An experimental strategy towards the proteome analysis of the parasitophorous vacuole in Plasmodium falciparum-infected erythrocytes [Elektronische Ressource] / Julius Nyalwidhe

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• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2003
• Nombre de lectures: 24
• Langue: English
• Poids de l'ouvrage: 8 Mo

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Philipps-Universität Marburg
Fachbereich Biologie
Abteilung Parasitologie
AG Lingelbach




An experimental strategy towards the proteome analysis of the
parasitophorous vacuole in Plasmodium falciparum-infected erythrocytes.


Dissertation
zur
Erlangung des Doktorgrades
Der Naturwissenschaften
(Dr.rer. nat)






Julius Nyalwidhe
aus Nairobi



Marburg/ Lahn 2003
i






An experimental startegy towards the proteome analysis of the
parasitophorous vacuole in Plasmodium falciparum infected erythrocytes


Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)



dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von
Julius O. Nyalwidhe
aus Nairobi


Marburg/Lahn 2003



Vom Fachbereich Biologie
der Philipps-Universität Marburg als Dissertation am 18 März 2003 angenommen.

Erstgutachter Prof.Dr. Klaus Lingelbach.
Zweitgutachter Prof. Dr. Uwe Maier.
Tag der mündlichen Prüfung am 18 März 2003.









ii Table of Contents
Page

List of Figures ………………………………………………………………........ v
List of Tables ……………………………………………………………........... vi
List of Abbreviations.............................................................................................. vii

I Introduction……………………………………………………………….......1

1.1 Morphology of Plasmodium falciparum and the infected host cell.....….... 1
1.1.1 The parasite.................................................................................………. .... 4
1.1.2 The infected erythrocyte…….…………......………………………….........5
1.2 Physiological alterations of the infected erythrocyte......………………......7
1.3 Secretory pathways in Plasmodium falciparum..........................................10
1.4 The parasitophorous vacuole- a transit compartment...................................15
1.5 Additional functions of the parasitophorous vacuole...................................19
1.6 From genomics to proteomics.......................................................................20
1.7 Biotin labeling of proteins.............................................................................22
1.8 Permeabilization of P.falciparum infected erythrocytes with SLO..............24
1.9 General objectives………………………………………………………….26

II Materials and Methods………………………………………………………....26
2.1 Materials…………………………………………………………………....26 2.1.1 Equipment………………………………………………………………….26
2.1.2 Disposable materials……………………..26
2.1.3 Chemicals and reagents……………………………………………………27
2.1.4 Solutions and buffers…………………………………29
2.1.5 Host cells and parasite isolates…………………………………………….32
2.1.6 Antibodies…………………………………32
2.2 Methods…………………………………………………………………….34
2.2.1 Parasite Cultures…………………………………………………………....34
2.2.2 SLO permeabilization of infected erythrocytes……………………....…....34


iii2.2.3 Biotinylation of SLO permeabilized iRBCs and extraction of soluble
proteins…………………………………………………….…………….....34
2.2.4 Biotin labeling of Bovine serum albumin (BSA)………...………………....35
2.2.5 Affinity purification of biotin labeled proteins…………………...………....35
2.2.6 Immunoprecipitation of the marker proteins………………....……………..36
2.2.7 Gel Electrophoresis………………….…………………………………….....36
2.2.8 Western Blot Analysis......................................................................................38
2.2.9 Indirect Immunofluorescence assay……...………………………………….39
2.2.10 Immunoelectromicroscopy………………………...………………………...39
2.2.11 Processing of proteins for analysis by MALDI-TOF.40
2.2.12 Peptide sequencing by tandem mass spectrometry…………………………..41
2.2.13 Protein identification in database searches by peptide mass fingerprinting…..41

III RESULTS…………………………………………………………….....................43
3.1 Experimental rationale.......................................................................................43
3.2 Permeabilisation of infected erythrocytes with SLO preserves the integrity of
the vacuolar membrane…………………………………………………….......45
3.3 Biotin accumulates in the parasitophorous vacuole in SLO permeabilized
infected erythrocytes…………………………………………………………...46
3.4 Streptavidin agarose beads specifically bind biotinylated parasite proteins…..48
3.5 Electrophoretic mobility of BSA is affected by biotinylation………………...49
3.6 The marker proteins SERP, GBP and PfALD are Biotinylatable……………..50
3.7 Biotinylation of SLO permeabilized infected erythrocytes is
Compartmentalized………………………………………………………….....51
3.8 The molecular chaperone PfBip interacts with biotinylated proteins………..54
3.9 Immunoelectronmicroscopy of SLO permeabilised and biotinylated IRBCs....58
3.10 Analysis of biotinylated soluble proteins by 1D SDS-PAGE…………….......60
3.11 Two-dimensional Gel electrophoretic analysis of Biotin labeled proteins…...64
3.12 Biotinylation pattern of the membrane proteins………………………….....69
3.13 Mass spectrometry analysis results……………………………………………72
3.13.1 Comparison of biotin labeled and non biotin labeled bovine serum albumin...72
3.13.2 Identification of P. falciparum proteins using peptide mass fingerprint data
from MALDI-TOF mass spectrometry to search protein databases………....76
3.14 Analysis of western blots from two dimensional gels…………...………….85
iv
3 .15 Localization of the heat shock proteins after cell fractionations……………..86

IV Discussion……………… ………………………………………………………….92
4.1 Experimental strategy...........................................................................................92
4.2 Specific Objectives...............................................................................................93
4.2.1 Removal of all of the cytosolic proteins of erythrocytes.....................................93
4.2.2 Verification of the existence of non-selective pores within the PVM..................93
4.2.3 Specific biotinylation of vacuolar proteins............................................................94
4.2.4 2D analysis and identification of biotinylated proteins........................................95
4.2.5 MALDI-TOF analysis of proteins........................................................................95
4.3 Limitations and the expectations of the study......................................................96
4.4 Characterization of novel vacuolar proteins and their possible functions............97
4.5 Conclusions.........................................................................................................101

References .........................................................................................................................102
Summary ............................................................................................................................120
Zusammenfassung..............................................................................................................122
Declaration..........................................................................................................................124
Acknowledgements..............................................................................................................125
Appendix
i) Julius Nyalwidhe, Stefan Baumeister, Alan R. Hibbs, Sallah Tawil, Janni Papakrivos, Uwe
Völker and Klaus Lingelbach (2002). A non-permeant biotin derivative gains access to the
parasitophorous vacuole in Plasmodium falciparum-infected erythrocytes permeabilized with
streptolysin 0. Journal of Biological Chemistry 277, 40005-40011.
ii) Sabine Wiek , Julius Nyalwidhe und Klaus Lingelbach (2002). Verteillung von
Parasitenproteinen Erythrozyten: Ein Schüsselereignis bei Pathogenese der Malaria. Bioforum
10, 678-680.





v List of Figures
Page
Figure 1. Life cycle of Plasmodium falciparum ..................................................................2
Figure 2. Asexual erythrocytic stages of Plasmodium falciparum ......................................3
Figure 3. Ultra structure of P.falciparum in permeablized erythrocytes..............................3
Figure 4. Destination of parasite proteins in Plasmodium falciparum infected
erythrocytes…........……….……………………………………………………...12
Figure 5. Structures of biotin derivatives and mech