SFB796 - Sub project B7


Functional analysis of bacterial Type VI effectors in EBI3-dependent regulation of enteric infection and inflammation


Project summary

During co-evolution with their hosts, enteric bacterial pathogens developed subtle ways to colonize the complex ecosystem of the gastrointestinal tract. Common strategies that subvert the regulatory pathways of the mucosal immune system of the host are often mediated by large multicomponent secretion systems that transport virulence factors through the inner and outer membranes of the bacteria. Recently, large-scale sequencing projects identified a novel secretion system (type VI or T6SS) in the genomes of many bacterial pathogens of plants, humans and animals. Although T6SSs have been shown to be major virulence factors regulating many aspects of bacterial pathogenesis, their physiological role and virulence mechanism remain poorly understood. Recent reports in the Helicobacter hepaticus model demonstrated that T6SS effectors directly modulate the magnitude of innate and adaptive immune responses and thus intestinal inflammation to confer long-term pathogen survival in the gut. Based on our studies identifying the immunoregulatory IL-12 family cytokine EBI3 as important modulator of Citrobacter rodentium-induced pathology, a model for EHEC/EPEC infections and inflammatory bowel disease in humans, we hypothesized that the cts1 T6SS of this strain may be a critical part of its strategy to survive in the gut. Therefore, luminescent T6SS mutant C. rodentium were generated and analyzed for their capacity to colonize the mouse gut by in vivo imaging. Interestingly, we found that mice infected with mutants with a defective T6SS display reduced bacterial burdens, diminished adherence to intestinal epithelial cells and develop less epithelial hyperplasia, the hallmark of C. rodentium-induced intestinal inflammation. In the second funding period we would like to further characterize molecular mechanisms of T6SS-dependent reprogramming of intestinal host cells. Besides the T6SS of C. rodentium, we will analyze the T6SS of Salmonella typhimurium, a potentially invasive intestinal pathogen. This T6SS is encoded in the Salmonella pathogenity island 6 (SPI-6) and sequence similarities to cts1 exist. In addition to in vivo studies to analyze how T6SSs and their effectors potentially modulate local immune responses and the regulatory functions of IL-12 related cytokines in the gut, we will directly analyze how T6SS effectors of these model organisms reprogram intestinal cell populations in vitro. We will focus here on the two proteins hcp and vgrG that are encoded in T6SS clusters of both C. rodentium and S. typhimurium and were described as the secreted T6SS effectors in other Gram-negative pathogens. To analyze hcp and vgrG functions we have introduced to our lab a recently published organoid culture system that recapitulates in three dimensions the crypt-villus architecture of the intestinal epithelium. This will allow for the first time in vitro studies of infection under near in vivo conditions. Furthermore hcp and vgrG functions will be analyzed in macrophage cell lines and primary dendritic cell subsets. Finally, bioinformatical methods and biochemical studies such as immunoprecipitation and mass spectrometry together with partners in the CRC796 will be used to identify potential molecular targets of T6SS effectors in their host cells.


Project relevant publications

  • Mahapatro M, Foersch S, Hefele M, He G, Giner-Ventura E, Mchedlidze T, Kindermann M, Vetrano S, Danese S, Günther C, Neurath MF*, Wirtz S*, Becker C*.   (2016).   Programming of the intestinal stem cell niche by IL-33 derived from pericryptal fibroblasts in response to systemic infection.   Cell reports   [accepted for publication]   *Shared senior authorship.

  • Mchedlidze T, Kindermann M, Vöhringer D, Neurath MF and Wirtz S.   (2016).   IL-27 suppresses type 2 immune responses in vivo via direct effects on group 2 innate lymphoid cells.   Mucosal immunology, doi: 10.1038/mi.2016.20.

  • Becker C, Neurath MF and Wirtz S.   (2015).   The Intestinal Microbiota in Inflammatory Bowel Disease.   ILAR journal / National Research Council, Institute of Laboratory Animal Resources 56: 192-204.

  • Wittkopf N, Pickert G, Billmeier U, Mahapatro M, Wirtz S, Martini E, Leppkes M, Neurath MF and Becker C.   (2015).   Activation of intestinal epithelial Stat3 orchestrates tissue defense during gastrointestinal infection.   PloS one 10: e0118401.

  • Gerlach K, Hwang Y, Nikolaev A, Atreya R, Dornhoff H, Steiner S, Lehr HA, Wirtz S, Vieth M, Waisman A, et al.   (2014).   TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells.   Nature immunology 15, 676-686.

  • Neurath MF   (2014).   Cytokines in inflammatory bowel disease.   Nature reviews. Immunology 14: 329-342.

  • Desel C, Werninghaus K, Ritter M, Jozefowski K, Wenzel J, Russkamp N, Schleicher U, Christensen D, Wirtz S, Kirschning C, Agger EM, Prazeres da Costa C, Lang R.   (2013).   The Mincle-activating adjuvant TDB induces MyD88-dependent Th1 and Th17 responses through IL-1R signaling.   PLoS One. 2013;8(1):e53531.

  • Mchedlidze T, Waldner M, Zopf S, Walker J, Rankin AL, Schuchmann M, Voehringer D, McKenzie AN, Neurath MF, Pflanz S, and Wirtz S.   (2013).   Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis.   Immunity 39: 357-371.

  • Wirtz, S., U. Billmeier, T. Mchedlidze, R. S. Blumberg, and Neurath, M.F.   (2011).   Interleukin-35 Mediates Mucosal Immune Responses That Protect Against T-Cell-Dependent Colitis.   Gastroenterology 141: 1875-1886.

  • Waldner, M. J., S. Wirtz, C. Neufert, C. Becker, and M. F. Neurath   (2011).   Confocal laser endomicroscopy and narrow-band imaging-aided endoscopy for in vivo imaging of colitis and colon cancer in mice.   Nature Protocols 6: 1471-1481.

  • Pickert, G., C. Neufert, M. Leppkes, Y. Zheng, N. Wittkopf, M. Warntjen, H. A. Lehr, S. Hirth, B. Weigmann, S. Wirtz, W. Ouyang, M. F. Neurath, and C. Becker   (2009).   STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing.   Journal of Experimental Medicine 206: 1465-1472.

  • Leppkes, M., C. Becker, I. I. Ivanov, S. Hirth, S. Wirtz, C. Neufert, S. Pouly, A. J. Murphy, D. M. Valenzuela, G. D. Yancopoulos, B. Becher, D. R. Littman, and Neurath, M.F.   (2009).   ROR gamma-Expressing Th17 Cells Induce Murine Chronic Intestinal Inflammation via Redundant Effects of IL-17A and IL-17F.   Gastroenterology 136: 257-267.

  • Goriely, S., Neurath, M.F. and Goldman, M.   (2008).   How microorganisms tip the balance between interleukin-12 family members.   Nat Rev Immunol 8, 81-86.

  • Wirtz, S., Neufert, C., Weigmann, B. and Neurath, M.F.   (2007).   Chemically induced mouse models of intestinal inflammation.   Nature Protocols 2, 541-546.

  • Wirtz, S. and Neurath, M.F.   (2007).   Mouse models of inflammatory bowel disease.   Adv Drug Deliv Rev 59: 1073-1083.

  • Wirtz, S., Tubbe, I., Galle, P.R., Schild, H.J., Birkenbach, M., Blumberg, R.S. and Neurath, M.F.   (2006).   Protection from lethal septic peritonitis by neutralizing the biological function of interleukin-27.   J Exp Med 203, 1875-1881.

  • Wirtz, S., Becker, C., Fantini, M.C., Nieuwenhuis, E.E., Tubbe, I., Galle, P.R., Schild, H.J., Birkenbach, M., Blumberg, R.S., and Neurath, M.F.   (2005).   EBV-induced gene 3 transcription is induced by TLR signaling in primary dendritic cells via NF-kappa B activation.   J Immunol 174, 2814-2824.