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In this investigation, we explored the diversity of melleolide-type meroterpenoids produced by Armillaria ostoyae, one of the largest and oldest organisms on Earth, using extracts from liquid and solid fermentation media. The study unveiled three unprecedented dimeric bismelleolides and three novel fatty-acid-substituted congeners, along with 11 new and 21 known derivatives. The structures were elucidated by 1D and 2D NMR spectroscopy and HRESI-MS, and ROESY spectral analysis for relative configurations. Absolute configurations were determined from crystal structures and through ECD spectra comparison. A compound library of melleolide-type meroterpenoids facilitated metabolomics-wide associations, revealing production patterns under different culture conditions. The library enabled assessments of antimicrobial and cytotoxic activities, revealing that the Δ2,4 double bond is not crucial for antifungal activity. Cytotoxicity was linked to the presence of an aldehyde at C1, but lost with hydroxylation at C13. Chemoinformatic analyses demonstrated the intricate interplay of chemical modifications on biological properties. This study marks the first systematic exploration of Armillaria spp. meroterpenoid diversity by MS-based untargeted metabolomics, offering insight into structure-activity relationships through innovative chemoinformatics.
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Antiinfecciosos , Estructura Molecular , Relación Estructura-Actividad , Antifúngicos , Espectroscopía de Resonancia MagnéticaRESUMEN
SUMMARY: Compound identification is one of the most eminent challenges in the untargeted analysis of complex mixtures of small molecules by mass spectrometry. Similarity of tandem mass spectra can provide valuable information on putative structural similarities between known and unknown analytes and hence aids feature identification in the bioanalytical sciences. We have developed CluMSID (Clustering of MS2 spectra for metabolite identification), an R package that enables researchers to make use of tandem mass spectra and neutral loss pattern similarities as a part of their metabolite annotation workflow. CluMSID offers functions for all analysis steps from import of raw data to data mining by unsupervised multivariate methods along with respective (interactive) visualizations. A detailed tutorial with example data is provided as supplementary information. AVAILABILITY AND IMPLEMENTATION: CluMSID is available as R package from https://github.com/tdepke/CluMSID/and from https://bioconductor.org/packages/CluMSID/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Metabolómica , Programas Informáticos , Análisis por Conglomerados , Minería de Datos , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: There continues to be a great need for better biomarkers and host-directed treatment targets for community-acquired pneumonia (CAP). Alterations in phospholipid metabolism may constitute a source of small molecule biomarkers for acute infections including CAP. Evidence from animal models of pulmonary infections and sepsis suggests that inhibiting acid sphingomyelinase (which releases ceramides from sphingomyelins) may reduce end-organ damage. METHODS: We measured concentrations of 105 phospholipids, 40 acylcarnitines, and 4 ceramides, as well as acid sphingomyelinase activity, in plasma from patients with CAP (n = 29, sampled on admission and 4 subsequent time points), chronic obstructive pulmonary disease exacerbation with infection (COPD, n = 13) as a clinically important disease control, and 33 age- and sex-matched controls. RESULTS: Phospholipid concentrations were greatly decreased in CAP and normalized along clinical improvement. Greatest changes were seen in phosphatidylcholines, followed by lysophosphatidylcholines, sphingomyelins and ceramides (three of which were upregulated), and were least in acylcarnitines. Changes in COPD were less pronounced, but also differed qualitatively, e.g. by increases in selected sphingomyelins. We identified highly accurate biomarkers for CAP (AUC ≤ 0.97) and COPD (AUC ≤ 0.93) vs. Controls, and moderately accurate biomarkers for CAP vs. COPD (AUC ≤ 0.83), all of which were phospholipids. Phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins were also markedly decreased in S. aureus-infected human A549 and differentiated THP1 cells. Correlations with C-reactive protein and procalcitonin were predominantly negative but only of mild-to-moderate extent, suggesting that these markers reflect more than merely inflammation. Consistent with the increased ceramide concentrations, increased acid sphingomyelinase activity accurately distinguished CAP (fold change = 2.8, AUC = 0.94) and COPD (1.75, 0.88) from Controls and normalized with clinical resolution. CONCLUSIONS: The results underscore the high potential of plasma phospholipids as biomarkers for CAP, begin to reveal differences in lipid dysregulation between CAP and infection-associated COPD exacerbation, and suggest that the decreases in plasma concentrations are at least partially determined by changes in host target cells. Furthermore, they provide validation in clinical blood samples of acid sphingomyelinase as a potential treatment target to improve clinical outcome of CAP.
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Fosfolípidos/sangre , Neumonía/sangre , Esfingomielina Fosfodiesterasa/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios de Casos y Controles , Ceramidas/sangre , Infecciones Comunitarias Adquiridas/sangre , Infecciones Comunitarias Adquiridas/diagnóstico , Femenino , Humanos , Mediadores de Inflamación/sangre , Lipidómica , Masculino , Persona de Mediana Edad , Neumonía/diagnóstico , Estudios Prospectivos , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Investigación Biomédica Traslacional , Adulto JovenRESUMEN
Sigma factors are essential global regulators of transcription initiation in bacteria which confer promoter recognition specificity to the RNA polymerase core enzyme. They provide effective mechanisms for simultaneously regulating expression of large numbers of genes in response to challenging conditions, and their presence has been linked to bacterial virulence and pathogenicity. In this study, we constructed nine his-tagged sigma factor expressing and/or deletion mutant strains in the opportunistic pathogen Pseudomonas aeruginosa. To uncover the direct and indirect sigma factor regulons, we performed mRNA profiling, as well as chromatin immunoprecipitation coupled to high-throughput sequencing. We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS). Our integrated approach revealed a highly modular network architecture which is composed of insulated functional sigma factor modules. Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes. Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.
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Interacciones Huésped-Parásitos/fisiología , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/fisiología , Factor sigma/genética , Transducción de Señal/fisiología , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Secuenciación de Nucleótidos de Alto Rendimiento , Infecciones por Pseudomonas/metabolismo , Receptor Cross-Talk/fisiología , Análisis de Secuencia de ARN , Factor sigma/metabolismo , TranscriptomaRESUMEN
Gold complexes with N-heterocyclic carbene (NHC) ligands represent a promising class of metallodrugs for the treatment of cancer or infectious diseases. In this report, the synthesis and the biological evaluation of halogen-containing NHC-AuI -Cl complexes are described. The complexes 1 and 5 a-5 f displayed good cytotoxic activity against tumor cells, and cellular uptake studies suggested that an intact Au-NHC fragment is essential for the accumulation of high amounts of both the metal and the NHC ligand. However, the bioavailability was negatively affected by serum components of the cell culture media and was influenced by likely transformations of the complex. One example (5 d) efficiently induced apoptosis in vincristine- and daunorubicin-resistant P-glycoprotein overexpressing Nalm-6 leukemia cells. Cellular uptake studies with this compound showed that both the wild-type and resistant Nalm-6 cells accumulated comparable amounts of gold, indicating that the gold drug was not excreted by P-glycoprotein or other efflux transporters. The effective inhibition of mammalian and bacterial thioredoxin reductases (TrxR) was confirmed for all of the gold complexes. Antibacterial screening of the gold complexes showed a particularly high activity against Gram-positive strains, reflecting their high dependence on an intact Trx/TrxR system. This result is of particular interest as the inhibition of bacterial TrxR represents a relatively little explored mechanism of new anti-infectives.
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Antibacterianos/farmacología , Antineoplásicos/química , Proteínas Bacterianas/antagonistas & inhibidores , Complejos de Coordinación/química , Oro/química , Bacterias Grampositivas/efectos de los fármacos , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Complejos de Coordinación/metabolismo , Complejos de Coordinación/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Heterocíclicos/química , Humanos , Metano/análogos & derivados , Metano/química , Pruebas de Sensibilidad Microbiana , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
A major reason for the low success rate in current drug development through chemical synthesis has been ascribed to the large fraction of quasi planar candidate molecules. Therefore, an "escape from flatland" strategy has been recommended for the generation of bioactive chemical entities. In a first attempt to test this recommendation, we synthesized a small collection of bridged bicyclic compounds possessing a rigid spherical core structure by combining a group of cyclic dienes with a collection of dienophiles. We started from planar biphenyl analogues and, by enzymatic dioxygenation, transformed them into hydroxylated diene structures. Using a small library of newly synthesized dienophiles, the dienes were converted into bridged bicycles via the Diels-Alder reaction. The resulting collection of 78 structures was first tested for bioactivity in a generic assay based on interference with the proliferation of mammalian cells. A more mechanism-targeted bioactivity profiling method, exploiting cellular impedance monitoring, was subsequently used to obtain suggestions for the mode of action exerted by those compounds that were the most active in the proliferation assay. Proteasome inhibition could be confirmed for 8 of a series of 9 respective candidates. Whilst 7 of these molecules showed relatively weak interference with proteasome activity, one candidate exerted a moderate but distinct inhibition. This result appears remarkable in view of the small size of the compound library, which was synthesized following a few basic considerations. It encourages the application of diverse synthetic approaches to further investigate the role of spherical shape for the success of compound libraries.
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Compuestos Bicíclicos con Puentes/síntesis química , Bibliotecas de Moléculas Pequeñas/síntesis química , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Reacción de Cicloadición , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/síntesis química , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , EstereoisomerismoRESUMEN
The soraphens are natural products that exhibit a molecular structure different from what would have been expected by following its polyketidal assembly line. The most significant differences are the presence of a hemiketal instead of a trisubstituted double bond and a double bond at C9 and C10 where a saturated carbon chain was expected. We were interested in the biological activity of the soraphens with architectures as described by the polyketide synthase since we hypothesized that these modifications reflect the evolutionary optimization of the soraphens. Herein we describe four additional derivatives of the so-called paleo-soraphens and their biological profiling to provide a picture of the hypothetical evolutionary optimization of this family of natural products. The syntheses required a unified and convergent strategy and their biological profiling was performed with the aid of impedance measurements. The results of these biological experiments are consistent with the proposed evolutionary optimization of the soraphens.
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Macrólidos/síntesis química , Macrólidos/farmacología , Animales , Línea Celular , Análisis por Conglomerados , Impedancia Eléctrica , Cinética , Macrólidos/química , Ratones , Conformación Molecular , EstereoisomerismoRESUMEN
The tedanolides are biologically active polyketides that exhibit a macrolactone constructed from a primary alcohol. Since polyketidal transformations only generate secondary alcohols, it has been hypothesized by Taylor that this unique lactone could arise from a postketidal transesterification. In order to probe this hypothesis and to investigate the biological profile of the putative precursor of all members of the tedanolide family, we embarked on the synthesis of desepoxyisotedanolide and its biological evaluation in comparison to desepoxytedanolide. The biological experiments unraveled a second target for desepoxytedanolide and provided evidence that the proposed transesterification indeed provides a survival advantage for the producing microorganism.
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Macrólidos/síntesis química , Macrólidos/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Eucariotas/efectos de los fármacos , Células Eucariotas/metabolismo , Fibroblastos/efectos de los fármacos , Humanos , Macrólidos/química , Ratones , Conformación Molecular , Poríferos/química , Conejos , Relación Estructura-ActividadRESUMEN
Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma (σ) factors. The largest group of alternative σ factors is that of the extracytoplasmic function (ECF) σ factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative σ factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF σ factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative σ factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa.
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Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Regulón , Factor sigma/metabolismo , Factores de Virulencia/biosíntesis , Sitios de Unión , Inmunoprecipitación de Cromatina , Medios de Cultivo/química , Eliminación de Gen , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Presión Osmótica , Unión Proteica , Factor sigma/genéticaRESUMEN
Hepatitis C virus (HCV) NS3-4A protease is essential for viral replication. All current small molecular weight drugs against NS3-4A are substrate peptidomimetics that have a similar binding and resistance profile. We developed inhibitory peptides (IPs) capping the active site and binding via a novel "tyrosine" finger at an alternative NS3-4A site that is of particular interest for further HCV drug development. The peptides are not cleaved due to a combination of geometrical constraints and impairment of the oxyanion hole function. Selection and optimization through combinatorial phagemid display, protein crystallography, and further modifications resulted in a 32-amino acid peptide with a K(i) of 0.53 nm. Inhibition of viral replication in cell culture was demonstrated by fusion to a cell-penetrating peptide. Negligible susceptibility to known (A156V and R155K) resistance mutations of the NS3-4A protease was observed. This work shows for the first time that antiviral peptides can target an intracellular site and reveals a novel druggable site on the HCV protease.
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Proteínas Portadoras/química , Mutación , Proteínas no Estructurales Virales/química , Antivirales/farmacología , Sitios de Unión , Péptidos de Penetración Celular/química , Cristalografía/métodos , Diseño de Fármacos , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Humanos , Concentración 50 Inhibidora , Péptidos y Proteínas de Señalización Intracelular , Cinética , Modelos Moleculares , Conformación Molecular , Biblioteca de Péptidos , Péptidos/química , Solventes/químicaRESUMEN
Synthesis can provide molecules such as paleo-soraphensâ A and B that are genetically encoded but not obtained from the natural source. Although it is unclear whether this is part of an evolutionary process or the consequence of the chemical synthesis, the biological evaluation of these genetically encoded natural products can shed light on how natural products are structurally optimized with respect to their biological profile.
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Macrólidos/síntesis química , Secuencia de Aminoácidos , Productos Biológicos , Macrólidos/química , Policétidos/químicaRESUMEN
Achieving cellular uptake is a central challenge for novel antibiotics targeting Gram-negative bacterial pathogens. One strategy is to hijack the bacterial iron transport system by siderophore-antibiotic conjugates that are actively imported into the cell. This was realized with the MECAM-ampicillin conjugate LP-600 we recently reported that was highly active against E. coli. In the present study, we investigate a paradoxical regrowth of E. coli upon treatment of LP-600 at concentrations 16-32 times above the minimum inhibitory concentration (MIC). The phenomenon, coined "Eagle-effect" in other systems, was not due to resistance formation, and it occurred for the siderophore conjugate but not for free ampicillin. To investigate the molecular imprint of the Eagle effect, a combined transcriptome and untargeted metabolome analysis was conducted. LP-600 induced the expression of genes involved in iron acquisition, SOS response, and the e14 prophage upon regrowth conditions. The Eagle effect was diminished in the presence of sulbactam, which we ascribe to a putative synergistic antibiotic action but not to ß-lactamase inhibition. The study highlights the relevance of the Eagle effect for siderophore conjugates. Through the first systematic -omics investigations, it also demonstrates that the Eagle effect manifests not only in a paradoxical growth but also in unique gene expression and metabolite profiles.
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Águilas , Sideróforos , Animales , Sideróforos/farmacología , Sideróforos/metabolismo , Escherichia coli , Águilas/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Ampicilina/farmacología , Hierro/metabolismoRESUMEN
IMPORTANCE: The integration of metabolomics-based approaches into the discovery pipeline has enabled improved mining and prioritization of prolific secondary metabolite producers such as endophytic fungi. However, relying on automated untargeted analysis tools might lead to misestimation of the chemical complexity harbored in these organisms. Our study emphasizes the importance of isolation and structure elucidation of the respective metabolites in addition to deep metabolome analysis for the correct interpretation of untargeted metabolomics approaches such as molecular networking. Additionally, it encourages the further exploration of endophytic fungi from traditional medicinal plants for the discovery of natural products.
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Plantas Medicinales , Policétidos , Endófitos , Lactonas/metabolismo , Policétidos/metabolismo , Metabolómica , Hongos/metabolismoRESUMEN
During a mycological survey of the Democratic Republic of the Congo, a fungal specimen that morphologically resembled the American species Hypoxylonpapillatum was encountered. A polyphasic approach including morphological and chemotaxonomic together with a multigene phylogenetic study (ITS, LSU, tub2, and rpb2) of Hypoxylon spp. and representatives of related genera revealed that this strain represents a new species of the Hypoxylaceae. However, the multi-locus phylogenetic inference indicated that the new fungus clustered with H.papillatum in a separate clade from the other species of Hypoxylon. Studies by ultrahigh performance liquid chromatography coupled to diode array detection and ion mobility tandem mass spectrometry (UHPLC-DAD-IM-MS/MS) were carried out on the stromatal extracts. In particular, the MS/MS spectra of the major stromatal metabolites of these species indicated the production of hitherto unreported azaphilone pigments with a similar core scaffold to the cohaerin-type metabolites, which are exclusively found in the Hypoxylaceae. Based on these results, the new genus Parahypoxylon is introduced herein. Aside from P.papillatum, the genus also includes P.ruwenzoriensesp. nov., which clustered together with the type species within a basal clade of the Hypoxylaceae together with its sister genus Durotheca.
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In this study, we investigated compounds of plant and mushroom origin belonging to Traditional Chinese Medicine (TCM) and to Traditional Tibetan Medicine (TTM): a sandy beige mushroom Trametes robiniophila Murr, commonly known as Huaier/TCM as well as Ershiwuwei Songshi Wan and Qiwei Honghua Shusheng Wan, which both belong to TTM. We aimed to study the efficacy of TTM and TCM in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) in vitro. TCM and TTM were tested either as a monotherapy, or in combination with standard therapeutics: sorafenib for HCC treatment and gemcitabine for CCA. We also discovered a protective mechanism behind the most successful therapeutic combinations. The results demonstrated that TCM and TTM inhibited the proliferation of cancer cells in a time- and dose-dependent manner. The results were compared to classical chemotherapeutics currently used in the clinic: sorafenib for HCC and gemcitabine for CCA. In HCC settings, a combination of Huaier (16 mg/ml) with half of the human plasma concentration of sorafenib, Qiwei Honghua Shusheng Wan (1 mg/ml) monotherapy as well as its combination with half or even a quarter dose of the human plasma concentration of sorafenib represented the most efficient treatments, inhibiting the growth of HCC cells more effectively than the standard therapy. The inhibitory mechanism relied on a strong induction of apoptosis. In CCA settings, Ershiwuwei Songshi Wan and Qiwei Honghua Shusheng Wan as monotherapies or in combination with very low doses of gemcitabine inhibited the growth of CCA cells more efficiently than the standard therapy. Importantly, Ershiwuwei Songshi Wan at the 8 and 16 mg/ml concentrations and Qiwei Honghua Shusheng Wan at the 4 mg/ml concentration were efficacious with gemcitabine applied at massively reduced concentrations. The protective mechanism in CCA relied on a strong induction of early and late apoptosis. Cellular senescence and necroptosis were not associated with protection against HCC/CCA. Combination therapy with TCM or TTM allowed for a dose reduction of standard chemotherapeutics. This is especially important as both chemotherapeutic drugs show strong side effects in patients. The reduction of chemotherapeutics and the synergistic effect observed while applying them in combination with TCM and TTM has strong perspectives for the clinic and patients suffering from HCC and CCA.
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BACKGROUND: Anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)-myopathy is a usually rapidly progressive form of immune-mediated necrotizing myopathy (IMNM). Rarer clinical courses show slow progression and resemble the phenotype of limb-girdle dystrophy (LGMD). OBJECTIVE: We demonstrate the difficulties in differentiating LGMD versus anti-HMGCR-myopathy. METHODS: We report on a 48-year-old patient with slowly progressive tetraparesis and hyperCKemia for more than 20 years. RESULTS: Due to myopathic changes in initial and second muscle biopsy and typical clinical presentation, the patient was diagnosed with LGMD 20 years ago; despite comprehensive genetic testing including exome diagnostics, the genetic cause of disease could not be identified. Finally, HMG-CoA reductase antibodies were detected, confirming the diagnosis of anti-HMGCR-myopathy. By re-work-up of a second muscle biopsy specimen from year 2009, the diagnosis of a IMNM was made in retrospect. Seven cycles of high-dose immunoglobulins were administered; patient reported outcome measures have mildly improved. CONCLUSION: Patients with clinical LGMD phenotype, degenerative changes in muscle biopsy but without genetic confirmation of the disease should be tested for HMG-CoA-myopathy, thereby allowing for an early start of treatment.
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Enfermedades Autoinmunes , Enfermedades Musculares , Distrofia Muscular de Cinturas , Miositis , Autoanticuerpos , Humanos , Enfermedades Musculares/patología , Distrofia Muscular de Cinturas/complicaciones , Distrofia Muscular de Cinturas/diagnóstico , Distrofia Muscular de Cinturas/genética , Miositis/patología , OxidorreductasasRESUMEN
Amino acids and their metabolites are key regulators of immune responses, and plasma levels may change profoundly during acute disease states. Using targeted metabolomics, we evaluated concentration changes in plasma amino acids and related metabolites in community-acquired pneumonia (CAP, n = 29; compared against healthy controls, n = 33) from presentation to hospital through convalescence. We further aimed to identify biomarkers for acute CAP vs. the clinically potentially similar infection-triggered COPD exacerbation (n = 13). Amino acid metabolism was globally dysregulated in both CAP and COPD. Levels of most amino acids were markedly depressed in acute CAP, and total amino acid concentrations on admission were an accurate biomarker for the differentiation from COPD (AUC = 0.93), as were reduced asparagine and threonine levels (both AUC = 0.92). Reduced tryptophan and histidine levels constituted the most accurate biomarkers for acute CAP vs. controls (AUC = 0.96, 0.94). Only kynurenine, symmetric dimethyl arginine, and phenylalanine levels were increased in acute CAP, and the kynurenine/tryptophan ratio correlated best with clinical recovery and resolution of inflammation. Several amino acids did not reach normal levels by the 6-week follow-up. Glutamate levels were reduced on admission but rose during convalescence to 1.7-fold above levels measured in healthy control. Our data suggest that dysregulated amino acid metabolism in CAP partially persists through clinical recovery and that amino acid metabolism constitutes a source of promising biomarkers for CAP. In particular, total amino acids, asparagine, and threonine may constitute plasma biomarker candidates for the differentiation between CAP and infection-triggered COPD exacerbation and, perhaps, the detection of pneumonia in COPD.
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Infecciones Comunitarias Adquiridas , Neumonía , Enfermedad Pulmonar Obstructiva Crónica , Asparagina , Biomarcadores , Infecciones Comunitarias Adquiridas/diagnóstico , Convalecencia , Humanos , Quinurenina , Treonina , TriptófanoRESUMEN
The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins, and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at subinhibitory concentrations could be distinguished clearly by RNA sequencing as well as metabolomics. We observed a strong (2.8- to 212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with extracellular DNA (eDNA) release. Gyrase B-binding aminocoumarins induced the most pronounced changes, including a strong downregulation of phenazine and rhamnolipid virulence factors. Cystobactamids led to a downregulation of a glucose catabolism pathway. The study implies that clustering cellular mechanisms of action according to the primary target needs to take class-dependent variances into account. IMPORTANCE Novel antibiotics are urgently needed to tackle the growing worldwide problem of antimicrobial resistance. Bacterial pathogens possess few privileged targets for a successful therapy: the majority of existing antibiotics as well as current candidates in development target the complex bacterial machinery for cell wall synthesis, protein synthesis, or DNA replication. An important mechanistic question addressed by this study is whether inhibiting such a complex target at different sites with different compounds has similar or differentiated cellular consequences. Using transcriptomics and metabolomics, we demonstrate that three different classes of gyrase inhibitors can be distinguished by their molecular signatures in P. aeruginosa. We describe the cellular effects of a promising, recently identified gyrase inhibitor class, the cystobactamids, in comparison to those of the established gyrase A-binding fluoroquinolones and the gyrase B-binding aminocoumarins. The study results have implications for mode-of-action discovery approaches based on target-specific reference compounds, as they highlight the intraclass variability of cellular compound effects.
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Mode of action (MoA) identification of bioactive compounds is very often a challenging and time-consuming task. We used a label-free kinetic profiling method based on an impedance readout to monitor the time-dependent cellular response profiles for the interaction of bioactive natural products and other small molecules with mammalian cells. Such approaches have been rarely used so far due to the lack of data mining tools to properly capture the characteristics of the impedance curves. We developed a data analysis pipeline for the xCELLigence Real-Time Cell Analysis detection platform to process the data, assess and score their reproducibility, and provide rank-based MoA predictions for a reference set of 60 bioactive compounds. The method can reveal additional, previously unknown targets, as exemplified by the identification of tubulin-destabilizing activities of the RNA synthesis inhibitor actinomycin D and the effects on DNA replication of vioprolide A. The data analysis pipeline is based on the statistical programming language R and is available to the scientific community through a GitHub repository.