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Cyanobakterien sind eine vielversprechende Quelle an strukturell diversen und biologisch hochaktiven Naturstoffen für die Entwicklung neuer Wirkstoffe. Bislang konnte die Strukturklasse der [7.7]Paracyclophane nur in fädigen Cyanobakterien der Gattungen Nostoc und Cylindrospermum nachgewiesen werden. Vorangegangene Arbeiten zeigten, dass gerade die Carbamidocyclophane chemisch und biologisch interessante Verbindungen darstellen. Im Rahmen dieser Arbeit wurden vor allem die Carbamidocyclophane produzierenden Cyanobakterien Nostoc sp. CAVN2 und Nostoc sp. CAVN10 unter besonderer Berücksichtigung der strukturellen Vielfalt an biosynthetisierten Metaboliten sowie deren antimikrobieller Aktivität umfassend charakterisiert. Um das biosynthetische Potenzial auf der metabolischen Ebene zu untersuchen, wurde im Vorfeld eine spezifische [7.7]Paracyclophan-Analytik etabliert, die skalierbare Methoden für alle Aufarbeitungsschritte beinhaltet. Die Optimierung endete in einem validierten sowie arbeits- und zeitsparenden einstufigen Extraktions- und Aufreinigungsverfahren mittels eines Zweiphasensystems und anschließender LC-UV-Analyse, um biologische Proben reproduzierbar zu analysieren und enthaltene Carbamidocyclophane zu quantifizieren. Kultivierungsstudien zum Einfluss der Temperatur an metabolisch aktiven und defizienten Nostoc-sp.-CAVN10-Kulturen ergaben einen direkten Zusammenhang zwischen der Biomassezunahme und der Temperaturerhöhung. Im Gegensatz dazu zeigten die einzelnen Carbamidocyclophan-Gehalte ein eher differenzierteres Bild über die verschiedenen Wachstumsphasen und Temperaturen hinweg. Da nur eine geringe Korrelation zwischen der spezifischen Wachstumsrate und der spezifischen Carbamidocyclophan-Produktionsrate ermittelt werden konnte, ist eine Relevanz dieser Verbindungen für den primären Zellstoffwechsel nicht ersichtlich. Bei Kultivierungsexperimenten an Nostoc sp. CAVN2 hatte der Zusatz von Chlorid- oder Bromid-Ionen eine drastische Erhöhung der Basalrate und Strukturdiversität der [7.7]Paracyclophane zur Folge. Das gleichzeitige Vorhandensein beider Halogenide im Medium zeigte kompetitive Effekte, wobei Chlorid als Substrat für den Halogenierungsprozess favorisiert wurde. Mit Hilfe eigens entwickelter Kultivierungsprozedere und Separierungsstrategien konnten insgesamt 25 Verbindungen aus Stamm CAVN2 isoliert und strukturell aufgeklärt werden. Dabei bilden die Carbamidocyclophane H–U neue chlorierte, bromierte und nicht halogenierte Naturstoffe. Zusätzlich konnten aus Stamm Cylindrospermum stagnale PCC 7417 neben den bekannten Cylindrocyclophanen A, B und D die drei neuen Cylindrofridine A–C erhalten werden. Diese stellen den Cylindrocyclophanen strukturell eng verwandte lineare Mono- und Dialkylresorcinole dar. Die vergleichende Evaluierung der Bioaktivität von 30 Reinsubstanzen ergab, dass viele Verbindungen sehr starke antimikrobielle Aktivität gegen grampositive Bakterien zeigen – besonders gegen Antibiotika-resistente Kokken mit minimalen Hemmkonzentrationen von oftmals deutlich unter 1 µM. Dabei bedingten die verschiedenen Substituenten (Carbamoyl- und Acetoxy-Reste sowie Hydroxygruppen oder Halogene) z.T. signifikante Aktivitätsunterschiede. Die Zytotoxizität der [7.7]Paracyclophane ist vor allem an das Vorhandensein des Makrozyklus gebunden, da lineare Derivate (Cylindrofridine B/C) kaum biologisch aktiv waren. Eine Ausnahme stellt dabei das nicht zytotoxische, aber antimikrobiell aktive Cylindrocyclophan-D-Monomer Cylindrofridin A dar. Die phylogenetische Analyse der 16S-rDNA-Daten bestätigte die morphologisch-taxonomische Identifizierung der Stämme CAVN2 und CAVN10 als Cyanobakterien der Gattung Nostoc und ergab weiterhin, dass alle Carbamido- und Cylindrocyclophane produzierenden Nostoc-Stämme Bestandteil einer monophyletischen Gruppe sind, die phylogenetisch distinkt zu anderen [7.7]Paracyclophan-Produzenten ist. Des Weiteren konnten keine Nukleotidunterschiede zwischen Stamm CAVN2 und CAVN10 auf den untersuchten Markergen-Sequenzen festgestellt werden, was beide auf der phylogenetischen Ebene als identisch erscheinen lässt und sie somit nur metabolisch aufgrund der strukturellen Diversität und Quantität an [7.7]Paracyclophanen differenzierbar sind. Mit Hilfe von molekulargenetischen Analyseverfahren und bioinformatorischer Auswertung konnte in Stamm CAVN2 das Carbamidocyclophan-Biosynthesegencluster mit einer Gesamtgröße von ca. 26,9 kbp identifiziert werden. Dieses beinhaltet 13 offene Leserahmen (cabA-cabM), wobei das Gen cabL für eine putative Carbamoyltransferase codiert. Ein neuer Halogenase-Typ in Verbindung mit einer Tandem-ACP-Domänen-Struktur in der Typ I Polyketidsynthase CabD könnte für die Ausbildung halogenierter Derivate verantwortlich sein. Der Nachweis eines codierenden Bereichs mit Rieske-Domäne (cabM) lässt eine direkte oxidative intermolekulare Makrozyklisierung bei der Assemblierung vermuten.
A highly stereoselective recombinant alcohol dehydrogenase aus 'Pseudomonas fluorescens' DSM50106
(2005)
The alcohol dehydrogenase was biochemically characterized. A broad range of arylaliphatic ketones is efficiently reduced to the corresponding optically active (R)-alcohols by a recombinant alcohol dehydrogenase (PF-ADH) produced by overexpression in 'Escherichia coli'. PF-ADH shows high activity and stereoselectivity in the reduction of acetophenone and various derivatives (45-99%), as well as in the reduction of 3-oxy-butyric acid methyl ester and 3-oxy-butyric acid methyl ester and 3-oxy-hexanoic acid ethyl ester (>99%). The highest activity was observed between 10 and 20°C. The copfactor NADH can be efficiently recycled by the addition of 10-20% of iso-propanol. A flow-through-polarimetry-based assay to determine oxidoreductase activity and stereoselectivity is described.
Understanding the fundamental mechanisms in the extracellular matrix of cells (ECM) is crucial for the development of drugs and biomaterials. Therefore, an atomistic model of the extracellular matrix is a cost-efficient way to observe influences of drugs, test the effect of mutations or misfolds in proteins or study the properties of fibril or network-forming peptides.
With this thesis, a refined molecular model of an adhesion complex is proposed that contains collagen, fibronectin and the cell receptor integrin. During the building of the model, major new insights are given for each of these proteins and a powerful protein-folding algorithm is
developed.
In their idealized forms, enzymes can facilitate complex reactions with extreme specificity and selectivity. Additionally, in this imaginative form, they only require mild reaction conditions, resulting in low energy consumption, and they are biodegradable, efficient, reusable, and sustainable. Unfortunately, this idealized form often deviates significantly from reality, where enzymes are more likely to be associated with marginal stability and low reaction rates, leaving them less than desirable for many industrial applications. As such, if we could master the process of engineering the configuration of a protein towards a given task, the implications could be staggering.
This thesis aims to contribute to the process of protein engineering, mainly how computational tools can be used to make the protein engineering process more efficient and accessible.
Article I explores the current state of the art in machine learning-guided directed evolution and serves as a foundation for Article II, which is a concrete application of these techniques to an engineering campaign. Despite successfully improving overall activity and selectivity, we also observe limitations and constraints within the methodology. Article III then delves into these drawbacks and attempts to lay the foundation for a more generalizable and, more importantly, efficient engineering workflow, balancing the strengths and weaknesses of computational techniques with advances in gene synthesis. We then validated this novel pipeline in Article IV, where we show the potential of this methodology. Article V describes a more standard protein engineering campaign on squalene-hopene cyclases for potentially interesting products in the flavor and fragrance industry. Lastly, Article VI outlines a PyMol plugin for molecular docking.
Im Rahmen dieser Arbeit wurden neuartige alpha-Phosphanylaminosäuren untersucht. Die Verbindungen wurden durch eine Dreikomponenten-Eintopfreaktion bei Raumtemperatur aus Diphenylphosphan, einem primären Amin und Glyoxylsäure hergestellt. Alle Verbindungen sind luftempfindlich und bilden in Lösung langsam Zersetzungsprodukte. Es wurden P-Sulfide, P-Oxide und P-Pentacarbonylmetall(0)komplexe hergestellt, Versuche zur Synthese von BH3-Addukten in Molverhältnis 1:1 und 1:3 durchgeführt. Das enantiomerenreine 1-(p-methoxyphenyl)ethyl-substituierte Phosphanylglycin wurde als Ligand auf Eignung in enantioselektiven, katalytischen Hydrierungen verschiedener alpha-, beta-ungesättigter Ketoverbindungen untersucht. Sieben Verbindungen aus verschiedenen Gruppen N-substituierter Phosphanylglycine wurden als Liganden mit Ni(COD)2 zu in situ in Katalysatoren umgesetzt und damit die Poly/Oligomerisation von Ethylen untersucht. Die meisten untersuchten Liganden bewirkten hohe katalytische Umsätze von Ethylen und zeigten somit gute Eignung als Liganden zur Stabilisierung aktiver Ni-Oligomerisationskatalysatoren.
The widespread use of natural and synthetic estrogens or chemicals with estrogenic activities is causing an increasing accumulation of estrogenic compounds in the environment. Already at very low concentrations these estrogenics can severely affect the wildlife, particularly in an aquatic environment. For these reasons measuring devices for detecting estrogen contaminations are in great demand. The majority of the analytical methods and bioassays on the market so far, lack semi-online adaptability, and usually cannot be used for automatic and continuous determination. Therefore, we have embarked on the development of new systems, which are able to fulfil those demands. The EstraMonitor combines recombinant A. adeninivorans G1212/YRC102-hERa-phyK yeast cells as the microbial component with an amperometric detection method to analyze estrogenic contaminations. A. adeninivorans G1212/YRC102-hERa-phyK was constructed by Kaiser et al. (2010). These cells were engineered to co-express the human estrogen receptor (hERa) gene and the inducible phytase (phyK, derived from Klebsiella sp. ASR1) reporter gene under control of a promoter with estrogen response elements (EREs). In the presence of estrogenic substances, such as 17ß -estradiol (E2), the phyK gene is expressed and recombinant phytase is secreted into the media. The level of phytase is quantified by amperometric detection using substrate p-aminophenyl phosphate (p-APP). Phytase dephosphorylates p-aminophenyl phosphate (p-APP) into an intermediate product p-aminophenol (p-AP). p-AP is electroactive and oxidized at the electrode. This generates electrons and produces a current which is proportional to the level of phytase activity. Since phytase activity is directly correlated to the E2 concentration, the estrogenic activity can thus be calculated from the current measured. The microbial component of the EstraMonitor, the non-immobilized A. adeninivorans G1212/YRC102-hERa-phyK, works well with the amperometric method in a quantitative manner. The optimal applied potential determined for amperometric measurements was 150 mV and provided a low background signal for the amperometric detection. The half maximal effective concentration (EC50) and limit of detection (LoD) values for E2 obtained from amperometric measurements with the EstraMonitor were 69.9 ng L-1 and 44.5 ng L-1, respectively. The measuring procedure of the EstraMonitor system including incubation of A. adeninivorans G1212/YRC102-hERa-phyK cells with E2, subsequently incubation with electrochemical substrate (p-APP), and signal recordation is completed within only 4 h and 10 min. Out of this total time, amperometric detection including substrate incubation and signals recordation takes only 10 min out of total time. The use of immobilized cells for a microbial biosensor is an essential advantage of the EstraMonitor system because it allows easy-handiness next to long-term stability and reusability. Immobilized A. adeninivorans G1212/YRC102-hERa-phyK cells revealed excellent properties which make them very suitable for semi-online, automatic and continuous monitoring. They were stable up to 30 days when stored at 4 °C. Furthermore, they could be reused up to 15 times. The EC50 and LoD values achieved for E2 using immobilized cells in combination with amperometric detection were 20.9 and 8.3 ng L-1, respectively. Furthermore, this application also removes the need to separate cells by centrifugation, to sterilize the samples as well as to cultivate repeatly. Additionally, both immobilized and non-immobilized A. adeninivorans G1212/YRC102-hERa-phyK cells remain fully functional in a wide range of untreated wastewater samples and in environments containing up to 5% NaCl. To enhance the sensitivity and reduce the time for estrogenic determination, an alternative A. adeninivorans G1214/YRC103-hERa-phyK strain was developed. This strain can produce a detectable amount of phytase within 2 h after induction with E2. It offers an improved microbial component in terms of sensitivity and time-effectiveness. In addition, to reduce the cost for estrogenic detection an alternative substrate, ascorbic acid 2-phosphate (AA2P), was tested. AA2P, which is both cheap and widely available, performed better than p-APP. The EC50 and LoD values for E2 obtained with AA2P were 15.69 and 0.92 ng L-1 versus 20.09 and 8.3 ng L-1 when examined with p-APP, respectively. Taken together, the EstraMonitor is an automated system with respect to sample cycling, sample measuring and calibration supplemented with an alarm function. This system makes it possible to control estrogenic activity semi-online, automatically and continuously. These are advantages of the EstraMonitor compared to other estrogenic detection systems. It can thus be concluded that, the EstraMonitor is a powerful and feasible semi-online device for monitoring estrogenic activity especially adapted for the use in sewage treatment plants.
In this thesis an artificial enzyme cascade consisting of an ADH from Lactobacillus kefir, a CHMO from Acinetobacter sp. NCIMB 9871 and lipase A from Candida antarctica has been investigated for the biocatalytic synthesis of the bulk chemical ε-caprolactone as well as several derivatives for their direct utilization as polymer building blocks. Due to major limitations, which hamper such a biocatalytic route, the first addressed demand in this work was the improvement of the stability of the CHMO. By structure-guided engineering, distinctively improved variants concerning the resistance against oxidation as well as temperature stability without compromising the catalytic activity were successfully created. Due to the incomplete knowledge of the mechanisms that lead to thermal and/or oxidative inactivation of enzymes, this study illustrates that the selection of mutations for increased protein stability is still hard to predict. Thus, these results can serve as a basis for further stability studies on this enzyme class to give better insights into the underlying mechanisms, which determine the stability of an enzyme. Such a highly stabilized biocatalyst will pave the way for the successful use of flavin-dependent enzymes for industrial applications. A further aim of this thesis was dedicated to the second major hurdle en route to polyester precursors represented by the product inhibition and enzyme deactivation caused by ε-caprolactone, particularly at higher concentrations. To overcome this limitation, we developed an elegant solution in which the ε-caprolactone produced by the one-pot two-step enzymatic method is directly subjected to ring-opening polymerization using the unique lipase A from Candida antarctica. Applying this enzyme cascade in a whole cell biocatalysis in combination with an improved cofactor regeneration approach, the problem of product inhibition problem was efficiently solved leading to the formation of oligo-ε-caprolactone at more than 20 g/L when starting from 200 mM cyclohexanol. By a process development approach through solvent engineering it was found that biotransformations proceed much faster in an isooctane-containing biphasic solvent system when using free enzymes. Finally, the improved enzyme cascade was applied for the synthesis of chiral substrates and provided access to functionalized chiral compounds in high yields (up to >99%) and optical purities (up to >99%ee). By subsequent enzymatic enantioselective ring-opening of the enantiopure monomers, oligomeric lactones were successfully synthesized, which can be directly serve as building blocks for the polymer industry.
Analyse der metabolischen Anpassung von Streptococcus pneumoniae an antimikrobielle Umwelteinflüsse
(2019)
Das Gram-positive Bakterium Streptococcus pneumoniae ist ein humanspezifisches Pathogen des oberen Respirationstraktes. Der opportunistische Krankheitserreger kann jedoch mehrere Organe befallen und tiefer in den Körper vordringen, was zu lokalen Entzündungen wie Sinusitis und Otitis media oder zu lebensbedrohlichen Infektionen wie Pneumonie, Meningitis oder Sepsis führen kann. Für das Bakterium S. pneumoniae wurden bisher kaum Metabolom-Daten erhoben. Daher war das Ziel dieser Dissertation eine umfassende Charakterisierung des Metaboloms von S. pneumoniae. In dieser Dissertation wurden als analytische Methoden die Gaschromatografie (GC) und Flüssigkeitschromatografie (LC) jeweils gekoppelt mit Massenspektrometrie (MS) sowie die Kernspinresonanzspektroskopie (NMR) verwendet, um die Metaboliten zu analysieren. Es sind mehrere Analysetechniken erforderlich, um den Großteil des Metaboloms mit seinen chemisch verschiedenen Metaboliten zu erfassen. Artikel I fasst die Literatur zu Untersuchungen des Metabolismus von S. pneumoniae in den letzten Jahren zusammen. Um eine Momentaufnahme des biologischen Systems zum jeweiligen Zeitpunkt zu erhalten, ist neben dem reproduzierbaren Wachstum während der Kultivierung auch die exakte Probenahme zu beachten. Aus diesem Grund wurde in dieser Dissertation ein Probenahmeprotokoll für das Endometabolom von S. pneumoniae etabliert (Artikel II). Mithilfe des optimierten Protokolls wurde eine umfassende Metabolomanalyse in einem chemisch definierten Medium durchgeführt (Artikel II). Um S. pneumoniae in einer Umgebung ähnlich der im Wirt zu untersuchen, wurde in einem modifizierten Zellkulturmedium kultiviert. Intermediate zentraler Stoffwechselwege von S. pneumoniae wurden analysiert. Das intrazelluläre Stoffwechselprofil wies auf einen hohen glykolytischen Flux hin und bot Einblicke in den Peptidoglykan-Stoffwechsel. Darüber hinaus widerspiegelten die Ergebnisse die biochemische Abhängigkeit von S. pneumoniae von aus dem Wirt stammenden Nährstoffen. Ein umfassendes Verständnis der Stoffwechselwege von Pathogenen ist wichtig, um Erkenntnisse über die Anpassungsstrategien während einer Infektion zu gewinnen und so neue Angriffspunkte für Wirkstoffe zu identifizieren.
Die zunehmende Verbreitung von resistenten S. pneumoniae-Stämmen zwingt zur Suche nach neuen antibiotisch wirksamen Substanzen. Im Zuge dessen wurde in Artikel III die metabolische Reaktion von S. pneumoniae während des Wachstums unter dem Einfluss antibakterieller Substanzen mit dem Ziel der Identifizierung metabolischer Anpassungsprozesse untersucht. Dabei wurden Antibiotika mit unterschiedlichen Wirkmechanismen verwendet, wie die Beeinflussung der Zellwandbiosynthese (Cefotaxim, Teixobactin-Arg10), der Proteinbiosynthese (Azithromycin) sowie Nukleotidsynthese (Moxifloxacin). Es konnten keine Wirkmechanismus-spezifischen Marker-Metaboliten identifiziert werden. Jedes Antibiotikum verursachte weitreichende Veränderungen im gesamten Metabolom von S. pneumoniae. Die Nukleotid- und Zellwandsynthese waren am stärksten betroffen. Besonders vielversprechend sind Antibiotika mit zwei Wirkorten wie Teixobactin-Arg10 und Kombinationen aus zwei Antibiotika. In dieser Dissertation wurde das erste Mal das synthetisch hergestellte Teixobactin-Arg10 mittels einer der modernen OMICS-Techniken untersucht. Die vorliegende umfassende Metabolom-Studie bietet wertvolle Erkenntnisse für Forscher, die an der Identifizierung neuer antibakterieller Substanzen arbeiten.
Insgesamt tragen die Ergebnisse der Dissertation zu einem besseren Verständnis der bakteriellen Physiologie bei.
Im Rahmen der vorliegenden Arbeit wurde ein auf dem Promotor Pdes basierendes Kälte-induzierbares Expressionssystem für B. subtilis konstruiert und sukzessive optimiert. Dazu wurden verschiedene Kälte-regulatorische DNA-Sequenzen aus B. subtilis an das entsprechende Zielgen fusioniert, was neben der Kälte-Induzierbarkeit in einem positiven Einfluss auf die Expressionsstärke durch eine effizientere Translation bzw. Stabilisierung der mRNA resultierte. Vorausgehend wurde in vergleichenden Versuchen die Eignung unterschiedlicher Galaktosidasen zur Verwendung als Reporterenzyme für B. subtilis untersucht. Hierbei wurde erstmals die heterologe Expression einer Kälte-angepassten β-Galaktosidase aus P. haloplanktis TAE79 in B. subtilis durchgeführt und diese durch die Integration der DB-Sequenz sowie einer stem-loop-Struktur aus der 5‘-UTR des B. subtilis cspB-Gens gesteigert. Somit konnte nachgewiesen werden dass sowohl die additiven Sequenzen der cspB-DB und der cspB-sl-UTR als auch des bkdB-Terminators zu einer deutlich erhöhten Synthese der entsprechenden Zielproteine führt. Anhand der Überexpression einer Xylanase aus B. subtilis sowie einer α-Glucosidase aus S. cerevisiae wurde abschließend die Eignung des konstruierten Systems für die sekretorische und intrazelluläre Proteinsynthese in B. subtilis demonstriert. Diese Ergebnisse bestätigen die Eignung von B. subtilis als Wirtsorganismus auch für die Überproduktion kritischer, schwer zu faltender Proteine.
Analysis of bioactive lipids from different infection models during bacterial and viral infections
(2021)
Bioactive lipids or lipid mediators influence numerous processes like the reproduction, the bone turnover, the pain perception, the cardiovascular function and the immune system. Eicosanoids and oxylipins are parts of the immunomodulatory lipid mediators, which can be synthesized from polyunsaturated fatty acids (PUFAs) by enzymatic and non-enzymatic reactions. Typical members of eicosanoids are prostaglandins and leukotrienes. The properties of bioactive lipids include the activation of inflammatory reactions as well as the support of resolution. Like hormones, they act locally restricted and in low concentrations. Further bioactive lipids exist i.e. intermediates of the sphingolipid class. The biosynthesis of some of these compounds like the prostaglandins can be influenced by different drugs whereas for other groups of lipid selective inhibitors are still missing. Their impact on inflammatory processes and against chronic diseases has already been analyzed, while studies in context with infection are largely limited. Infection of the upper respiratory tract caused by viral and bacterial pathogens constitute a huge burden for the human healthcare. The main pathogens are the Influenza A virus (IAV), Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae) and Streptococcus pyogenes (S. pyogenes). Besides mono-infection with one of these pathogens, frequently occurring bacto-viral co-infections exist, which negatively influence the etiopathology. The main task of the immune system is the detection and the elimination of pathogens, which can essentially be affected by lipid mediators. Their instability due to oxidizability, the existence of regioisomers and the low abundance of eicosanoids and other oxylipins are the main problems for their analytical measurement.
The mayor objective of this dissertation was the establishment of a suitable analytical method for selected lipid mediators and the detection of infection-related changes. The separation and detection was performed by using high-performance liquid chromatography (HPLC) coupled with triple quad mass spectrometry. This combination is called tandem mass spectrometry (MS/MS). The MS parameters were optimized for approximately 30 lipid mediators by use of chemical standards and the detection was achieved by dynamic multiple reaction monitoring (MRM). Furthermore, the spatial resolution of selected sphingolipids was analyzed in tissue samples using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MS-Imaging). Concerning the HPLC-MS/MS detection, an MS method was established and optimized with standard compounds. Another crucial part of the establishment was the extraction of bioactive lipids from the different sampling materials. Whereas well tested protocols exist for the extraction and detection of lipid mediators, such protocols for MALDI-MS-Imaging are still limited due to the novelty of this measurement. Ultimately, robust and reproducible protocols for both techniques that were used for the analysis of a broad array of samples from infection experiments were established for both techniques. The analyses of infected cell culture, mice and pigs revealed infection-related perturbations of host lipid mediator levels. Depending on the scientific issue, the sample types cell pellets, lungs, spleens, livers, blood plasmas, pawns including bones or bronchoalveolar lavages were analyzed. For MALDI-MS-Imaging, the spatial distribution of sphingolipids in lung and spleen was detected.
The present dissertation includes four coherent research scopes, in which the pathogen impact on host-derived lipid mediators was detected with the above mentioned analytical methods. The infection models epithelial cells (article II), mouse (article III and IV) and pig (article I) – the latter as the most human like model - showed different aspects of the host-pathogen interaction. The analysis of samples from IAV infection for all three hosts revealed a couple of similarities for some oxylipins that were also described in human infections. Additionally, cell culture and mouse samples from mono-infections as well as co-infections with the pathogens S. aureus and S. pneumoniae were measured. In particular for the bacterial mono- and co-infections, these are the first published results with aspects of infection related changes of lipid mediators. The additional spatial resolution of the sphingolipid intermediates sphingosine 1-phosphate and ceramide 1-phosphate revealed important new insights into their tissue distribution and changes during co-infection.
Article I describes the IAV-specific oxylipin changes in the pig (german landrace) as infection model. Therefore, the sample types lung, spleen, blood plasma, and bronchoalveolar lavage from infected animals at different time points after infection were analyzed and compared with samples from uninfected pigs. Mainly in the lung and the spleen, increased amounts of certain lipid mediators were observed. These changes coincide well with already described alterations in humans and mice. Furthermore, the analysis of different sample material provided an overview about appropriate sample types. Surprisingly, many perturbations were detected in the spleen, which itself was uninfected. Based on the local reaction of lipid mediators, most studies concentrate on sample material with close contact to side of infection. Therefore, this dissertation reveals new insights into a form of systemic immune response. Besides the use of animals with a complex immune system for infection experiments, human bronchial epithelial cells (16HBE) were mono- and co-infected with the pathogens S. aureus, S. pneumoniae and IAV as described in article II. Such cells are the initial barrier for and first contact site with pathogens and thus the comprehension of this host-pathogen interaction is of essential importance. Most changes were detected during pneumococcal infection. Furthermore, the analyzed infections with bacterial pathogens differed from IAV infection by an increased synthesis of 5-hydroxyeicosatetraenoic acid (HETE). For further infections with the above mentioned pathogens, the mouse was used as an infection model. Besides infections affecting the respiratory tract, also the impact of an S. pyogenes infection in different mice strains was analyzed and described in article III. Infection-related changes in prostaglandins, which are involved in bone turnover in swollen pawns as well as enhanced amounts of sepsis- and arthritis-associated lipid mediators were detected, in case arthritis had been induced prior to infection. Furthermore, increased amounts of 20-HETE could be observed for such severe infections. An enhanced biosynthesis of 20-HETE was further confirmed in a high-pathogenic S. aureus LUG2012 infection in article IV for all examined sample types. In this last article of this dissertation, bacterial and viral infections in mice were analyzed similar to those described in article II. Mainly IAV-specific lipid mediator alterations were detected, which are in accordance with the findings of the infected pigs. The additional MALDI-MS-Imaging measurements revealed so far unknown accumulation of ceramide 1-phosphate in lung and spleen as well as enrichment in the red pulp of the spleen.
In summary, this dissertation provides substantial lipid mediator profiles for infections in three different model systems with selected bacterial and viral pathogens. The obtained data constitute a suitable basis for continuative research projects, in which the influence of single bioactive lipids on the course of infection could be examined in more detail.