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GRK 1482 Jahrbuch 2011-2014

Publications [1] Pacheco, A.R., et al., Fucose sensing regulates bacterial intestinal colonization. Nature, 2012. [2] Sekirov, I., et al., Gut microbiota in health and disease. Physiol Rev, 2010. 90(3): p. 859-904. [3] Martins dos Santos, V., M. Muller, and W.M. de Vos, Sys- tems biology of the gut: the interplay of food, microbiota and host at the mucosal interface. Curr Opin Biotechnol, 2010. 21(4): p. 539-50. [4] Stecher, B. and W.D. Hardt, Mechanisms controlling pa- thogen colonization of the gut. Curr Opin Microbiol, 2011. 14(1): p. 82-91. [5] Backhed, F., et al., Host-bacterial mutualism in the hu- man intestine. Science, 2005. 307(5717): p. 1915-20. Harvey, P.C., et al., Salmonella enterica serovar typhimu- rium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolismgenes.InfectImmun,2011.79(10):p.4105-21. [6] Juge, N., Microbial adhesins to gastrointestinal mucus. Trends Microbiol, 2012. 20(1): p. 30-9. [7] Johansson, M.E., et al., Composition and functional role of the mucus layers in the intestine. Cell Mol Life Sci, 2011. 68(22): p. 3635-41. [8] Johansson, M.E., J.M. Larsson, and G.C. Hansson, The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host- microbial interactions. Proc Natl Acad Sci U S A, 2011. 108 Suppl 1: p. 4659-65. ASSOCIATED FELLOWS GRK Progress Report 2011-2014 | Page 77 Aim In this project, we seek for further metabolic pathways of Sal- monella Typhimurium providing growth and colonization ad- vantages within the GIT, especially during interaction with the mucosa and the epithelial surface. Possible results are genetic determinants that allow Salmonella to establish a metabolic ni- che in the densely populated GIT. A long time goal is to unravel metabolic interdependencies between an enteric pathogen and its host. Methods and Results To monitor changes in the gene expression of S. Typhimurium after contact with host cells, in this case Caco-2 and HT29/ B6 human colon carcinoma cells. The reason for using HT29/ B6 besides Caco-2 cells is the mucus secretion of the HT29/ B6 cell line, allowing a more realistic mimicry od the infection process than using Caco-2 cells not secreting mucus. In the genetic background of an invasion deficient (∆invA) S. Typhi- murium mutant a transposon-luciferase library will be const- ructed. Via horizontal gene transfer a vector carrying a Tn5-lux cassette will be transferred into the ∆invA mutant. After random insertion of the lux-cassette into the genome luminescence will be monitored in cell culture assays. Interesting phenotypes will be further characterized and be confirmed in the wild type by deletion and complementation assays. To further characterize the 1,2-propanediol (pdu) and L-fucose degradation pathway Salmonella reporter strains will be const- ructed to detect promoter activity in different media, cell culture and in vivo. Anaerobic culture methods will also be established to determine the operon structure of the pdu operon via RT-PCR and qRT-PCR. Isotopological analysis with labeled compounds might be applied as well as in vivo studies of the strains. Outlook After construction of the invasion deficient Salmonella Typhi- murium mutant the Tn5-lux mutant library will be constructed and screened in cell culture assays. Interesting phenotypes will be further characterized and tested in the wild type. In the fol- lowing reporter strains can be constructed and being investi- gated in in vitro approaches as well as in model organisms. C13 labeled 1,2-propanediol or L-fucose could serve as substrates for isotopological analysis of the respective metabolic pathway. Figure 1: Invasion assay of S. Typhimurium wild type compared to S. Ty- phimurium ∆invA spectinomycinR mutant in Caco-2 and HT29/B6 cells. Invasion of the mutant is significantly reduced in HT29/B6 cells compared to the wild type. Supervisors Prof. Thilo M. Fuchs I TUM I Microbial Ecology Prof. Hannelore Daniel I TUM I Physiology of Human Nutrition Start of project: January 2012 Academic background: Studies of Molecular Biotechnology at Rheinisch-Westfälische Technische Hochschule Aachen