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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.

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.

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.

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-HPO HZ11), 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-HPO HZ11 can have divergent consequences, eliciting a detoxifying effect for strain HZ11 while simultaneously enhancing antibiotic activity against other bacteria.

Bile salts such as cholate are surface-active steroid compounds with functions for digestion and signaling in vertebrates. Upon excretion into soil and water bile salts are an electron- and carbon-rich growth substrate for environmental bacteria. Degradation of bile salts proceeds via intermediates with a 3-keto-Δ1,4-diene structure of the steroid skeleton as shown for e.g. Pseudomonas spp. Recently, we isolated bacteria degrading cholate via intermediates with a 3-keto-7-deoxy-Δ4,6-structure of the steroid skeleton suggesting the existence of a second pathway for cholate degradation. This potential new pathway was investigated with Novosphingobium sp. strain Chol11. A 7α-hydroxysteroid dehydratase encoded by hsh2 was identified, which was required for the formation of 3-keto-7-deoxy-Δ4,6-metabolites. A hsh2 deletion mutant could still grow with cholate but showed impaired growth. Cholate degradation of this mutant proceeded via 3-keto-Δ1,4-diene metabolites. Heterologous expression of Hsh2 in the bile salt-degrading Pseudomonas sp. strain Chol1 led to formation of a dead-end steroid with a 3-keto-7-deoxy-Δ4,6-diene structure. Hsh2 is the first steroid dehydratase with an important function in a metabolic pathway of bacteria that use bile salts as growth substrates. This pathway contributes to a broad metabolic repertoire of Novosphingobium strain Chol11 that may be advantageous in competition with other bile salt-degrading bacteria. This article is protected by copyright. All rights reserved.

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.

Summary Pseudomonas aeruginosa uses the alkylquinolones PQS (2-heptyl-3-hydroxy-4(1 H )-quinolone) and HHQ (2-heptyl-4(1 H )-quinolone) as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. The biosynthesis of HHQ is generally accepted to require the pqsABCD gene products. We now reconstitute the biosynthetic pathway in vitro, and demonstrate that in addition to PqsABCD, PqsE has a role in HHQ synthesis. PqsE acts as thioesterase, hydrolyzing the biosynthetic intermediate 2-aminobenzoylacetyl-coenzyme A to form 2-aminobenzoylacetate, the precursor of HHQ and 2-aminoacetophenone. The role of PqsE can be taken over to some extent by the broad-specificity thioesterase TesB, explaining why the pqsE deletion mutant of P. aeruginosa still synthesizes HHQ. Interestingly, the pqsE mutant produces increased levels of 2,4-dihydroxyquinoline, resulting from intramolecular cyclization of 2-aminobenzoylacetyl-coenzyme A. Overall, our data suggest that PqsE promotes the efficiency of alkylquinolone signal molecule biosynthesis in P. aeruginosa and balances the levels of secondary metabolites deriving from the alkylquinolone biosynthetic pathway.

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.

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.

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.