Porins of lyme disease and relapsing fever spirochetes [Elektronische Ressource] / vorgelegt von Marcus Thein

Marcus Thein

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

PORINS OF
LYME DISEASE AND RELAPSING FEVER
SPIROCHETES





Dissertation
zur Erlangung des naturwissenschaftlichen Doktorgrades
der Fakultät für Biologie
an der Bayerischen Julius-Maximilians-Universität Würzburg





vorgelegt von
Marcus Thein
aus Würzburg





Würzburg, 2009
























Eingereicht am: …………………………………………..




Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. ………………….
1. Gutachter: Prof. Dr. Dr. h.c. Roland Benz
2. Gutachter: Prof. Dr. Sven Bergström




Tag des Promotionskolloquiums: …………………………………………..



Doktorurkunde ausgehändigt am:






























Diese Dissertation wurde von mir selbständig und nur mit den
angegebenen Quellen und Hilfsmitteln angefertigt.

Die von mir vorgelegte Dissertation hat noch in keinem früheren
Prüfungsverfahren in ähnlicher oder gleicher Form vorgelegen.

Ich habe zu keinem früheren Zeitpunkt versucht,
einen akademischen Grad zu erwerben.





Würzburg, ……………. …………………………

































"Natürlicher Verstand kann fast jeden Grad von Bildung ersetzen,
aber keine Bildung den natürlichen Verstand."



Arthur Schopenhauer
Publications



Pinne M., Thein M., Denker K., Benz R., Coburn J. and Bergström S. (2007). Elimination
of channel-forming activity by insertional inactivation of the p66 gene in Borrelia burgdorferi. FEMS
Microbiol Lett 266:241-9.

Thein M., Bunikis I., Denker K., Larsson C., Cutler S., Drancourt M., Schwan T. G.,
Mentele R., Lottspeich F., Bergström S. and Benz R. (2008). Oms38 is the first identified
pore-forming protein in the outer membrane of relapsing fever spirochetes. J Bacteriol
190(21):7035-42.

Thein M., Bonde M., Bunikis I., Denker K., Sickmann A., Bergström S. and Benz, R.
(2008). DipA, a pore-forming protein in the outer membrane of Lyme disease Borrelia exhibits
specificity for the permeation of dicarboxylates. Manuscript.

Thein M., Bárcena-Uribarri I., Sacher A., Bunikis I., Bergström S. and Benz R. (2008). The
P66 porin is present in both Lyme disease and relapsing fever species: a comparison of the
biophysical properties of six P66 species. Manuscript.

Thein M., Bárcena-Uribarri I., Maier E., Bonde M., Bergström S. and Benz R. (2008). The
use of nonelectrolytes as molecular tools reveals the channel size and the oligomeric constitution
of the Borrelia burgdorferi porin P66. Manuscript.

7
Table of contents

Publications ........................................................................................................................................... 7

Table of contents..... 9

Summary................. 15

Zusammenfassung ................................................................................................................................ 18

Chapter 1 – Introduction ......................................................................................................... 23
1.1. The bacterial genus Borrelia ...................................................................... 23
1.1.1. Classification ............................................................................................... 23 1.1.2. General characteristics ............................................................................. 24 1.1.3. Vectors, hosts and life cycle .................................................................... 25
1.1.4. Borrelia and pathogenicity26 1.1.5. Species included in this work ................................................................. 27
1.2. The Gram-negative cell-envelope ........................................................... 28
1.2.1. General features of pore-forming outer-membrane proteins (Porins) ....................................................................................... 29
1.3. The Borrelia cell-envelope .......................................................................... 30
1.3.1. Biogenesis and structure of spirochetal outer
membrane proteins .................................................................................. 31
1.3.2. Outer surface lipoproteins of Borrelia ................................................. 32
1.3.3. Pore-forming proteins in the outer membrane of Borrelia burgdorferi ................................................................................. 33
1.4. Aims of this work .............................................................................................. 35

Chapter 2 – Oms38 is the first identified pore-forming protein in
the outer membrane of relapsing fever spirochetes ..................... 37
2.1. Summary ................................................................................................................ 37
2.2. Introduction ........................................................................................................ 38
2.3. Materials and Methods ................................................................................. 40
9 Table of contents
2.3.1. Bacterial strains and growth conditions ............................................. 40
2.3.2. Isolation of outer membrane proteins and
purification of the 38 kDa protein ....................................................... 40
2.3.3. Protein electrophoresis ............................................................................ 40
2.3.4. Protein sequencing ................................................................................... 41
2.3.5. Planar lipid bilayer assays ....................................................................... 41
2.4. Results .................................................................................................................... 42
2.4.1. Pore-forming activities in the outer membrane
fractions of B. duttonii, B. hermsii and B. recurrentis .................. 42
2.4.2. Purification and identification of a 38 kDa, pore-forming
protein in the outer membranes of B. duttonii, B. hermsii and B. recurrentis ..................................................................................... 43
2.4.3. Analysis of the amino acid sequences of Oms38 of
B. duttonii, B. hermsii, B. recurrentis and B. turicatae ................ 44
2.4.4. Single-channel measurements of Oms38 ........................................... 46
2.4.5. Voltage dependence ................................................................................. 48
2.4.6. Selectivity measurements ........................................................................ 48
2.5. Discussion ............................................................................................................ 50
2.5.1. Lipid bilayer experiments with outer membrane fractions
(OMFs) of RF species suggest the presence of porins ................. 50
2.5.2. Purification and identification of Oms38 of B. duttonii,
B. hermsii and B. recurrentis ................................................................ 50
2.5.3. Deduced amino acid sequences of Oms38 of B. duttonii, B. hermsii, B. recurrentis and B. turicatae ....................................... 51
2.5.4. Biophysical properties of Oms38 .......................................................... 52

Chapter 3 – DipA, a pore-forming protein in the outer membrane
of Lyme disease spirochetes exhibits specificity for the
permeation of dicarboxylates .................................................................... 55
3.1. Summary ................................................................................................................ 55
3.2. Introduction ......................................................................................................... 56
3.3. Materials and Methods ................................................................................. 58
3.3.1. Bacterial strains and growth conditions ............................................. 58
3.3.2. Isolation of outer membrane proteins and
purification of the 36 kDa protein ....................................................... 58
3.3.3. SDS-PAGE and Immunoblotting ......................................................... 58 3.3.4. Overexpression of a recombinant fragment of DipA ...................... 59 3.3.5. Antiserum .................................................................................................... 59
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