Novel phospholipases A of Pseudomonas aeruginosa [Elektronische Ressource] : biochemical characterisation and cellular localisation / Filip Kovačić

Kovacic

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2011
• Nombre de lectures: 24
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Novel phospholipases A of Pseudomonas aeruginosa

biochemical characterisation and cellular localisation




















Filip Kovačić

Thesis for doctoral degree









Novel phospholipases A of Pseudomonas aeruginosa:
biochemical characterisation and cellular localisation




Inaugural-Dissertation

zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf

vorgelegt von

Filip Kovačid
aus Čakovec in Kroatien


Düsseldorf, June 2010


Aus dem Institut für Molekulare Enzymtechnologie
der Heinrich-Heine Universität Düsseldorf
im Forschungszentrum Jülich





Diese Arbeit wurde gefördert durch ein Stipendium der Europäische Union im Rahmen des Marie-
Curie „Early stage research Antibiotarget project“ (MEST-CT-2005-020278).















Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-
Heine-Universität Düsseldorf



Referent: Prof. Dr. Karl-Erich Jaeger
Koreferent: Prof. Dr. Lutz Schmitt




Tag der mündlichen Prüfung: 23. 06. 201























to Lina and Diana
Acknowledgments
Acknowledgments

Writing an acknowledgment section is not an easy task as too many amazing people have
contributed in various ways in bringing this dissertation about. So let me start by thanking all those
people who have helped make my life so amusing and thrilling during the last years.

I would like to thank Dr. Karl-Erich Jaeger for giving me the opportunity to join a group and to
explore the world of science. Thank you for all the scientific discussions, recommendations and
personal understanding.

I want to thank Dr. Lutz Schmitt for the kind acceptance of the duty to be the co-referee.

I can’t thank my supervisors, Dr. Susanne Wilhelm and Dr. Frank Rosenau, enough for their bountiful
scientific advice and inspiring support. I would also like to thank them for their suggestions which
assisted me in preparing the thesis and for the numerous discussions about Pseudomonas, life and
how to be a good scientist. Simply, thank you for being such great mentors and good people.

I would like to thank Dr. Romé Voulhoux from the Laboratoire d'Ingénierie des Systèmes
Macromoléculaires, (IBSM, CNRS, Marseille) for hosting me in his laboratory, providing me with
supervision in localization of proteins and for making my stay in Marseille scientifically and socially an
outstanding experience. Also I would like to thank Geneviève Ball (IBSM, CNRS) for helping in the
construction of deletion mutants and for assisting in the laboratory. Thank you Melissa Sivaneson
and Astrid Wahl for enriching my stay in Marseille.

Sincere thanks goes to Renu Batra-Safferling and Joachim Granzin from the Institute of Structural
Biology II (FZ Juelich) for help in the crystallization and the solving of TesA protein structure.

I would like to thank Dr. Sophie Bleves and Richard Salacha from IBSM (CNRS Marseile) for
productive discussions about the secretion and virulence of Pseudomonas aeruginosa.

Thanks goes out to Rien Hoge from IMET (FZ Juelich) who tested virulence of my P. aeruginosa
strains in Dr. Laurence Rahme laboratory at the Harvard Medical School, Boston.

I would also like to thank Dr. Melanie Brocker from the Institute of Biotechnology (FZ Juelich) for
help in the identification of proteins by MALDI MS.

My special thanks goes out to Dr. Silke Isenhardt-Heckmann (IMET, FZ Juelich) for valuable
discussions and commentaries about my Ph. D. thesis and to my colleagues, Rien Hoge, Sebnem
Bukavaz and Franco Circolone (IMET, FZ Juelich) who have been contributing in the development of
my scientific thoughts and for helping me in moments of professional and personal happiness. Thank
you very much.

I would like to thank all of my fellows and supervisors and all persons involved in the Antibiotarget
project for the productive, beautiful and funny moments during the project meetings. It was great to
meet you all.

I thank all my friends for making my Ph. D. “adventure” easier.

Last but absolutely not least I would like to thank my family, mother, father, brothers, wife and my
small angels, daughters Lina and Diana, who have been supporting me all the time and giving me the
best advice coming deeply from their hearts.
Publications
Publications in frame of PhD thesis

Articles
Accepted: R. Salacha, F. Kovačić, B.-A. Celine, S. Wilhelm, J. Tommassen, A. Filloux, R. Voulhoux,
and S. Bleves (2010) The Pseudomonas patatin-like protein PlpD is the archetype of a
novel Type V Secretion System, Environmental Microbiology, Environmental
Microbiology. 12(6): 1498-1512.
: I. Leščid Ašler, N. Ivid, F. Kovačić, S. Schell, J. Knorr, U. Krauss, S. Wilhelm, B. Kojid-Prodid
and K.-E. Jaeger (2010) Probing enzyme promiscuity of SGNH – hydrolases,
ChemBioChem. 11(15): 2158-67.
In preparation: F. Kovačić, F. Rosenau, S. Wilhelm, K.-E. Jaeger, Diversity of novel phospholipases A
from Pseudomonas aeruginosa.
: F. Kovačić, R. Batra-Safferling, F. Rosenau, J. Granzin, B. Kojid-Prodid, K.-E. Jaeger, S.
Wilhelm, Structural and functional characterisation of TesA, a periplasmic
lysophospholipase from opportunistic pathogen Pseudomonas aeruginosa

Poster presentations
April 2007, Conference of German Association for General and Applied Microbiology, Osnabrueck,
Germany, F. Kovačić, S. Wilhelm, F. Rosenau and K.-E. Jaeger, Characterisation of
the lipolytic system of Pseudomonas aeruginosa.
June 2007, Tag des wissenschaftlischen nachwuches, Düsseldorf, Germany, F. Kovačić, S. Wilhelm, F.
Rosenau and K.-E. Jaeger, Characterisation of lipolytic system of the pathogenic
bacterium P. aeruginosa.
August 2009, XII International Pseudomonas Conference, Hannover, F. Kovačić, A. Mandrysch, S.
Wilhelm, F. Rosenau, K.-E. Jaeger, Pseudomonas aeruginosa PAO1 expresses
enzymes with phospholipase A and phospholipase B activity.
August 2009, XII International Pseudomonas Conference, Hannover, R. Salacha, F. Kovačić, J.
Tommassen, A. Filloux, R. Voulhoux, and S. Bleves, An original mechanism for the
secretion of a patatin-like protein by Pseudomonas aeruginosa.

Oral presentations
November 2006, First Antibiotarget symposium, Nottingham, England, F. Kovačić, S. Wilhelm, F.
Rosenau and K.-E. Jaeger, Characterisation of the lipolytic system of Pseudomonas
aeruginosa.
October 2007, Second Antibiotarget symposium, Jülich, F. Kovačić, S. Wilhelm, F. Rosenau and K.-E.
Jaeger, Characterisation of the lipolytic system of Pseudomonas aeruginosa.
May 2008, Third Antibiotarget symposium, Marseilles, France, F. Kovačić, S. Wilhelm, F. Rosenau and
K.-E. Jaeger, Characterisation of the lipolytic system of Pseudomonas aeruginosa.
December 2009, Fifth Antibiotarget symposium, London, England, F. Kovačić, S. Wilhelm, F. Rosenau
and K.-E. Jaeger, Characterisation of the lipolytic system of Pseudomonas
aeruginosa.

I Contents
Contents

Publications in frame of PhD thesis ..................................................................................... I
Contents ........................................................... II
List of Tables .................... VI
List of figures ................... VII

1. Introduction ............................................................................................................. 01

1.1. Pseudomonas aeruginosa, a medical relevant bacterium ................ 1

1.2. Lipolytic enzymes of P. aeruginosa involved in pathogenesis .......................................... 2

1.3. Phospholipases .............................................................................. 3
1.3.1. Definition and classification of phospholipases ...... 3
1.3.2. Three-dimensional structures of prokaryotic phospholipases A ............................................ 5
1.3.3. Physiological functions of bacterial phospholipases A ........................... 7
1.3.4. Role of bacterial phospholipases A in pathogenesis ............................................................... 9

1.4. Phospholipases from GDSL-hydrolases family 13

1.5. Aims of this study ........................................................................................................ 18

2. Materials ................................................................................. 19

2.1. Chemicals and Enzymes ............................................................... 19

2.2. Bacterial strains and plasmids .................