Rational pathogen design [Elektronische Ressource] : extending the host range of Listeria monocytogenes by thermodynamically re-engineering the internalin, E-cadherin interface / von Thomas Wollert

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• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2007
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 15 Mo

Extrait

Rational Pathogen Design:
Extending the Host Range of Listeria monocytogenes
by Thermodynamically Re-engineering the
Internalin / E-Cadherin Interface


Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades eines

Doktors der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n

















von Thomas Wollert
aus Magdeburg












































Honorarprofessor Dr. Dirk Heinz 1. Referent:
Professor Dr. Petra Dersch 2. Referentin:
21.05.2007 eingereicht am:
17.07.2007 mündliche Prüfung (Disputation) am:

Druckjahr 2007 Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab
veröffentlicht:

Publikationen

Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A.D.,
Heinz, D.W., Lengeling, A. & Schubert, W.-D. (2007) Extending the host range of Listeria
monocytogenes by rational protein design. Cell 129 (5), 891-902.

Wollert, T., Heinz, D.W. & Schubert, W.-D. (2007) Thermodynamically re-engineering the
listerial invasion complex InlA / E-cadherin. PNAS, doi:10.1073/pnas.0702199104.

Tagungsbeiträge

Wollert, T., Heinz, D.W. & Schubert, W.-D.: Strengthening the binding of Internalin to
human E-cadherin. (Poster) Structural Biology at Crossroads, EMBL Hamburg, Germany
(2004). Poster-Prize.

Wollert, T., Heinz, D.W. & Schubert, W.-D.: Structure based re-engineering of Internalin –
the intestinal invasin of Listeria monocytogenes. (Vortrag) Murnau Conference on Structural
Biology of Molecular Recognition, Murnau , Germany (2005).

Wollert, T., Heinz, D.W. & Schubert, W.-D.: Structure based re-engineering of Internalin –
the intestinal invasin of Listeria monocytogenes. (Vortrag) Eighth Heart of European Bio-
Crystallography Meeting, Karlovy Vary, Czech Republic (2005). Best Talk Prize.

Wollert, T., Heinz, D.W. & Schubert, W.-D.: Structure based re-engineering of Internalin –
the intestinal invasin of Listeria monocytogenes. (Poster) Meeting of the Network of
Excellence (NoE) EuroPathoGenomics, Universität Würzburg, Germany (2005).



Wollert, T., Heinz, D.W. & Schubert, W.-D.: Structure based re-engineering of Internalin –
the intestinal invasin of Listeria monocytogenes. (Vortrag) Jahrestagung der Deutschen
Gesellschaft für Kristallographie, Universität Freiburg, Germany (2006).

Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A.D.,
Heinz, D.W., Lengeling, A. & Schubert, W.-D.: Structure based re-engineering of Internalin –
rd
an invasin of Listeria monocytogenes. (Poster) 23 European Crystallographic Meeting,
University of Leuven, Belgium (2006).

Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A.D.,
Heinz, D.W., Lengeling, A. & Schubert, W.-D.: Structure-based pathogen design – a new
murine listeriosis model. (Vortrag) Cold Spring Harbor Laboratory Meeting, Genomic
Perspectives to Host-Pathogen Interactions, Welcome-Trust Conference Centre, Hinxton, UK,
(2006).

Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A.D.,
Heinz, D.W., Lengeling, A. & Schubert, W.-D.: Structure based pathogen design – extending
the host specificity of Listeria monocytogenes. (Vortrag) Structural Biology of Pathogens,
University of Birmingham, UK (2006).

Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A.D.,
Heinz, D.W., Lengeling, A. & Schubert, W.-D.: Structure based re-engineering of Internalin –
th
an invasin of Listeria monocytogenes. (Poster) 58 Mosbacher Colloquium, Mosbach,
Germany (2007).

Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A.D.,
Heinz, D.W., Lengeling, A. & Schubert, W.-D.: Structure based pathogen design – extending
the host range of Listeria monocytogenes. (Poster) Annual Meeting of the American
Crystallographic Association, Salt Lake City, Utah, USA (2007).
CONTENTS 3
Contents
Contents...................................................................................................................................... 3
Abbreviations ............................................................................................................................. 6
Summary .................................................................................................................................... 7
1 Introduction........................................................................................................................ 8
1.1 Pathophysiology of L. monocytogenes............................................................................. 8
1.1.1 Virulence factors and intracellular infection cycle of L. monocytogenes ................. 9
1.2 Invasion proteins – key to enter host cells ..................................................................... 10
1.2.1 The leucine-rich-repeat family of proteins.............................................................. 11
1.2.2 Structural insights into the InlA / hEC1 recognition complex................................ 12
1.3 Bacterial invasion strategies........................................................................................... 14
1.4 Host specificity of Listeria monocytogenes ................................................................... 16
1.5 Aim of the work ............................................................................................................. 17
2 Methods............................................................................................................................ 19
2.1 Protein production.......................................................................................................... 19
2.1.1 Generation of InlA and E-Cadherin constructs....................................................... 19
2.1.2 Protein expression and purification......................................................................... 20
2.2 X-ray analysis................................................................................................................. 21
2.2.1 Co-crystallization of InlA-variants and receptors ................................................... 21
2.2.2 Data collection, structure determination and refinement ........................................ 22
2.3 Isothermal titration calorimetry...................................................................................... 23
2.3.1 Sample preparation.................................................................................................. 23
2.3.2 Experimental setup 24
2.3.3 Data evaluation........................................................................................................ 24
2.4 Generation of transgenic L. monocytogenes strains....................................................... 25
2.4.1 Cloning strategy to create pPL2 and pAUL-A integration vectors......................... 25
2.4.2 Generation of pPL2-inlAB constructs ..................................................................... 27
2.4.3 Generation of competent listerial cells.................................................................... 27
m 5’2.4.4 pAUL-A inlA -inlB -gfp integration and homologous recombination .................. 27
2.4.5 pPL2 integration...................................................................................................... 29
4 CONTENTS
2.4.6 Protein preparations for Western-blot analysis........................................................29
2.5 Adhesion and invasion assays ........................................................................................30
2.5.1 Immunofluorescent staining of infected Caco-2 cells .............................................31
2.6 Mouse infection experiments..........................................................................................32
2.6.1 Oral infection...........................................................................................................32
2.6.2 Intravenous infection ...............................................................................................32
2.6.3 Infection of pregnant mice.......................................................................................32
2.6.4 Bacterial counts in organs, placentae and embryos .................................................33
2.6.5 Statistical analysis....................................................................................................33
2.6.6 Histology and immunohistochemistry.................