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GRK 1482 Jahrbuch 2011-2014

Publications [1] Jostins L, Ripke S, Weersma RK, Duerr RH, McGovern DP, Hui KY, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature. 1. November 2012, 491(7422):119–24. [2] Kim SC, Tonkonogy SL, Albright CA, Tsang J, Balish EJ, Braun J, Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria. Gastroenterology. April 2005, 128(4):891–906. [3] Steck N, Hoffmann M, Sava IG, Kim SC, Hahne H, ..., Haller D. Enterococcus faecalis metalloprotease com- promises epithelial barrier and contributes to intes- tinal inflammation. Gastroenterology. September 2011, 141(3):959–71. [4] SteckN,MuellerK,SchemannM,HallerD.Bacterialprote- asesinIBDandIBS.Gut.November2012,61(11):1610–8. Supervisors Prof. Dr. Dirk Haller | TUM | Nutrition and Immunology Prof. Dr. Wolfgang Liebl | TUM | Microbiology Start of project: March 2011 Academic background: Studies of Nutritional Science at Technische Universität München PhD FELLOWS GRK Progress Report 2011-2014 | Page 39 Aim The aim of this study is to further assign structure-based func- tions of the model organism E. faecalis under normal and chro- nically inflamed conditions in order to investigate the impact of microbe-host interactions in chronic intestinal inflammation. Methods and Results In this study, we characterized enterococcal virulence in vitro and in vivo using newly generated epaB deletion (TX5692) and lgt deletion mutants. Both ∆epaB and ∆lgt mutants showed the same morphological phenotype with single rounded cocci. A deletion of epaB resulted in diminished in vitro biofilm gene- ration under static and flow conditions including attenuated formation of micro-colonies and impaired adhesion to epithelial cells in vitro. Moreover, the loss of epaB significantly reduced vi- rulence of E. faecalis in infection models of Caenorhabditis ele- gans and Galleria mellonella in comparison to wild-type strain OG1RF. While the deletion of lgt had no impact on biofilm formation or adhesion of E. faecalis in vitro, ∆lgt mutants showed attenuated virulence in infection models of C. elegans and G. mellonella in comparison to wild-type strain OG1RF. To further analyze microbe-host interaction, germ-free Mandu- ca sexta larvae, a natural host to E. faecalis, were mono-asso- ciated with E. faecalis mutant and wild-type strains. Compared to wild-type OG1RF, the ∆epaB and ∆lgt mutants showed at- tenuated intestinal colonization. Analyzed by confocal micro- scopy, ∆epaB mutants revealed altered adhesion to the larval intestinal epithelium in vivo. In addition, mono-association of M. sexta larvae with the ∆epaB mutant led to reduced intestinal expression levels of peptidoglycan-recognition protein (PGRP). In a next step, we investigated the impact of ∆epaB and ∆lgt mutants on intestinal pathogenesis in the IL-10-/- mouse mo- del of chronic intestinal inflammation. Germ-free wild-type and IL-10-/- mice were mono-associated with wild-type OG1RF or ∆epaB or ∆lgt mutant strains and sacrificed after 16 weeks. Compared to IL-10-/- mice that were associated with wild-type OG1RF, mice associated with ∆epaB or ∆lgt mutants showed diminished levels of chronic inflammation markers such as lower weight of spleen and mesenteric lymph nodes and less increased colon length. Outlook Our results suggest a prominent role of epaB and lgt as viru- lence factors mediating the interaction of E. faecalis with the host in multiple ways. To bring these results in context with chronic intestinal inflammation, we recently performed mono- association experiments in a mouse model of experimental colitis. By using E. faecalis as commensal model organism, we hope to unravel the unclear functional relationship between commensal gut microbiota and host in chronic intestinal in- flammation. Biofilm formation of E. faecalis wild-type OG1RF (top) and ∆epaB mutant strain (bottom) on biotic surface after 20h of incubation (60x magnifica- tion). E. faecalis (green) from ∆epaB mutant strain form less micro-co- lonies on a fixed layer of differentiated murine intestinal epithelial PTK-6 cells (Nuclei=blue, E-cadherin=red).