I. Genomic Approaches to Unravel the Molecular Mechanisms of Pathogenicity in the Human Fungal Pathogen Candida glabrata - FunPath

• Programm: ERA-NET PathoGenoMics
• Förderung: BMBF
• Laufzeit: 01.02.2007 bis 31.07.2010
• Partner:
  Karl Kuchler, Medizinische Universität Wien
  Christoph Schüller, Universität Wien
  Christophe d’Enfert, Institut Pasteur, Paris
  Dominique Ferrandon, IBMC du CNRS, Strasbourg
  Bernhard Hube, HKI, Jena
  Toni Gabaldon, Bioinformatics Department, Valencia

Bei immunsupprimierten Patienten können pathogene Pilze lebensbedrohende Infektionen mit hoher Letalität auslösen, für die nur begrenzte diagnostische und therapeutische Möglichkeiten existieren. Das Ziel der geplanten Arbeiten ist es molekulare Mechanismen dieser Pilzinfektionen aufzuklären. Dies soll durch Untersuchungen der humanpathogenen Hefe, Candida glabrata, erreicht werden. In einem genomweiten Ansatz werden Gendeletionsstudien eingesetzt um zentrale Virulenzgene zu identifizieren. Aufeinander aufbauende in vitro und in vivo Assays zur Bewertung der Virulenz der erzeugten C. glabrata Stämme werden zur Charakterisierung der Stämme eingesetzt. Molekular werden dieser Stämme über Transkriptionsprofile charakterisiert. Daraus lassen sich grundlegende Pathogenitätsmechanismen pathogener Pilze ableiten. Gene mit zentraler Funktion bei der Virulenz sind ideale Targets für die Entwicklung von Antimykotika oder für die Diagnostik. Durch die Entwicklung von entsprechenden Assayssytemen sollen diese Targets als Diagnostika bzw. zum Wirkstoffscreening verwertet werden. Zudem sind die gewonnen Erkenntnisse geeignet zur Prävention von Mykosen bei Risikopatienten beizutragen.

II. Dr. Jekyll and Mr. Hyde: A Systems Approach to the Therapy of Nosocomial Infections Caused by Candida albicans: A Commensal Organism Switches to a Deadly Pathogen

• Programm: Medizinische Systembiologie
• Förderung: BMBF
• Laufzeit: 02/09-01/12
• Partner:
• Helmholtz-Zentrum für Infektionsforschung (HZI)
  Prof. Dr. Ursula Bilitewski, Target identification (TI)
  Dr. Dipl-Ing. Vitor Martins dos Santos, Systems and Synthetic Biology Group (SSBI)
• Klinikum der Universität Tübingen (UT)
  Prof. Dr. Martin Schaller, Universitäts-Hautklinik (HK)
  PD Dr. Klaus Schröppel, Institut für medizinische Mikrobiologie und Hygiene (IMH)
• Centre for Systems Biology, Universität Stuttgart (CSB)
  Prof. Dr. – Ing. Dr. h.c. Matthias Reuss, Institut für Bioverfahrenstechnik
• German Cancer Research Center (DKFZ)
  Prof. Dr. Thomas Höfer, Research Group Modeling of Biological Systems
• Genedata Bionformatik GmbH (GD)
  Dr. Thomas Hartsch
• Insilico Biotechnology AG (IS)
  Dr.-Ing. Dirk Müller
• BIOBASE – Biological Databases GmbH (BB) (subcontractor)
  Dr. Alexander Kel
  

The fungus Candida albicans resides asymptomatically on the skin and the mucosa of healthy people but causes serious invasive diseases in immunocompromised patients. Whereas conventional diagnostic and therapeutic strategies focus on the identification and elimination of the pathogen, the contribution of the host is almost neglected. However, in an in vitro system it was shown that cytokines are produced by host cells in response to the presence of C. albicans and that they are essential for the protection of the host, i.e. maintenance of the commensal state of C. albicans. Removal of these cytokines, e.g. of tumor necrosis factor α(TNF α ), lead to the destruction of the epithelial layer by invading C. albicans. However, the precise protective mechanisms are not yet identified, but are related to the enhanced expression of toll-like receptor 4 (TLR4) and probably to the secretion of antimicrobial peptides by host cells. Thus, it is anticipated that an improved understanding of the protective and defense mechanisms of the host will lead to new diagnostic biomarkers and also to new therapeutic strategies. As illustrated in the scheme below, this project will focus in a multidisciplinary, integrated approach on host-pathogen interactions by assessing under commensal and pathogenic conditions, from a Systems Biology perspective, both the immune response of the host to the presence of the opportunistic pathogen and the response of the pathogen to the host.

• Wirt-Pathogen-Interaktionsmodelle
• Adhäsions und Invasions Assays in Infektionsmodellen
• genomweite Analysen zum Target-Screening
• Wirkstoff-Screening in Infektionsmodellen
• Zellwand-Analytik pathogener Pilze
• Herpes-Simplex-Infektionsmodell
• Reporter-Zelllinien zur Identifikation von Pyrogenen (PAMPS) und Pathogenen (angeborenes Immunsystem im Reagenzglas) nach GLP

• Zellkultur Laboratorien
• mikrobiologische und molekularbiologische Laboratorien nach S2 Standards
• Proteom Analytik: Nano-LCMS-Analytik (MALDI-TOF/TOF + ESI-MS), 2D-SDS-PAGE
• Microarray facility (Biorobotic Microgrit II Arrayer und Array Scanner)
• Lightcycler 480 (Roche)
• Laserscanning und Fluoreszenz Mikroskopie

Burger-Kentischer, A., Finkelmeier, D., Bauer, J., Eickhoff, H., Kleymann, G., Rayyan, A., Schröppel, K., Wiesmüller, K.H. and Rupp, S. (2011) A screening assay based on host-pathogen interaction models identifies of a set of novel antifungal benzimidazole derivatives. Antimicrob. Agents Chemotherapy, accepted.

Luo S, Blom AM, Rupp S, Hipler UC, Hube B, Skerka C, Zipfel PF. (2011)The pH-regulated antigen 1 of Candida albicans binds the human complement inhibitor C4b-binding protein and mediates fungal complement evasion. J Biol Chem., 286(10):8021-9.

Roetzer A, Klopf E, Gratz N, Marcet-Houben M, Hiller E, Rupp S, Gabaldón T, Kovarik P, Schüller C. (2011) Regulation of Candida glabrata oxidative stress resistance is adapted to host environment. FEBS Lett., 585(2):319-27.

Schild L, Heyken A, de Groot PW, Hiller E, Mock M, de Koster C, Horn U, Rupp S, Hube B. (2011) Proteolytic cleavage of covalently linked cell wall proteins by Candida albicans Sap9 and Sap10. Eukaryot Cell, 10(1):98-109.

Calderon, J., Zravel, M., Ragni, E., Fonzi, W.A., Rupp, S., and Popolo, L. (2010) Cell wall glucan remodeling is required for Candida albicans adhesion and invasion. Microbiology, 156: 2484-94

Burger-Kentischer, A., Abele, I.S., Finkelmeier, D., Wiesmüller, K.H. and Rupp, S. (2010) A new cell-based innate immune receptor assay for the examination of receptor activity, ligand specificity, signalling pathways and the detection of pyrogens. Journal of Immunological Methods, 358(1-2):93-103.

Lindemann, E., Rohde, B., Rupp, S., Regenbogen, J. and Sohn, K. (2010)
A Multidimensional Electrophoretic System of Separation for the Analysis of Gene Expression (MESSAGE). Electrophoresis, 31(8):1330-43.

Brunke, S.; Seider, K.;  Almeida, R.S.; Heyken, A.; Fleck, C.B.; Brock, M.; Barz, D.; Rupp, S. and Hube B. (2010) Candida glabrata tryptophan-based pigment production via the Ehrlich pathway. Mol Microbiol, 76: 25-47.

Luo, S., Poltermann, S., Kunert, A., Rupp, S. and Zipfel PF. (2009). Immune evasion of the human pathogenic yeast Candida albicans: Pra1 is a Factor H, FHL-1 and plasminogen binding surface protein. Mol. Immunol. 47(2-3):541-50.

Xiong, X.; Ghosh, R.; Hiller, E.; Drepper, F.; Knapp, B.; Brunner, H. and  Rupp, S. (2009). A new procedure for rapid, high yield purification of Type I collagen for tissue engineering. Process Biochemistry 44 (11): 1200-1212.

Hauser, N.C., Dukalska, M., Fellenberg, K. and S. Rupp (2009) From experimental setup to data analysis in transcriptomics: copper metabolism in the human pathogen Candida albicans. Journal of Biophotonics, 2(4):262-8.

Bareiss, P.M., M. Metzger, K. Sohn, S. Rupp, J.S. Frick, I.B. Autenrieth, F. Lang, H. Schwarz, T. Skutella, and L. Just (2008) Organotypical tissue cultures from adult murine colon as an in vitro model of intestinal mucosa. Histochem Cell Biol. 129(6): 795-804.

Wilson, D., A. Tutulan-Cunita, W. Jung, N.C. Hauser, R. Hernandez, T. Williamson, K. Piekarska, S. Rupp, T. Young, and L. Stateva (2007) Deletion of the high-affinity cAMP phosphodiesterase encoded by PDE2 affects stress responses and virulence in Candida albicans. Mol. Microbiol 65(4): 841-56.

Hiller E, Heine S, Brunner H, Rupp S (2007) Candida albicans Sun41p, a Putative Glycosidase, Is Involved in Morphogenesis, Cell Wall Biogenesis, and Biofilm Formation. Eukaryot. Cell 6: 2056-2065

Hauser, N.C., Martinez, R., Jacob, A., Rupp, S., Hoheisel, J.D. and Matysiak, S. (2006) Utilising the left-helical conformation of L-DNA for analysing different marker types on a single universal microarray platform. Nucleic Acids Res, 34: 5101-5111.

Borchers, K., Weber, A., Hiller, E., Rupp, S., Brunner, H. and Tovar, G.E.M. (2006) Nanoparticle-based Diagnostic 3D-Protein-Biochip for Candida albicans. PMSE Preprints, 95, 1016-1017.

Sohn, K., Senyürek, I., Fertey, J., Königsdoefer, A., Joffroy, C., Hauser, N., Zelt, G., Brunner, H., and Rupp, S. (2006) An in vitro-assay to study the transcriptional response during adherence of Candida albicans to different human epithelia. FEMS Yeast Research, 6: 1085-93.

Sohn, K., Hauser, N., Brunner, H., and Rupp, S. (2006) Cell wall dynamics and transcriptional response in Candida albicans during morphogenesis and adhesion to mammalian epithelia. Mycoses, 5: 352.

Sohn K., Schwenk J., Urban C., Lechner J., Schweikert M., Rupp S. (2006). Getting in touch with Candida albicans the cell wall of a fungal pathogen. Curr Drug Targets, 7(4):505-12.

Carter, G. Rupp, S. Fink, G., Galitski, T. (2006). Disentangling inform-ation flow in the Ras-cAMP signaling network. Genome, 16(4):520-6.

Sohn, K., Roehm, M., Urban, C., Saunders, N., Rothenstein, D., Lottspeich, F., Schröppel, K., Brunner, H. and Rupp, S. (2005). Identification and Characterization of Cor33p, a protein implicated in tolerance towards oxidative stress in Candida albicans, Eukaryotic cell, 4(12):2160-2169.

Urban, C., Xiong, X., Sohn, K., Schröppel, K., Brunner, H. and Rupp, S. (2005) The Moonlighting protein Tsa1p is implicated in oxidative stress response and in cell wall biogenesis in Candida albicans, Mol Microbiol, 57(5):1318-41.

Maidan, M.M., De Rop, L., Serneels, J., Exler, S., Rupp, S., Tournu,
H., Thevelein, J.M., and Van Dijck, P. (2005) The G Protein-coupled Receptor Gpr1 and the G{a} Protein Gpa2 Act through the cAMP-PKA Pathway to Induce Morphogenesis in Candida albicans. Mol Biol Cell. 16 (4): 1971-86.

Lotz, H., Sohn, K., Brunner, H., Mühlschlegel F.A., and Rupp, S. (2004) RBR1, a novel pH regulated cell wall gene of Candida albicans, is repressed by RIM101 and activated by NRG1. Eukaryotic Cell, 3, 776-784.

Hauser, N.C., Teifel, S., Buck, M., Fellenberg, K., Wajant, H., Rupp, S. and Knabbe, C. (2003) DNA-Chip for diagnostic purposes in breast cancer. Clin Chem and Lab Med, 41(10): 97.

Urban, C., Sohn, K., Lottspeich, F., Brunner, H. and Rupp, S. (2003) Identification of cell surface determinants in Candida albicans reveals Tsa1p, a protein differentially localized in the cell, FEBS Lett., 544, 229-236.

Rottmann, M., Dieter, S., Brunner, H. and Rupp, S. (2003) CaMCM1 is an essential gene in C. albicans, crucial for morphogenesis. Mol Microbiol, 47: 943-59.

Sohn, K., Urban, C., Brunner, H. and Rupp, S. (2003) EFG1 is a major regulator of cell wall dynamics in Candida albicans as revealed by DNA microarrays. Mol Microbiol, 47: 89-102.

Dieterich, C., Schandar, M., Noll, M, Johannes, F.-J., Brunner, H., Graeve, T. and Rupp, S. (2002). In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion. Microbiology, 148: 497-506.

Schweizer, A., Rupp, S., Taylor, B. N., Röllinghoff, M. and Schröppel, K. (2000). The TEA/ATTS transcriptionfactor CaTec1p regulates hyphal development and virulence in Candida albicans. Mol Microbiol, 38: 435-45.

Joos, T. O., Schrenk, M., Hopfl, P., Kroger, K., Chowdhury, U., Stoll, D., Schorner, D., Durr, M., Herick, K., Rupp, S., Sohn, K. and Hammerle, H. (2000). A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics. Electrophoresis, 21: 2641-50.

Kontoyiannis, D. P. and Rupp, S. (2000). Cyclic AMP and fluconazole resistance in Saccharomyces cerevisiae. Antimicrob Agents Chemother, 44: 1743-4.

Rupp, S., Summers, E., Lo, H. J., Madhani, H., and Fink, G. (1999). MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene. Embo J, 18: 1257-69.

Rupp, S., An approach to characterize the membrane proteome of Candida albicans. (2010): Future Microbiol. 5(2): 147-151.

Rupp, S., (2007) Interactions of the fungal pathogen C. albicans with the host,
Future Medicine, 2: 141-151.

Sohn, K., Schwenk, J., Urban, C., Lechner, J., Schweikert, M. Rupp, S. (2006) Getting in touch with Candida albicans: the cell wall of a fungal pathogen. Current Drug Targets, 7: 505-12.

Rupp, S. und Hauser, N.C. (2005) DNA-Chip Technologien zur Untersuchung der Wirt-Pathogen-Interaktion bei C. albicans. BIO Spektrum, 11: 513-515.

Rupp S. (2004a) Proteomics on its way to study host-pathogen interaction.
Current Opinion in Microbiology 7: 330-335

Rupp, S. (2004b) Die Zellwand von C. albicans als Schnittstelle zwischen Wirt und Pathogen. Hygiene und Mikrobiologie, 2: 46-51.

Hauser, N., Weber, A., Tovar, G. & Rupp, S. (2003) Nanopartikel-Biochips zur Untersuchung von C. albicans. Biospektrum, 9: 710-712

Hauser, N. C., Fellenberg, K. and Rupp, S. (2002). How to Discover Pathogenic Mechanisms - New Evaluation Tools Towards Drug Discovery. Screening, 3: 28-31.

Rupp, S. (2000). Identifizierung von Virulenzfaktoren in Hefen, Bioworld, 3: 21-23.

Sohn, K. and S. Rupp, Human epithelial model systems for the study of Candida infections in vitro: part I. Adhesion to epithelial models. Methods Mol Biol, 2009. 470: 95-104.

Rupp, S., Introduction: fungal pathogens. Methods Mol Biol, 2009. 470: 69-70.

Hernandez, R. and S. Rupp, Human epithelial model systems for the study of Candida infections in vitro: part II. Histologic methods for studying fungal invasion. Methods Mol Biol, 2009. 470: 105-23.

Sohn, K, Hiller, E , Rupp, S.,  2008, Proteomics Sample Preparation, Sample Preparation for the Cell-Wall Proteome Analysis of Yeast and Fungi, J. von Hagen, Wiley-Vch. 371-378.

Rupp, S. (2008) Transcriptomics of the fungal pathogens focusing on C. albicans, The Mycota, 7, 2^nd Edition, “Human and Animal Relationships”, 187-222.

Sohn, K., Hauser, N. C. and Rupp, S. (2002). Proteomics, genomics and molecular genetics to study fungal development, Recent Res. Devel. in Mol. Microbiol., 1: 45-70, Research Signpost, Ed. S.G. Pandalai, Kerala, India.

Rupp, S. (2002) LacZ assays in Yeast, Methods in Enzymology, 350: 112-131.

Rupp, S., Johannes, F.-J. und Komischke, K., (2001): Biochips – Moderne Werkzeuge der Biotechnologie, Analysen und Expertisen zur Wehrtechnischen Vorausschau, FhG INT, Euskirchen.

Host-Pathogen Interactions, (2009), Methods Mol Biol, Humana Press, Editors Steffen Rupp and Kai Sohn.