various experimental approaches, we demonstrate that catalysis depends on a protein-mediated photoreduction of the flavin cofactor, which proceeds via a radical mechanism and a transient semiquinone intermediate.
Assuntos
Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/metabolismo , Oxigenases de Função Mista/metabolismo , NAD/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Cristalografia por Raios X , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Luz , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Moleculares , NADP/metabolismo , Oxirredução , Processos Fotoquímicos , Pseudomonas aeruginosa/genéticaRESUMO
Pseudomonas aeruginosa is an important nosocomial pathogen that is frequently recalcitrant to available antibiotics, underlining the urgent need for alternative therapeutic options against this pathogen. Targeting virulence functions is a promising alternative strategy as it is expected to generate less-selective resistance to treatment compared to antibiotics. Capitalizing on our nonligand-based benzamide-benzimidazole (BB) core structure compounds reported to efficiently block the activity of the P. aeruginosa multiple virulence factor regulator MvfR, here we report the first class of inhibitors shown to interfere with PqsBC enzyme activity, responsible for the synthesis of the MvfR activating ligands HHQ and PQS, and the first to target simultaneously MvfR and PqsBC activity. The use of these compounds reveals that inhibiting PqsBC is sufficient to block P. aeruginosa's acute virulence functions, as the synthesis of MvfR ligands is inhibited. Our results show that MvfR remains the best target of this QS pathway, as we show that antagonists of this target block both acute and persistence-related functions. The structural properties of the compounds reported in this study provide several insights that are instrumental for the design of improved MvfR regulon inhibitors against both acute and persistent P. aeruginosa infections. Moreover, the data presented offer the possibility of a polypharmacology approach of simultaneous silencing two targets in the same pathway. Such a combined antivirulence strategy holds promise in increasing therapeutic efficacy and providing alternatives in the event of a single target's resistance development.
Assuntos
Polifarmacologia , Pseudomonas aeruginosa/genética , Regulon/efeitos dos fármacos , Tolerância a Medicamentos , Inibidores Enzimáticos/farmacologia , Terapia de Alvo Molecular/métodos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/enzimologia , Virulência/efeitos dos fármacos , Fatores de VirulênciaRESUMO
Pseudomonas aeruginosa uses quorum sensing (QS) as a cell-to-cell communication system to orchestrate the expression of virulence determinants. The biosynthesis of the important Pseudomonas quinolone signal (PQS) requires the pqsABCDE operon. Here, PqsE acts as a pathway-specific thioesterase, but it also contributes to the regulation of bacterial virulence via an unknown mechanism. In this manuscript, we report the discovery of PqsE inhibitors as tool compounds to gain further insights into its different functions. Differential scanning fluorimetry (DSF) was used to screen a fragment library, and isothermal titration calorimetry (ITC) was employed as a secondary filter. As proven by X-ray crystallography, hit molecules bound to the active center inhibiting PqsE's thioesterase activity in cell-based and in vitro assays. Notably, the ligands did not affect the levels of the PqsE-regulated virulence factor pyocyanin. These findings indicate that the regulatory function of PqsE is not linked to its thioesterase activity and must be encoded outside of the active center. This study highlights the potential of fragment-based screening for the discovery of tool compounds. This approach provided novel insight into complex biological systems, which could not be obtained by knockout studies.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Ácidos Carboxílicos/farmacologia , Pseudomonas aeruginosa/fisiologia , Tioléster Hidrolases/antagonistas & inibidores , Benzoatos/farmacologia , Cristalografia por Raios X , Descoberta de Drogas , Fluorometria , Piocianina/biossíntese , Piridinas/farmacologia , Pirróis/farmacologia , Quinolonas/metabolismo , Percepção de Quorum , Tiofenos/farmacologia , Virulência/efeitos dos fármacos , Fatores de Virulência/biossínteseRESUMO
The study by Dulcey and colleagues in this issue of Chemistry & Biology changes our perception of the pathway of 2-alkyl-4-hydroxyquinoline biosynthesis by the opportunistic pathogen Pseudomonas aeruginosa and suggests that the biosynthetic protein complex PqsBC is a potential antibacterial target.