Molekulare Analysen der bakteriellen Gemeinschaften in Trinkwasser und Biofilmen - Säsonale Dynamik, Viabilität und Zusammensetzung der Bakteriellen Gemeinschaften von kaltem und heißem Trinkwasser

Although water treatment and disinfection measures aim at eliminating all harmful mircroorganisms, the water quality at the customer tap does not necessarily provide the same quality as the water leaving the waterworks facility. It still provides an environment for a regrowing microflora. The objective of the present thesis was to get insights into the bacterial drinking water communities and their associated habitats (e.g biofilms and hot water). Therefore, it is of relevance, which environmental factors are influencing the presence and activity of the members of these communities and what interactions exist between these communities. In the first two parts of the thesis we investigated the bacterial dynamics in the drinking water of Braunschweig, Germany, over the timeframe of 18 month. Using SSCP-fingerprints and sequencing major bands we analysed the seasonal dynamics of the community composition and its activity of cold and hot drinking water ot the the campus of the Helmholtz Centre for Infection Research (HZI) in relation to meteorological data of the catchment area. For cold drinking water it was demonstrated that the precipitation was the major impact influencing the amount and activity of bacteria. The hot drinking was mainly composed of thermophilic and thermotolerant bacteria. In the third part a detailed analysis of the bacterial community in biofilms and corresponding bulk water was done. The bacteria in all biofilm samples showed a highly different structure with no overlap to the phylotypes observed in bulk water. Biofilms sampled on nearby sampling points showed similar communities in spite of different support materials. In the fourth part we investigated which bacterial species are present in drinking water. Therefore a combination of Live/Dead-staining, FACS and community fingerprinting was used for the analysis of drinking water samples. The developed approach enabled the assessment of the physiological state of taxonomic groups of interest.Obwohl die üblichen Aufbereitungsmaßnahmen alle schädlichen Mikroorganismen eliminieren wollen, erreicht die Wasserqualität beim Endverbraucher nicht dieselbe Qualität wie nach der Wasseraufbereitung. Trinkwasser bietet immer noch einen Lebensraum für eine nachwachsende Mikroflora. Die Zielsetzung dieser Dissertation war, Einblicke in die bakteriellen Gemeinschaften von Trinkwasser und seinen assoziierten Habitaten (z.B. Biofilme, Heißwasser) zu bekommen. Dazu war die Fragestellung, welche Umweltfaktoren die Präsenz und die Aktivität der Mitglieder dieser bakteriellen Gemeinschaften beeinflussen und welche Interaktionen zwischen diesen Gemeinschaften existieren. In den ersten beiden Teilen der Dissertation untersuchten wir die bakterielle Dynamik im Trinkwasser innerhalb von 18 Monaten. Mit SSCP-Fingerprints und dem Sequenzieren der Hauptbanden wurde die saisonale Dynamik der bakteriellen Gemeinschaft und ihrer Aktivität im kalten und heißem Trinkwasser des Campus des Helmholtzzentrums für Infektionsforschung untersucht. Durch die Relation zu den meteorologischen Daten des Einzugsgebiets der Talsperren wurde für das kalte Trinkwasser gezeigt, dass die Bakterienzahl und -aktivität hauptsächlich durch den Niederschlag beeinflußt wurde. Im heißen Trinkwasser fanden sich primär thermophile und thermotolerante Bakterien. Im dritten Teil wurde eine detaillierte Analyse der bakteriellen Gemeinschaft in Trinkwasser-Biofilmen und dem dazugehörigen Wasser untersucht. Alle Biofilme zeigten eine eigene, einzigartige Struktur ohne gemeinsame Phylotypen zum Trinkwasser. Trotz unterschiedlichen Oberflächenmaterials wiesen benachbarte Biofilme ähnliche bakterielle Gemeinschaften auf. Im vierten Teil wurde untersucht, welche Bakterien lebendig im Trinkwasser zu finden sind. Hierzu wurde ein kombinierter Ansatz aus Lebend/Tot-Färbung und Durchflußzytometrie verwendet. Dieser Ansatz ermöglichte eine Einschätzung des physiologischen Zustandes der bedeutenden taxonomischen Gruppen

Itaconate controls its own synthesis via feedback-inhibition of reverse TCA cycle activity at IDH2.

peer reviewedMacrophages undergo extensive metabolic reprogramming during classical pro-inflammatory polarization (M1-like). The accumulation of itaconate has been recognized as both a consequence and mediator of the inflammatory response. In this study we first examined the specific functions of itaconate inside fractionated mitochondria. We show that M1 macrophages produce itaconate de novo via aconitase decarboxylase 1 (ACOD1) inside mitochondria. The carbon for this reaction is not only supplied by oxidative TCA cycling, but also through the reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase (IDH). While macrophages are capable of sustaining a certain degree of itaconate production during hypoxia by augmenting the activity of IDH-dependent reductive carboxylation, we demonstrate that sufficient itaconate synthesis requires a balance of reductive and oxidative TCA cycle metabolism in mouse macrophages. In comparison, human macrophages increase itaconate accumulation under hypoxic conditions by augmenting reductive carboxylation activity. We further demonstrated that itaconate attenuates reductive carboxylation at IDH2, restricting its own production and the accumulation of the immunomodulatory metabolites citrate and 2-hydroxyglutarate. In line with this, reductive carboxylation is enhanced in ACOD1-depleted macrophages. Mechanistically, the inhibition of IDH2 by itaconate is linked to the alteration of the mitochondrial NADP+/NADPH ratio and competitive succinate dehydrogenase inhibition. Taken together, our findings extend the current model of TCA cycle reprogramming during pro-inflammatory macrophage activation and identified novel regulatory properties of itaconate

Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages.

peer reviewedSince its discovery in inflammatory macrophages, itaconate has attracted much attention due to its antimicrobial and immunomodulatory activity1-3. However, instead of investigating itaconate itself, most studies used derivatized forms of itaconate and thus the role of non-derivatized itaconate needs to be scrutinized. Mesaconate, a metabolite structurally very close to itaconate, has never been implicated in mammalian cells. Here we show that mesaconate is synthesized in inflammatory macrophages from itaconate. We find that both, non-derivatized itaconate and mesaconate dampen the glycolytic activity to a similar extent, whereas only itaconate is able to repress tricarboxylic acid cycle activity and cellular respiration. In contrast to itaconate, mesaconate does not inhibit succinate dehydrogenase. Despite their distinct impact on metabolism, both metabolites exert similar immunomodulatory effects in pro-inflammatory macrophages, specifically a reduction of interleukin (IL)-6 and IL-12 secretion and an increase of CXCL10 production in a manner that is independent of NRF2 and ATF3. We show that a treatment with neither mesaconate nor itaconate impairs IL-1β secretion and inflammasome activation. In summary, our results identify mesaconate as an immunomodulatory metabolite in macrophages, which interferes to a lesser extent with cellular metabolism than itaconate

Pyruvate dehydrogenase fuels a critical citrate pool that is essential for Th17 cell effector functions

Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in T helper 17 (Th17) cells still remains to be studied. Here, we show that PDH is essential for the generation of a glucose-derived citrate pool needed for Th17 cell proliferation, survival, and effector function. In vivo, mice harboring a T cell-specific deletion of PDH are less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increases glutaminolysis, glycolysis, and lipid uptake in a mammalian target of rapamycin (mTOR)-dependent manner. However, cellular citrate remains critically low in mutant Th17 cells, which interferes with oxidative phosphorylation (OXPHOS), lipid synthesis, and histone acetylation, crucial for transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restores their metabolism and function, identifying a metabolic feedback loop within the central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity

Integration of JAK/STAT receptor-ligand trafficking, signalling and gene expression in Drosophila melanogaster cells

The JAK/STAT pathway is an essential signalling cascade required for multiple processes during development and for adult homeostasis. A key question in understanding this pathway is how it is regulated in different cell contexts. Here we have examined how endocytic processing contributes to signalling by the single cytokine receptor, Domeless, in Drosophila melanogaster cells. We identify an evolutionarily conserved di-Leu motif that is required for Domeless internalisation and show that endocytosis is required for activation of a subset of Domeless targets. Our data indicate that endocytosis both qualitatively and quantitatively regulates Domeless signalling. STAT92E, the single STAT transcription factor in Drosophila, appears to be the target of endocytic regulation and our studies show that phosphorylation of STAT92E on Tyr704, while necessary, is not always sufficient for target transcription. Finally, we identify a conserved residue, Thr702, which is essential for Tyr704 phosphorylation. Taken together, our findings identify previously unknown aspects of JAK/STAT pathway regulation likely to play key roles in the spatial and temporal regulation of signalling in vivo

Seasonal dynamics of bacterial community structure and composition in cold and hot drinking water derived from surface water reservoirs.

In temperate regions, seasonal variability of environmental factors affects the bacterial community in source water and finished drinking water. Therefore, the bacterial core community and its seasonal variability in cold and the respective hot drinking water was investigated. The bacterial core community was studied by 16S rRNA-based SSCP fingerprint analyses and band sequencing of DNA and RNA extracts of cold and hot water (60 °C). The bacterial communities of cold and hot drinking water showed a highly different structure and phylogenetic composition both for RNA and DNA extracts. For cold drinking water substantial seasonal dynamics of the bacterial community was observed related to environmental factors such as temperature and precipitation affecting source and drinking water. Phylogenetic analyses of the cold water community indicated that the majority of phylotypes were very closely affiliated with those detected in former studies of the same drinking water supply system (DWSS) in the preceding 6 years, indicating a high stability over time. The hot water community was very stable over time and seasons and highly distinct from the cold water with respect to structure and composition. The hot water community displayed a lower diversity and its phylotypes were mostly affiliated with bacteria of high temperature habitats with high growth rates indicated by their high RNA content. The conversion of the cold to the hot water bacterial community is considered as occurring within a few hours by the following two processes, i) by decay of most of the cold water bacteria due to heating, and ii) rapid growth of the high temperature adapted bacteria present in the hot water (co-heated with the cold water in the same device) using the nutrients released from the decaying cold water bacteria. The high temperature adapted bacteria originated partially from low abundant but beforehand detected members of the cold water; additionally, the rare members ("seed bank ") of the cold water are considered as a source

Analysis of structure and composition of bacterial core communities in mature drinking water biofilms and bulk water of a citywide network in Germany.

The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity