Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120327-1091821-1-5 Inhaltsverzeichnis |
| Item Description: | Zsfassung in dt. Sprache |
| Physical Description: | VIII, 91 Bl., Bl. X - XXXI Ill., graph. Darst. |
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| 100 | 1 | |a Steck, Natalie Marie Annabel |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation |c Natalie Marie Annabel Steck |
| 264 | 1 | |c 2012 | |
| 300 | |a VIII, 91 Bl., Bl. X - XXXI |b Ill., graph. Darst. | ||
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| 337 | |b n |2 rdamedia | ||
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| 500 | |a Zsfassung in dt. Sprache | ||
| 502 | |a München, Techn. Univ., Diss., 2012 | ||
| 650 | 0 | 7 | |a Streptococcus faecalis |0 (DE-588)4183659-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Chronische Darmentzündung |0 (DE-588)4273697-3 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Metalloproteinasen |0 (DE-588)4284137-9 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Toxizität |0 (DE-588)4078372-8 |2 gnd |9 rswk-swf |
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| 689 | 0 | 0 | |a Chronische Darmentzündung |0 (DE-588)4273697-3 |D s |
| 689 | 0 | 1 | |a Streptococcus faecalis |0 (DE-588)4183659-5 |D s |
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Record in the Search Index
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| adam_text | IMAGE 1
TABLE OF CONTENTS
PUBLICATIONS AND PRESENTATIONS I
TABLE OF CONTENTS IV
ABSTRACT VII
ZUSAMMENFASSUNG VIII
1 INTRODUCTION 1
1.1 THE IMPORTANCE OF PROTEASES 1
1.1.1 GENERAL CHARACTERISTICS AND FUNCTIONS OF PROTEASES 1
1.1.2 HOST-DERIVED AND BACTERIAL PROTEASES IN THE GUT - AN UNEXPLORED
WORLD OF COMPLEXITY. 2
1.2 INFLAMMATORY BOWEL DISEASES 3
1.2.1 PROTEASES IN CHRONIC INTESTINAL INFLAMMATION 4
1.2.2 INTESTINAL MUCOSAL BARRIER DYSFUNCTION AS A CENTRAL KEY PLAYER IN
THE PATHOGENESIS OF IBD 5
1.2.3 BACTERIA-HOST INTERACTION: COMMENSAL GUT MICROBIOTA IN IBD 7
1.2.4 GASTROINTESTINAL INFECTIONS AS CRITICAL TRIGGER FOR IBD 9
1.2.5 BACTERIAL PROTEASES IN IBD 10
1.2.6 RECEPTORS FOR BACTERIAL PROTEASES IN THE GUT 13
1.3 ENTEROCOCCI: A NARROW PATH BETWEEN COMMENSALISM AND PATHOGENICITY 15
1.3.1 CHARACTERISTICS, DISTRIBUTION AND ASSOCIATION WITH DISEASE 16
1.3.2 PUTATIVE VIRULENCE FACTORS IN ENTEROCOCCI 19
2 STUDY OBJECTIVE 23
3 MATERIAL AND METHODS 24
3.1 BACTERIA 24
3.1.1 GENERATION OF THE EPAB DELETION MUTANTS 25
3.1.2 CULTIVATION AND PREPARATION OF CONCENTRATED CONDITIONED MEDIA 25
3.1.3 ASSESSMENT OF THE GELATINOLYTIC ACTIVITY BY CULTIVATION 26
3.1.4 ASSESSMENT OF GELE ACTIVITY WITH AN AZOCASEIN ASSAY 26
3.1.5 DETERMINATION OF GELATINOLYTIC ACTIVITY WITH ZYMOGRAPHY 26
3.1.6 BIOFILM FORMATION 26
3.1.7 ASSESSMENT OF GENE EXPRESSION ON TRANSCRIPTIONAL LEVEL USING
QUANTITATIVE REAL TIME PCR (QRT-PCR) 27
3.1.8 INFECTION MODEL GALLERIA MELLONELLA (G. MELLONELLA) 28
3.1.9 INFECTION MODEL CAENORHABDITIS ELEGANS (C. ELEGANS) 29
3.2 PROTEIN PURIFICATION 29
3.2.1 GELE PURIFICATION FROM THE CULTURE SUPERNATANT OF ENTEROCOCCI
STRAINS 29
3.2.2 PURIFICATION OF THE E. FAECALIS LIPOPROTEIN EF1362 30
3.3 CELL CULTURE 30
3.3.1 TRANSWELL EXPERIMENTS: MEASUREMENT OF THE TRANSEPITHELIAL
ELECTRICAL RESISTANCE AND TRANSLOCATION ACROSS POLARIZED INTESTINAL
EPITHELIAL CELLS 30
3.3.2 TRANSLOCATION EXPERIMENTS WITH E. FAECALIS 31
3.4 USSING CHAMBER EXPERIMENTS 31
IV
HTTP://D-NB.INFO/1025589793
IMAGE 2
3.5 HISTOLOGICAL AND IMMUNOHISTOCHEMICAL METHODS 32
3.5.1 H&E STAINING AND HISTOLOGICAL SCORING 32
3.5.2 ALCIAN BLUE STAINING 32
3.5.3 IMMUNOFLUORESCENCE STAINING IN VIVO AND IN VITRO 32
3.6 MICE, BACTERIAL MONOASSOCIATION AND ISOLATION OF PRIMARY IEC 33
3.7 WESTERN BLOT ANALYSIS 34
3.8 RNA ISOLATION, REVERSE TRANSCRIPTION AND QRT-PCR 34
3.9 DEGRADATION OF RECOMBINANT E-CADHERIN 35
3.10 IDENTIFICATION OF PROTEINS AND CLEAVAGE SITES USING LC-MS/MS
ANALYSIS 35
3.11 DETERMINATION OF GELE ANTIGENICITY 36
3.12 DNA LADDERING 36
3.13 WNT REPORTER ASSAY 37
3.14 STATISTICAL ANALYSIS 37
4 RESULTS 38
4.1 CHARACTERIZATION OF THE GELATINOLYTIC ABILITY OF E. FAECALIS 38
4.1.1 GELE EXPRESSION IN E. FAECALIS OG1RF, ITS ISOGENIC MUTANT STRAINS
AND NONCHARACTERIZED ISOLATES FROM IBD PATIENTS 38
4.1.2 GELE PURIFICATION USING ANION EXCHANGE CHROMATOGRAPHY 41
4.1.3 INHIBITION OF GELE ACTIVITY BY EDTA, HEAT TREATMENT AND MARIMASTAT
43
4.1.4 E. FAECALIS GELE IS NOT INDUCING APOPTOSIS AND IS NOT AN ANTIGENIC
STRUCTURE 45
4.1.5 E. FAECALIS GELE DOES NOT POSSESS A MUCOLYTIC ACTIVITY 46
4.2 E. FAECALIS GELE IMPAIRS BARRIER INTEGRITY OF INTESTINAL EPITHELIAL
CELLS 48
4.2.1 GELE DISRUPT BARRIER FUNCTION OF IEC 48
4.2.2 THE GELE-MEDIATED LOSS OF BARRIER FUNCTION IN IEC IS ASSOCIATED
WITH A REDUCTION OF TIGHT JUNCTION PROTEIN OCCLUDIN 53
4.2.3 SYNERGISTIC EFFECTS OF E. FAECALIS GELE AND PRO-INFLAMMATORY
CYTOKINES 55
4.3 INTERACTION BETWEEN E. FAECALIS GELE AND HOST-DERIVED GELATINASES 56
4.4 IMPACT OF E. FAECALIS GELE ON JUNCTION PROTEINS IN VIVO 57
4.4.1 E. FAECALIS GELE MEDIATES LOSS OF EXTRACELLULAR DOMAIN OF AJ
PROTEIN E-CADHERIN 57
4.4.2 INDUCTION OF SIGNALLING PATHWAYS BY E. FAECALIS GELE 61
4.4.3 E-CADHERIN CAN BE DIRECTLY DEGRADED BY E. FAECALIS GELE 63
4.5 E. FAECALIS GELE REDUCE BARRIER FUNCTION IN IBD MOUSE MODELS 65
4.6 ADDITIVE EFFECTS OF GELE AND EPAB ON E. FAECALIS VIRULENCE 71
4.6.1 GENERATION OF EPAB, GELE-EPAB AND FSRB-EPAB DELETION MUTANTS 71
4.6.2 CHARACTERIZATION OF EPAB, GELE-EPAB AND FSRB-EPAB MUTANTS 73
4.6.3 SYNERGISTIC EFFECTS OF EPAB AND GELE IN THE CONTEXT OF
ENTEROCOCCAL VIRULENCE 77
5 DISCUSSION 82
5.1 E. FAECALIS GELE IMPAIRS INTESTINAL BARRIER FUNCTION THROUGH
DEGRADATION OF JUNCTION PROTEINS 82
5.2 COMPARISON BETWEEN ENDOGENOUS GELATINASES AND E. FAECALIS GELE 84
5.3 RECEPTOR ACTIVATION BY E. FAECALIS GELE 85
5.4 CLINICAL IMPLICATIONS FOR BACTERIA-DERIVED PROTEASES 86
5.5 POSSIBLE ROLE FOR EPAB IN E. FAECALIS- MEDIATED CHRONIC INTESTINAL
INFLAMMATION 88
V
IMAGE 3
5.6 CONCLUSION AND PERSPECTIVE 89
6 APPENDIX 91
LIST OF FIGURES X
LIST OF TABLES XII
ABBREVIATIONS XIII
REFERENCES XVI
ACKNOWLEDGEMENTS XXIX
CURRICULUM VITAE XXX
ERKLARUNG XXXI
V I
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| discipline | Biologie Medizin |
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| spelling | Steck, Natalie Marie Annabel Verfasser aut Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation Natalie Marie Annabel Steck 2012 VIII, 91 Bl., Bl. X - XXXI Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache München, Techn. Univ., Diss., 2012 Streptococcus faecalis (DE-588)4183659-5 gnd rswk-swf Chronische Darmentzündung (DE-588)4273697-3 gnd rswk-swf Metalloproteinasen (DE-588)4284137-9 gnd rswk-swf Toxizität (DE-588)4078372-8 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Chronische Darmentzündung (DE-588)4273697-3 s Streptococcus faecalis (DE-588)4183659-5 s Metalloproteinasen (DE-588)4284137-9 s Toxizität (DE-588)4078372-8 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20120327-1091821-1-5 http://mediatum.ub.tum.de/node?id=1091821 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120327-1091821-1-5 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025087579&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Steck, Natalie Marie Annabel Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation Streptococcus faecalis (DE-588)4183659-5 gnd Chronische Darmentzündung (DE-588)4273697-3 gnd Metalloproteinasen (DE-588)4284137-9 gnd Toxizität (DE-588)4078372-8 gnd |
| subject_GND | (DE-588)4183659-5 (DE-588)4273697-3 (DE-588)4284137-9 (DE-588)4078372-8 (DE-588)4113937-9 |
| title | Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation |
| title_auth | Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation |
| title_exact_search | Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation |
| title_full | Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation Natalie Marie Annabel Steck |
| title_fullStr | Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation Natalie Marie Annabel Steck |
| title_full_unstemmed | Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation Natalie Marie Annabel Steck |
| title_short | Mechanistic impact of Enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation |
| title_sort | mechanistic impact of enterococcus faecalis metalloprotease on bacterial virulence in the context of chronic intestinal inflammation |
| topic | Streptococcus faecalis (DE-588)4183659-5 gnd Chronische Darmentzündung (DE-588)4273697-3 gnd Metalloproteinasen (DE-588)4284137-9 gnd Toxizität (DE-588)4078372-8 gnd |
| topic_facet | Streptococcus faecalis Chronische Darmentzündung Metalloproteinasen Toxizität Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1091821 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120327-1091821-1-5 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025087579&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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