Browsing by Subject "Quorum sensing"

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Interaktion des bakteriellen Quorum Sensing Moleküls N-(3-oxododecanoyl)-L-Homoserin-Lacton (AHL-12) mit humanen neutrophilen Granulozyten
(2013) Kahle, Nadine; Hänsch, Gertrud Maria
To evade immune-defense mechanisms of the host, Pseudomonas aeruginosa form so called biofilms. To coordinate this complex process, the bacteria communicate via quorum sensing signals, such as N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12). AHL-12 is also recognized by human polymorphonuclear neutrophils (PMN). It is chemotactic for PMN and thus might cause their directed migration to the site of infection in order to eliminate infiltrated microorganisms. Through different approaches, the aim of this work was to find out in which way AHL-12 interacts with PMN. As a first step, an AHL-12 induced signaling-pathway should be elucidated in in vitro experiments using primary, human PMN. In a further approach using radioactive- (3H-AHL-12) and fluorescence-labeled (AHL-12-FITC) AHL-molecules, the interaction of AHL-12 with PMN should be examined, especially with regard to the expression of a specific receptor for AHL-12 on PMN. In this work it was confirmed that AHL-12 induced chemotaxis of human PMN. After stimulation of PMN with AHL-12, MAPK-p38 was phosphorylated and hence activated. Furthermore, it was shown that MAPK-p38 phosphorylated MAPKAP-Kinase 2, which in turn phosphorylated and hence activated leukocyte specific protein 1 (LSP1). LSP1 finally affected chemotaxis via binding to F-Actin and by modulating the intensity of integrin-mediated adherence. The AHL-12 induced chemotaxis was inhibited by SB203580, an inhibitor of MAPK-p38. The question, whether there is a specific receptor for AHL-12 in PMN, could not be answered conclusively, because it was not possible to isolate a protein with receptor function using conventional methods such as immunoprecipitation and crosslinking. In uptake studies with 3H-AHL-12 and AHL-12-FITC, it was shown, that the major portion of AHL-12-FITC bound to the cell membrane. However, another portion got into the cells, which raises the possibility that an intracellular receptor or an intracellular binding protein for AHL-12, for example IQGAP1, exists. Taken together these data provide evidence that AHL-12 is chemotactic for PMN. Assuming that AHL-12 is released in the early phase of biofilm formation, before bacteria are embedded in the EPS, PMN have the chance to kill bacteria and thus could contribute to prevent biofilm formation.
Modeling the time course of ComX: towards molecular process control for Bacillus wild-type cultivations
(2021) Treinen, Chantal; Magosch, Olivia; Hoffmann, Mareen; Klausmann, Peter; Würtz, Berit; Pfannstiel, Jens; Morabbi Heravi, Kambiz; Lilge, Lars; Hausmann, Rudolf; Henkel, Marius
Wild-type cultivations are of invaluable relevance for industrial biotechnology when it comes to the agricultural or food sector. Here, genetic engineering is hardly applicable due to legal barriers and consumer’s demand for GMO-free products. An important pillar for wild-type cultivations displays the genus Bacillus. One of the challenges for Bacillus cultivations is the global ComX-dependent quorum sensing system. Here, molecular process control can serve as a tool to optimize the production process without genetic engineering. To realize this approach, quantitative knowledge of the mechanism is essential, which, however, is often available only to a limited extent. The presented work provides a case study based on the production of cyclic lipopeptide surfactin, whose expression is in dependence of ComX, using natural producer B. subtilis DSM 10 T. First, a surfactin reference process with 40 g/L of glucose was performed as batch fermentation in a pilot scale bioreactor system to gain novel insights into kinetic behavior of ComX in relation to surfactin production. Interestingly, the specific surfactin productivity did not increase linearly with ComX activity. The data were then used to derive a mathematic model for the time course of ComX in dependence of existing biomass, biomass growth as well as a putative ComX-specific protease. The newly adapted model was validated and transferred to other batch fermentations, employing 20 and 60 g/L glucose. The applied approach can serve as a model system for molecular process control strategies, which can thus be extended to other quorum sensing dependent wild-type cultivations.
Molecular characterization of the interaction of lactobacilli with food environments and enterohemorrhagic Escherichia coli O157:H7
(2009) Hüfner, Eric; Hertel, Christian
The first part of this thesis focuses on the gene expression of Lactobacillus sakei and Lactobacillus reuteri in food fermentation using in vivo expression technology (IVET) and DNA microarray hybridization analysis, respectively. Both technologies allow the identification of regulated genes in a specific environment, which are likely to contribute to the ecological performance of the organism. Thus, the obtained results provide a basis for the development of new strategies to improve the fermentation process, as it was demonstrated by the development of an efficient method for the improvement of sausage fermentation using L. sakei. To obtain hygienically safe products, the function of starter cultures mostly relies on the ability to acidify and produce other antimicrobial principles. However, it was recently demonstrated that the interaction with pathogens also can take place on another level, apart from killing or growth inhibition. Lactobacilli have been shown to influence the virulence gene expression of enterohemorrhagic Escherichia coli (EHEC) via the bacterial communication system termed quorum sensing. The second part of the thesis explores the impact of quorum sensing between Lactobacillus reuteri strains and EHEC O157:H7 on EHEC virulence gene expression. By using a green fluorescent protein reporter gene assay, it was demonstrated for the first time that the transcription of the ler virulence regulator gene is significantly reduced by secreted substances of L. reuteri in a strain- and quorum sensing-dependent manner.