Cloning, functional characterisation and deletion of UDP-galactopyranose-mutase of Leishmania major [Elektronische Ressource] / von Barbara Kleczka

Barabara Kleczka

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2006
• Nombre de lectures: 45
• Poids de l'ouvrage: 2 Mo

Extrait

Cloning, Functional Characterisation

and Deletion of UDP-Galactopyranose-Mutase

of Leishmania major










Von der Naturwissenschaftlichen Fakultät
der Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades einer

Doktorin der Naturwissenschaften
Dr. rer. nat.

genehmigte Dissertation










von

Dipl.-Biol. Barbara Kleczka

geboren am 13.01.1978 in Hannover


2006
























Referentin: Prof. Dr. rer. nat. Françoise Routier
Institut Zelluläre Chemie, Medizinische Hochschule Hannover

Korreferent: Prof. Dr. rer. nat. Dieter Steinhagen
Fachgebiet Fischkrankheiten, Zentrum für Infektionsmedizin
Tierärztliche Hochschule Hannover

Tag der Promotion: 19.07.2006 Content

Abstract……………………………………………………………………..……….………...…1

Zusammenfassung………….…………………………………………………………….…....3

1 Introduction..............................................................................................................5
1.1 Leishmania and Leishmaniases ....................................................................5
1.2 surface..........................................................................................9
1.2.1 Composition of Leishmania glycocalyx...........................................................9
1.2.2 Function of the surface glycoconjugates of L. major in the survival and
proliferation...................................................................................................12
1.3 Galactofuranose containing glycoconjugates.............................................17
1.3.1 Occurrence of Galactofuranose ......................................................................17
1.3.2 Galf biosynthetic pathway ...............................................................................17
1.4 Aim of this study ............................................................................................18

2 Materials and Methods...........................................................................................20
2.1 Materials..........................................................................................................20
2.1.1 Laboratory animals.......................................................................................20
2.1.2 Eukaryotic cell line........................................................................................20
2.1.3 Bacterial strains............................................................................................20
2.1.4 Phage...........................................................................................................21
2.1.5 Plasmids.......................................................................................................21
2.1.6 Oligonucleotides24
2.1.7 Antibodies.....................................................................................................25
2.1.8 Molecular weight markers.............................................................................26
2.1.9 Enzymes27
2.1.10 Culture media and additives.........................................................................27
2.1.11 Kits and further materials..............................................................................27
2.1.12 Standard buffer and media...........................................................................28
2.1.13 Chemicals.....................................................................................................29
2.1.14 Laboratory Equipment..................................................................................30
2.2 Cell biological approach................................................................................31
2.2.1 Leishmania major culture conditions ............................................................31
2.2.2 In vitro growth...............................................................................................32
2.2.3 Electroporation conditions for L. major.........................................................32
2.2.4 Fluorescence and Immunofluorescence microscopy ...................................32
2.2.5 Mice infection model.....................................................................................33
2.3 Biochemical techniques................................................................................33
i Content
2.3.1 Assignation of protein concentration ............................................................33
2.3.2 Activity assays of UGM.................................................................................34
2.3.3 Preparation of total cell lysates for GP63 and LPG analysis ........................35
2.3.4 Preparation of lysates for GIPLs analysis.....................................................35
2.3.5 HPTLC analysis............................................................................................36
2.3.6 Matrix assistance laser desorption (MALDI) mass spectrometry .................36
2.3.7 HPAEC analysis...........................................................................................36
2.3.8 Immunoprecipitation.....................................................................................37
2.3.9 SDS-PAGE...................................................................................................37
2.3.10 Coomassie staining of SDS gels ..................................................................37
2.3.11 LPS Silver staining .......................................................................................38
2.3.12 Western Blot.................................................................................................38
2.3.13 Immunostaining of Western Blots.................................................................38
2.4 Molecular biology techniques.......................................................................39
2.4.1 Nucleic acids precipitation............................................................................39
2.4.2 Phenol Chloroform extraction39
2.4.3 Determination of DNA concentrations ..........................................................39
2.4.4 DNA electrophoresis on agarose gels40
2.4.5 General cloning approaches.........................................................................40
2.4.6 Extraction of genomic DNA from L. major ....................................................45
2.4.7 Southern Blotting..........................................................................................45

3 Results....................................................................................................................47
3.1 Identification and Characterisation of the UDP- galactopyranose mutase
from Leishmania major..................................................................................47
3.1.1 Analysis of the amino acid sequence ...........................................................47
3.1.2 In vivo activity assay.....................................................................................49
3.1.3 In vitro testing of L. major UGM....................................................................50
3.2 Generation of a L. major GLF gene deletion mutant...................................51
3.2.1 GLF is a single copy gene in L. major 5ASKH .............................................52
3.2.2 Cloning of the targeting constructs...............................................................53
3.2.3 Targeted gene deletion of L.major GLF........................................................54
3.2.4 Re-expression of UDP-galactopyranose mutase in L. major ∆glf mutant ....59
3.3 Characterisation of L. major ∆glf mutant.....................................................60
3.3.1 In vitro growth of ∆glf mutant........................................................................60
3.3.2 Characterisation of L. major ∆glf cell surface glycoconjugates ....................61
3.4 Cellular localisation of UGM..........................................................................72
ii Content
3.5 Experimental mice infection study with ∆glf mutant ..................................73

4 Discussion..............................................................................................................76
4.1 Identification and partial characterisation of L. major UDP-
galactopyranose mutase…..……………………………………………………...76
4.2 Generation and characterisation of a L. major GLF gene deletion mutant
80
4.2.1 Characterisation of L. major GLF gene deletion mutants .............................80
4.2.2 Virulence......................................................