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GRK 1482 Jahrbuch 2011-2014

Publications [1] Benner J, Daniel H, Spanier B. A glutathione peroxidase, intracellular peptidases and the TOR complexes regu- late peptide transporter PEPT-1 in C. elegans. PLoS One. 2011, 6(9):e25624. [2] Spanier B, Lasch K, Marsch S, Benner J, Liao W, Hu H, Kienberger H, Eisenreich W, Daniel H. How the intestinal peptide transporter PEPT-1 contributes to an obesity pheno-type in Caenorhabditits elegans. PLoS One. 2009, 4(7):e6279. [3] Chavez V, Mohri-Shiomi A, and Garsin DA. Ce-Duox1/ BLI-3 generates reactive oxygen species as a protective innate immune mechanism in Caenorhabditis elegans. Infection and Immunity. 2009, 77(11): 4983-9. [4] Wang R, Dashwood WM, Nian H, Löhr CV, Fischer KA, Tsuchiya N, Nakagama H, Ashktorab H, Dashwood RH. NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2-amino- 1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Int J Cancer. 2011, 128(11): 2581-90. PhD FELLOWS GRK Progress Report 2011-2014 | Page 47 Aim We hypothesize that Duox1/2 activity is closely interconnected to the action of NHE3, which provides protons at the luminal side of the intestine. Additionally, we have good evidence that proton transport by PEPT1 is coupled to NHE3 and vice versa allowing pHin homeostasis. In the present project we will functionally analyze the interplay of the three brush border membrane proteins Duox1/2, NHE3 and PEPT1 and define their role in host defense against selec- ted pathogenic bacteria such as Enterococcus faecalis and Sal- monella enterica serovarTyphimurium. Methods and Results In C. elegans strains with gene deletions or with RNAi gene silencing for PEPT-1, NHX-2, DUOX/BLI-3 we will assess the protein contribution to H2O2 production and infection rate by pathogenic bacteria. Experiments with the human colon carcinoma cell line Caco-2 will be performed to study the interplay of the homologous mammalian proteins by analysis of H2O2 production, DUOX expression and functional coupling to acid-base homeostasis. Therefore, the Amplex Red assay was established to mea- sure the H2O2 production of 14 days post-confluent Caco-2 cells after treatment with different PEPT1 substrates or NHE3 inhibitors. The H2O2 produced by the Caco-2 cells was signifi- cantly lower after treatment with the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI), as well as with the specific NHE3 inhibitor S1611. Moreover, we were able to show that the supplementation with glycyl-glutamine as a PEPT1 substrate leads to an increased H2O2 production in Caco-2 cells. Western Blot analysis and immunohistochemistry as well as H2O2 measurements will show whether DUOX abundance and function in small intestine and colon from Pept-1-/- and infla- med mice are altered in comparison to healthy littermates. In initial immunohistochemical stainings we were able to detect DUOX at the apical membrane in the distal colon of wild type mice (see Figure 2). Outlook Future experiments will focus on immunohistochemical ana- lysis of DUOX2 expression in Pept1-/- and Nhe3-/- mice. The susceptibility of various C. elegans strains for infection with E. faecalis and their corresponding H2O2-based defence will be analysed. Genetically modified Caco-2 cell with DUOX loss-of- function and gain-of-function background are under construc- tion and will be analyzed as a stable model for modulators of DUOX. Figure 2: Immunohistochemistry of colonic tissue of wild type mice. Duox expression (red) is visible at the apical brushborder membrane. Nuclei were counterstained with DAPI. Supervisors Prof. Dr. Hannelore Daniel I TUM I Physiology of Human Nutrition Dr. Britta Spanier I TUM I Physiology of Human Nutrition Prof. Dr. Thilo Fuchs I TUM I Microbial Ecology Start of project: August 2011 Academic background: Studies of Biology at Technische Universität München