Metabolome-based studies of virulence factors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen and an important causative agent of potentially life-threatening nosocomial infections in predisposed patients. The Gram-negative bacterium produces a large and diverse repertoire of small-molecule secondary metabolites that serve as regulators and effectors of its virulence. In this study, a range of mass spectrometry-based bacterial metabolomics approaches was used to investigate these small-molecule virulence factors and their interplay with pseudomonal metabolism as well as with phenotypic traits related to virulence. The groundwork was laid by exploring the metabolite inventory of P. aeruginosa and improving the coverage of its metabolome by the application of a custom software named CluMSID, that clusters analytes based on similarities of their MS² spectra. CluMSID led to the annotation of, i.a., 27 novel members of the class of alkylquinolone quorum sensing signalling molecules, which represent crucial players in the highly complex network that regulates pseudomonal virulence. The tool was developed towards a versatile and user-friendly R package hosted on Bioconductor, whose functionalities and benefits are described in detail. The new findings on the alkylquinolone chemodiversity led to further studies with a mechanistic focus that probed the substrate specificity of the enzyme complex PqsBC. It was demonstrated that PqsBC accepts different medium-chain acyl-coenzyme A substrates for the condensation with 2-aminobenzoylacetate and thereby produces alkylquinolones with various side chain lengths, whose distribution is a function of substrate specificity and substrate availability. Moreover, it was shown that PqsBC also synthesises alkylquinolones with unsaturated side chains. The focus was further broadened from metabolite and pathway-centred questions to a more global perspective on pseudomonal virulence and metabolism, which directed attention at PrmC, an enzyme with a partially unknown function indispensable for in vivo virulence. An untargeted metabolomics experiment yielded insights into the role of PrmC and its influence on the pseudomonal endo- and exometabolome. Finally, clinical P. aeruginosa strains with different virulence phenotypes were examined by untargeted metabolomics in order to disclose metabolic variation and interconnections between virulence and metabolism. The analysis resulted in the discovery of a putative virulence biomarker and enabled the construction of a random forest classification model for certain virulence phenotypes based only on metabolomics data. In summary, this study demonstrated the potential of metabolomics for the investigation of P. aeruginosa virulence factors and thereby contributed towards the comprehension of the complex interplay of metabolism and virulence in this important pathogen.Pseudomonas aeruginosa ist ein wichtiger opportunistischer Erreger potenziell lebensbedrohlicher nosokomialer Infektionen bei prädisponierten Patienten. Das Gram-negative Bakterium produziert ein vielfältiges Repertoire an niedermolekularen Sekundärmetaboliten, die als Regulatoren und Effektoren seiner Virulenz dienen. In dieser Studie wurde eine Reihe von Massenspektrometrie-basierten Ansätzen der bakteriellen Metabolomik verwendet, um diese niedermolekularen Virulenzfaktoren und ihre Wechselwirkungen mit dem pseudomonalen Metabolismus sowie mit virulenzassoziierten phänotypischen Merkmalen zu untersuchen. Die Grundlage bilden die Untersuchung des Metaboliteninventars von P. aeruginosa und die Verbesserung der analytischen Abdeckung des Metaboloms durch die Anwendung einer selbstentwickelten Software namens CluMSID, die MS²-Spektren nach Ähnlichkeit clustert. CluMSID führte zur Annotation von u.a. 27 neuen Mitgliedern der Klasse der Alkylchinolone, die als Quorum-Sensing-Signalmoleküle entscheidende Akteure im hochkomplexen Netzwerk der Virulenzregulation darstellen. Das Tool wurde zu einem R-Paket entwickelt, das auf Bioconductor verfügbar ist und dessen Funktionalitäten und Vorteile ausführlich beschrieben werden. Die neuen Erkenntnisse über die Chemodiversität der Alkylchinolone führten zu weiteren Studien mit mechanistischem Schwerpunkt, die die Substratspezifität des Enzymkomplexes PqsBC untersuchten. Es wurde nachgewiesen, dass PqsBC verschiedene mittelkettige Acyl-Coenzym-A-Substrate für die Kondensation mit 2-Aminobenzoylacetat akzeptiert und dadurch Alkylchinolone mit verschiedenen Seitenkettenlängen produziert, deren Verteilung eine Funktion der Substratspezifität und der Substratverfügbarkeit ist. Zudem konnte gezeigt werden, dass PqsBC auch Alkylchinolone mit ungesättigten Seitenketten synthetisiert. Im Weiteren wurde der Fokus von Metaboliten- und Stoffwechselweg-zentrierten Fragen hin zu einer globaleren Perspektive der pseudomonalen Virulenz und des Metabolismus erweitert, was die Aufmerksamkeit auf PrmC lenkte, ein Enzym mit teilweise unbekannter, für die in vivo-Virulenz unverzichtbarer Funktion. Ein globales Metabolomik-Experiment lieferte Einblicke in die Rolle von PrmC und seinen Einfluss auf das pseudomonale Endo- und Exometabolom. Schließlich wurden klinische P. aeruginosa-Stämme mit unterschiedlichen Virulenzphänotypen mittels ungerichteter Metabolomik untersucht, um metabolische Variationen und Zusammenhänge zwischen Virulenz und Metabolismus aufzudecken. Die Analyse resultierte in der Entdeckung eines putativen Virulenzbiomarkers und ermöglichte die Konstruktion eines Random-Forest-Klassifikationsmodells für bestimmte Virulenzphänotypen, das nur auf Metabolomik-Daten basiert. Zusammenfassend hat diese Studie das Potenzial der Metabolomik für die Untersuchung der Virulenzfaktoren von P. aeruginosa aufgezeigt und damit zum Verständnis des komplexen Zusammenspiels von Metabolismus und Virulenz bei diesem wichtigen Pathogen beigetragen

Observing Short-Term Geomorphic Change in a Human-Modified River Using Terrestrial Repeat Photographs and Traditional Surveys: Uncompahgre River, Colorado, USA

The Uncompahgre River in Ouray, CO, was modified in 1996 from a braided river system to a meandering river channel. Large boulders of riprap were placed along designed meanders to prevent erosion and enable the development of permanent human structures on the flood plain. Deposition of gravel bars in the modified channel occurs annually during the summer. This gravel is "mined" by the City of Ouray; however, the effects of this excavation and the original modification were never assessed. This study provides an assessment by quantifying cross-sectional area change, cumulative grain-size distributions, shear stresses, slopes, and sinuosities using traditional survey methods. In addition, volume change of a gravel bar inside the modified channel was estimated using extreme oblique photographs (>45 degrees from nadir) that were obtained from nearby cliffs. Close-range photogrammetry was used in the natural channel downstream to evaluate photogrammetric methods using different lenses, image sensors, and camera geometries. Both traditional and photogrammetric methods clearly indicated significant deposition in the modified channel, whereas erosion occurred directly downstream from the modified channel, but did not occur at a reach 1.5 km downstream. In the natural channel, no cross-sectional area change occurred, grains were poorly sorted, and the longitudinal slope was ~four times steeper than the modified channel. Shear stress ratios were used as an erosion threshold, which did not correlate with actual cross-sectional area change, but a decrease in shear stress ratios from May 2011 to September 2011 were associated with erosion. Average RMSE values for DEMs created from extremeoblique photographs of a gravel bar in May 2011 and September 2011 were 0.140 m and 0.324 m, respectively. Using a DEM of difference with a t-statistic filter revealed that 115m3 of gravel was deposited. The Uncompahgre River showed similar geomorphic characteristics to other rivers in southwest Colorado, however, the slope of the natural and modified channels were much steeper than other rivers. Extreme-oblique photography and unconventional sensors both yielded reliable results, showing that these atypical techniques can be used in terrestrial photogrammetric applications such as, post-restoration assessments, as long as proper base-to-height ratios are achieved

Creating tangible and intangible hospitality products with a sustainable value – The case of the Altes Land apples

This paper focuses on a specific sub-part of hospitality, namely sustainable product creation via regional integration. According to the research question “To what extent does the integration of local apple products contribute to regional sustainable value creation in Altes Land in regard to tourism stakeholders?” this research examined the sustainable value – defined as people, planet, profit – created by one specific regional food speciality, the apples of Altes Land. The region Altes Land, situated close to Hamburg, Germany, is known for its apple products and promotes them as part of the cultural heritage of the region. For this purpose, the concepts were defined as regional value creation, sustainable value creation and regional food products. The primary research was conducted in the form of thirteen interviews with farmers, inhabitants and domestic visitors. In addition, observation was conducted in the area with a focus on the farms. The outcomes of the research highlight multiple values created, where all the different types of interviewees perceived values on all three levels. The apples and their production are seen as both of a high cultural value and also as making an economic and environmental contribution to the region. In economic terms, both visitors and inhabitants are also willing to pay more and specifically look out for these apples when shopping, hence a special value is also attached to them.Keywords: sustainable value creation, local food product

Observing Short-Term Geomorphic Change in a Human-Modified River Using Terrestrial Repeat Photographs and Traditional Surveys: Uncompahgre River, Colorado, USA

The Uncompahgre River in Ouray, CO, was modified in 1996 from a braided river system to a meandering river channel. Large boulders of riprap were placed along designed meanders to prevent erosion and enable the development of permanent human structures on the flood plain. Deposition of gravel bars in the modified channel occurs annually during the summer. This gravel is "mined" by the City of Ouray; however, the effects of this excavation and the original modification were never assessed. This study provides an assessment by quantifying cross-sectional area change, cumulative grain-size distributions, shear stresses, slopes, and sinuosities using traditional survey methods. In addition, volume change of a gravel bar inside the modified channel was estimated using extreme oblique photographs (>45 degrees from nadir) that were obtained from nearby cliffs. Close-range photogrammetry was used in the natural channel downstream to evaluate photogrammetric methods using different lenses, image sensors, and camera geometries. Both traditional and photogrammetric methods clearly indicated significant deposition in the modified channel, whereas erosion occurred directly downstream from the modified channel, but did not occur at a reach 1.5 km downstream. In the natural channel, no cross-sectional area change occurred, grains were poorly sorted, and the longitudinal slope was ~four times steeper than the modified channel. Shear stress ratios were used as an erosion threshold, which did not correlate with actual cross-sectional area change, but a decrease in shear stress ratios from May 2011 to September 2011 were associated with erosion. Average RMSE values for DEMs created from extremeoblique photographs of a gravel bar in May 2011 and September 2011 were 0.140 m and 0.324 m, respectively. Using a DEM of difference with a t-statistic filter revealed that 115m3 of gravel was deposited. The Uncompahgre River showed similar geomorphic characteristics to other rivers in southwest Colorado, however, the slope of the natural and modified channels were much steeper than other rivers. Extreme-oblique photography and unconventional sensors both yielded reliable results, showing that these atypical techniques can be used in terrestrial photogrammetric applications such as, post-restoration assessments, as long as proper base-to-height ratios are achieved

Chronic unpredictable stress regulates visceral adipocyte-mediated glucose metabolism and inflammatory circuits in male rats

Chronic psychological stress is a prominent risk factor involved in the pathogenesis of many complex diseases, including major depression, obesity, and type II diabetes. Visceral adipose tissue is a key endocrine organ involved in the regulation of insulin action and an important component in the development of insulin resistance. Here, we examined for the first time the changes on visceral adipose tissue physiology and on adipocyte-associated insulin sensitivity and function after chronic unpredictable stress in rats. Male rats were subjected to chronic unpredictable stress for 35 days. Total body and visceral fat was measured. Cytokines and activated intracellular kinase levels were determined using high-throughput multiplex assays. Adipocyte function was assessed via tritiated glucose uptake assay. Stressed rats showed no weight gain, and their fat/lean mass ratio increased dramatically compared to control animals. Stressed rats had significantly higher mesenteric fat content and epididymal fat pad weight and demonstrated reduced serum glucose clearing capacity following glucose challenge. Alterations in fat depot size were mainly due to changes in adipocyte numbers and not size. High-throughput molecular screening in adipocytes isolated from stressed rats revealed activation of intracellular inflammatory, glucose metabolism, and MAPK networks compared to controls, as well as significantly reduced glucose uptake capacity in response to insulin stimulation. Our study identifies the adipocyte as a key regulator of the effects of chronic stress on insulin resistance, and glucose metabolism, with important ramifications in the pathophysiology of several stress-related disease states

Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

The thyroid hormone derivative 3-iodothyronamine (3-T1AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs

In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens

<i>Staphylococcus aureus </i>Transcriptome Architecture:From Laboratory to Infection-Mimicking Conditions

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 1