Antibiotic mechanisms of invertebrate and mammalian defensins

109 Pages·2013·3.47 MB·English

by  Miriam Wilmes

Antibiotic mechanisms of
invertebrate and mammalian
defensins

Dissertation

zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn

vorgelegt von
Miriam Wilmes
aus
Werdohl

Bonn 2012

III

Die vorliegende Arbeit wurde mit Genehmigung der Mathematisch-
Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität
Bonn angefertigt.

Erstgutachter: Prof. Dr. Hans-Georg Sahl

Zweitgutachterin: apl. Prof. Dr. Christiane Dahl

Tag der Promotion: 17.12.2012

Erscheinungsjahr: 2013

V

Table of contents
Table of contents .............................................................................................................. V
Abbreviations .................................................................................................................. VII
1 Introduction ................................................................................................................... 1
1.1 Host Defence Peptides ........................................................................................ 1
1.2 Defensins ............................................................................................................ 3
1.3 Antimicrobial mode of action of HDPs ................................................................. 7
1.4 Immunomodulatory functions of HDPs .............................................................. 10
1.5 HDP expression and human disorders .............................................................. 12
1.6 Resistance towards HDPs ................................................................................. 14
1.7 Objectives of this work ....................................................................................... 15
2 Material and Methods ................................................................................................. 17
2.1 Chemicals and solvents ..................................................................................... 17
2.2 Antibiotics and antimicrobial peptides ................................................................ 18
2.3 Microbiological methods .................................................................................... 19
2.3.1 Bacterial strains, culture media and growth conditions ........................... 19
2.3.2 Sterilisation of media, equipment and bacterial cultures ......................... 21
2.3.3 Determination of the optical density of a bacterial culture ....................... 21
2.3.4 Determination of the minimal inhibitory concentration ............................ 21
2.3.5 Antagonisation of putative target molecules ........................................... 22
2.3.6 Bacterial killing kinetics .......................................................................... 22
2.3.7 Growth kinetic measurement .................................................................. 22
2.3.8 Potassium release from whole cells ....................................................... 23
2.3.9 Determination of the membrane potential using tetraphenyl-
phosphonium bromide ............................................................................ 23
2.3.10 Incorporation of radio-labelled metabolites ............................................. 24
2.3.11 Membrane permeabilisation assay ......................................................... 24
2.3.12 Intracellular accumulation of the final soluble cell wall precursor
UDP-N-acetylmuramyl-pentapeptide ...................................................... 25
2.4 Methods in molecular genetics .......................................................................... 26
2.4.1 Determination of concentration and purity of nucleic acids ..................... 26
2.4.2 Agarose gel electrophoresis ................................................................... 26
2.4.3 Isolation of plasmid DNA ........................................................................ 26
2.4.4 Preparation of electrocompetent E. coli cells .......................................... 26
2.4.5 Transformation of E. coli by electroporation ........................................... 26
2.5 Protein and biochemical methods ...................................................................... 27
2.5.1 Heterologous expression and purification of His-tagged PBP2 ............... 27
2.5.2 Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis ................... 27
2.5.3 Zymogram analysis ................................................................................ 28
2.5.4 Determination of peptide and protein concentration ............................... 29
2.5.5 Peptide quantification by spectrometry ................................................... 29
2.5.6 In vitro lipid II synthesis and purification ................................................. 29
2.5.7 Phosphate determination ....................................................................... 31
2.5.8 Inhibition of PBP2-catalysed reaction in vitro.......................................... 32
2.5.9 Complexation of lipid II ........................................................................... 32
2.5.10 Transmission electron microscopy ......................................................... 32
VI

2.5.11 Biomolecular interaction analysis by surface plasmon resonance .......... 33
2.5.12 Chromatographic purification of lipoteichoic acid .................................... 34
2.5.13 Determination of glucose concentration ................................................. 35
2.5.14 CF-efflux from LTA-containing liposomes ............................................... 35
3 Results ....................................................................................................................... 37
3.1 Part 1: Insight into invertebrate defensin mode of action ................................... 37
3.1.1 Antibacterial activity spectrum of oyster defensins ................................. 38
3.1.2 Impact of oyster defensins on the membrane integrity of S. aureus ....... 38
3.1.3 Accumulation of the soluble cell wall precursor UDP-N-
acetylmuramyl-pentapeptide in oyster defensin treated cells .................. 39
3.1.4 Antagonisation of antibacterial activity of oyster defensins ..................... 41
3.1.5 Binding of oyster defensins to lipid II ...................................................... 41
3.2 Part 2: Insight into the antistaphylococcal mode of action of θ-defensins ........... 45
3.2.1 Antibacterial activity of θ-defensins against staphylococci ...................... 46
3.2.2 Impact of RTDs on staphylococcal cell wall biosynthesis ....................... 46
3.2.3 Impact of RTDs on macromolecular synthesis ....................................... 47
3.2.4 Impact of RTDs on the membrane integrity ............................................ 48
3.2.5 Morphological changes of RTD-2 treated S. aureus cells ....................... 51
3.2.6 Release of cell wall lytic enzymes by RTDs ........................................... 53
3.2.7 Antagonisation of antibacterial activity of θ-defensins ............................ 56
3.2.8 Release of carboxyfluorescein from LTA-containing liposomes .............. 56
3.2.9 Susceptibility testing of a PepI producing strain ..................................... 57
3.3 Part 3: Insight into the Gram-negative mode of action of defensins ................... 59
3.3.1 Antibacterial activity of hBD3 against E. coli ........................................... 60
3.3.2 Growth kinetic measurements in presence of hBD3 ............................... 60
3.3.3 Permeabilisation of E. coli outer and inner membrane ........................... 61
3.3.4 Morphological changes of hBD3 treated E. coli cells .............................. 62
3.3.5 Activity of hBD3 against E. coli strains with different LPS structures ...... 63
4 Discussion .................................................................................................................. 65
4.1 Part 1: The central cell wall building block lipid II as target for defensins ........... 65
4.2 Part 2: Induction of autolysis as mode of action of defensins ............................. 70
4.3 Part 3: Interaction of hBD3 with the Gram-negative cell envelope ..................... 74
4.4 Conclusion ........................................................................................................ 76
4.5 Outlook .............................................................................................................. 77
5 Summary .................................................................................................................... 79
6 References ................................................................................................................. 81
7 Publications ................................................................................................................ 97
8 Declaration (Eidesstattliche Erklärung) ....................................................................... 99
VII

Abbreviations
A absorbance at a wavelength of λ
λ
ABC ATP binding cassette
ad fill up to
AD atopic dermatitis
AM amidase
APS ammonium persulfate
ATCC American Type Culture Collection
BCA bicinchoninic acid
BHI brain heart infusion (complex medium)
BSA bovine serum albumin
°C degree Celsius
C -P undecaprenyl phosphate (lipid carrier)
55
CCCP carbonyl cyanide m-chlorophenylhydrazone
CD Crohn´s disease
CENTATM chromogenic cephalosporin (β-lactamase substrate)
CF carboxyfluorescein
CFU colony forming unit(s)
Cg-Def defensin from the oyster Crassostrea gigas
CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
hydrate
Ci Curie
cpm counts per minute
CSαβ cysteine-stabilised α-helix β-sheet (motif)
3D three-dimensional
Da Dalton
DAG diacylglycerol
DMSO dimethyl sulfoxide
DMSZ German Research Center for Microorganisms and Cell Culture
DNA deoxyribonucleic acid
DNase deoxyribonuclease
DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
EDTA ethylenediaminetetraacetic acid
et al. et alii, and others
GL glucosaminidase
GlcCer glucosylceramide
GlcNAc N-acetylglucosamine
VIII

h hour
hBD human β-defensin
HD human defensin (α-defensin)
HDP host defence peptide
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV human immunodeficiency virus
HNP human neutrophil peptide (α-defensin)
IC half maximal inhibitory concentration
50
IFN interferon
IL interleukin
IPTG isopropyl-β-D-galactopyranoside
l liter
LB lysogeny broth (complex medium)
lipid II undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide-GlcNAc
LPS lipopolysaccharides
LTA lipoteichoic acid
M mol
MALDI-TOF MS matrix-assisted laser desorption/ionisation time-of-flight mass
spectrometry
MES 2-(N-morpholino)ethanesulfonic acid
MIC minimal inhibitory concentration
MilliQ ultrapure water (type I)
min minute
MRSA methicillin-resistant Staphylococcus aureus
MurNAc N-acetylmuramic acid
MW molecular weight
MWCO molecular weight cut-off
Ni-NTA nickel-nitrilotriacetic acid
NMR nuclear magnetic resonance
OD optical density at a wavelength of 600 nm
600
ONPG o-nitrophenyl-β-D-galactopyranoside
PBP penicillin binding protein
pH pondus hydrogenii, hydrogen ion concentration
PMA phosphomolybdic acid
R resistance
RNA ribonucleic acid
IX

RNase ribonuclease
RP-HPLC reversed-phase high performance liquid chromatography
rpm rounds per minute
RT room temperature
RTD Rhesus macaque θ-defensin
RU response unit(s)
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SPB sodium phosphate buffer
SPR surface plasmon resonance
SUV small unilamellar vesicle
TAE Tris-acetate-EDTA buffer
TEMED N,N,N',N'-tetramethylethylenediamine
TLC thin layer chromatography
TLR Toll-like receptor
TNF tumour necrosis factor
TPP+ tetraphenylphosphonium bromide
Tris tris(hydroxymethyl-)aminomethan
Triton X-100 polyethylene glycol tert-octylphenyl ether
TSB tryptic soy broth (complex medium)
UDP uridine diphosphate
UDP-MurNAc-pp UDP-N-acetylmuramyl-pentapeptide
V volt
VRE vancomycin-resistant enterococci
v/v volume per volume
w/v weight per volume

The abbreviations of the amino acids follow the IUPAC regulations. For time, length and
mass description the standard IS units were used.