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Secondary cell wall polysaccharides in Bacillus anthracis and Bacillus cereus strains [Elektronische Ressource] / von Christine Leoff

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208 pages
Secondary cell wall polysaccharides in Bacillus anthracis and Bacillus cereus strains der Fakultät für Biologie der EBERHARD KARLS UNIVERSITÄT TÜBINGEN zur Erlangung des Grades eines Doktors der Naturwissenschaften von Christine Leoff aus Saarlouis vorgelegte Dissertation 2009 ______________________________________________________________________ Tag der mündlichen Prüfung: 01. August 2008 Dekan: Prof. Dr. H. A. Mallot 1. Berichterstatter: Prof. Dr. E. Kannenberg 2. Berichterstatter: Prof. Dr. A. Peschel 2 3SCWPs in B. anthracis and B. cereus strains________________________________ Table of Contents: Abbreviations ............................................................................................................... 7 Summary .................................................................................................................10 Chapter 1: General introduction ............................................................................... 14 ∗ The Bacillus cereus group of bacteria ........................................................... 14 ∗ The taxonomy of the B. cereus group of bacteria.......................................... 14 ∗ Pathogenicity of the group............................................................. 16 ∗ Virulence factors of B. anthracis...................................................................
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Secondary cell wall polysaccharides in
Bacillus anthracis and Bacillus cereus
strains


der Fakultät für Biologie
der EBERHARD KARLS UNIVERSITÄT TÜBINGEN

zur Erlangung des Grades eines Doktors
der Naturwissenschaften

von

Christine Leoff
aus Saarlouis

vorgelegte

Dissertation


2009
______________________________________________________________________



























Tag der mündlichen Prüfung: 01. August 2008

Dekan: Prof. Dr. H. A. Mallot
1. Berichterstatter: Prof. Dr. E. Kannenberg
2. Berichterstatter: Prof. Dr. A. Peschel
2


3SCWPs in B. anthracis and B. cereus strains________________________________
Table of Contents:

Abbreviations ............................................................................................................... 7
Summary .................................................................................................................10
Chapter 1: General introduction ............................................................................... 14
∗ The Bacillus cereus group of bacteria ........................................................... 14
∗ The taxonomy of the B. cereus group of bacteria.......................................... 14
∗ Pathogenicity of the group............................................................. 16
∗ Virulence factors of B. anthracis................................................................... 17
∗ Treatment and prevention of anthrax 18
∗ Scope of medically related uses of carbohydrates ......................................... 19
∗ Architecture and carbohydrates of the Gram positive bacilli cell wall.......... 20
∗ Questions and objectives addressed in this work........................................... 28
Chapter 2: Cell wall carbohydrate compositions of strains from the Bacillus cereus
group of species correlate with phylogenetic relatedness
Journal of Bacteriology 190 [1]: 112-121, (2008) .................................. 30
Chapter 3: The Structure of the major cell wall polysaccharide of
Bacillus anthracis is species specific
Journal of Biological Chemistry 281 [38], 27932-41, (2006)................. 60
Chapter 4: Structural elucidation of the non-classical secondary cell wall
polysaccharide from B. cereus ATCC 10987 and comparison with the arides from B. anthracis and B. cereus type strain ATCC 14579
reveals a common structural theme with varying modifications
Submitted to Journal of Biological Chemistry........................................ 91
Chapter 5: Secondary cell wall polysaccharides in B. anthracis and pathogenic B.
cereus strains are structurally closely related ....................................... 122
Chapter 6: saccharide of Bacillus anthracis and Bacillus
cereus strains are antigens that display both common and strain-specific
epitopes
Manuscript in preparation ..................................................................... 140
Chapter 7: Resume and conclusions....................................................................... 170
∗ Differences in cell wall glycosyl composition correlate with
phylogenetic relatedness and possibly with pathogenic functions .............. 170
4______________________________________________________________________
∗ The non-classical secondary cell wall polysaccharide of B. cereus
group members............................................................................................172
∗The immunochemical properties of the HF-PS structure ............................ 177
∗ Implications of the results for the viability and virulence functions of
the HF-PS and future research perspectives................................................ 180
References (General introduction)............................................................................186
References (Resume and conclusions) .....................................................................200
List of Publications ...................................................................................................205
Acknowledgements206
Curriculum vitae ......................................................................................................208


5SCWPs in B. anthracis and B. cereus strains________________________________
6__________________________________________________________Abbreviations
ABBREVIATIONS


ATCC American Type Cultre Collection
Ba B. anthracis
Bc B. cereus
BHI agar Brain heart infusion agar
Bovine albumine serum BSA
CDAP 1-cyano-4-dimethylaminopyridinium tetrafluroborate
CHO Carbohydrates
CM Cytoplasmic membrane
CPS Capsular polysaccharide
Crystal protein (B. thuringiensis) Cry
CWS delipidated, deproteinized, cell wall skeleton
Cyt Cytolytic protein
CytK Cytotoxin K
DI water Deionized water
Deoxyribonucleic acid DNA
EA1 Extractable antigen 1
EdTx Edema toxin
EF Edema factor
FAO Food and Agriculture Organization of the United Nations
Galactose Gal
GalNAc N-acetylgalactosamine
GC-MS Gas chromatography mass spectrometry
Glc Glucose
GlcN Glucosamine
N-acetylglucosamine GlcNAc
Hbl Hemolysin BL
IgG Immune globuline G
kb kilo base pair
7SCWPs in B. anthracis and B. cereus strains________________________________
kDa kilo Dalton
KLH Keyhole limp hemocyanine
Lethal toxin LeTx
LF Lethal factor
LPS Lipopolysaccharide
LTA Lipoteichoic acid
mAb Monoclonal antibody
N-acetylmannosamine ManNAc
MAPKK Mitogen activated protein kinase kinase
MPL Monophosphoryl lipid A a derivative of diphosphoryl lipid A from
Salmonella minnesota R595
MurN Muramic acid
MurNAc N-acetylmuramic acid
neural cell adhesion molecule N-CAM
Nhe Non hemolytic enterotoxin
OD Optical density
OIE World Organisation for Animal Health
OMe O-methyl group
Open reading frame ORF
PA broth Phage assay broth
PG Peptidoglycan
Pyr Pyruvate
rRNA Ribosomal Ribonucleic acid
Surface array protein Sap
SCWP Secondary cell wall polymer
SLH Surface layer homology
TA Teichuronic acid
TDM trehalose dimycolate purified cord factor from mycobacterium phlei
T-cell independent 1 TI-1
TI-2 T-cell independent 2
8__________________________________________________________Abbreviations
WHO World Health Organisation
WTA Wall teichoic acid

Figures are numbered for each chapter separately. If not otherwise stated, the
mentioned figure numbers refer to the figures in the same chapter.






9SCWPs in B. anthracis and B. cereus strains________________________________
SUMMARY

The Bacillus cereus group of bacteria is comprised of the closely related species
B. cereus, B. anthracis, and B. thuringiensis. As animal or human pathogens, all three
species are of high biological relevance. A comparison of the cell wall glycosyl (CWG)
composition of species from the B. cereus group has, to date, not been performed.
This thesis presents a systematic comparison of cell walls from closely related B.
cereus, B. thuringiensis and B. anthracis strains. The strains used in this study have
phylogentically been grouped into two clades and four lineages. Our findings showed
that the CWG compositions varied significantly both qualitatively and quantitatively
among the investigated strains. The cell walls from B. anthracis strains (Clade
1/Anthracis) contained glucose (Glc), galactose (Gal), N-acetylmannosamine
(ManNAc), and N-acetylglucosamine (GlcNAc). Qualitatively, B. cereus strains
belonging to Clade 1/Cereus IV (such as B. cereus G9241 and B. cereus 03BB87) had
the same CWG composition as strains belonging to Clade 1/Anthracis. The cell walls
from strains of Clade 1/Cereus III (e.g. B. cereus 03BB102) differed from those of
Clade 1/Anthracis in that they contained an additional mannose (Man) residue. Strains
belonging to Clade I/Cereus I (e.g. B. cereus ATCC 10987) differed from B. anthracis
in that their cell walls additionally contained N-acetylgalactosamine (GalNAc). The cell
walls from strains belonging to Clade 2 (e.g. the B. cereus type strain and B.
thuringiensis strains) lacked Gal and contained GalNAc. Our findings suggest the CWG
composition in the various B. cereus group strains display differences that correlate with
their phylogenetic relatedness and that the carbohydrate profiles may have diagnostic
value for strain recognition (Chapter 2/{Leoff, 2008 #10326}).
The treatment of the cell walls with aqueous hydrofluoric acid (HF) released a
polysaccharide structure (named HF-PS). A comparison of the glycosyl compositions
from the released and purified HF-PS structures of the different strains closely matched
the differences that were observed for the cell wall compositions, suggesting that this
cell wall fragment is likely a major or the major carbohydrate structure in these bacterial
strains. Moreover, shifts in the relative amounts of the various sugars in the cell walls
from B. anthracis strains with different plasmid content showed a possible impact of the
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