Strain-specific interactions of Staphylococcus aureus with human endothelium

Staphylococcus aureus ist ein weit verbreitetes Pathogen, dass durch seine Virulenz eine Vielfalt an lebensbedrohlichen Krankheiten verursacht. Von besonderer Bedeutung ist die Interaktion der Bakterien mit dem Endothel, hier am Beispiel von HUVECs gezeigt. In dieser Arbeit wurden S. aureus Isolate...

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Weiterer Titel:Strain specific interactions of Staphylococcus aureus with human endothelium
Verfasser: Sinitski, Dzmitry
Weitere Beteiligte: Schnittler, Hans-Joachim (Gutachter)
FB/Einrichtung:FB 13: Biologie
Dokumenttypen:Dissertation/Habilitation
Medientypen:Text
Erscheinungsdatum:2016
Publikation in MIAMI:22.02.2016
Datum der letzten Änderung:16.09.2016
Angaben zur Ausgabe:[Electronic ed.]
Schlagwörter:Staphylococcus aureus; HUVEC; intrazelluläres Überleben; Genexpression; Apoptose; Signalübertragung; Signalweg Stapylococcus aureus; HUVEC; intracellular survival; gene expression; apoptosis; signalling pathway
Fachgebiet (DDC):570: Biowissenschaften; Biologie
Lizenz:InC 1.0
Sprache:English
Format:PDF-Dokument
URN:urn:nbn:de:hbz:6-96279584246
Permalink:https://nbn-resolving.de/urn:nbn:de:hbz:6-96279584246
Onlinezugriff:diss_sinitski.pdf
Inhaltsverzeichnis:
  • Table of Contents .................................................................... I
  • Abbreviations .................................................................................................................................. IV
  • 1. Introduction ............................................................................................................................... 1
  • 1.1. Principles of bacteria-host interaction and microbial virulence ...................................... 1
  • 1.2. Staphylococcus aureus. .......................................................................................................... 3
  • 1.2.1. S. aureus is a human pathogen ...................................................................................... 3
  • 1.2.2. Antibiotic resistance of S. aureus .................................................................................. 4
  • 1.3. S. aureus virulence factors and their role in infection process ........................................ 5
  • 1.4. The innate immune system response to S. aureus infections .......................................... 9
  • 1.4.1. Endothelium in the innate immunity ........................................................................... 11
  • 1.4.2. Endothelium – S. aureus infection ............................................................................... 12
  • 1.5. Different strains of S. aureus cause diverse responses in endothelium ........................ 17
  • 1.6. Aim of the study .................................................................................................................... 20
  • 2. Materials and methods ........................................................................................................ 21
  • 2.1. Materials ................................................................................................................................ 21
  • 2.1.1. Reagents and materials ................................................................................................. 21
  • 2.1.2. Equipment ....................................................................................................................... 22
  • 2.1.3. Solutions, buffers, medium .......................................................................................... 24
  • 2.1.4. Commercial reagent kits ............................................................................................... 25
  • 2.1.5. Antibodies/reagents ...................................................................................................... 25
  • 2.1.6. Bacteria strains ............................................................................................................... 26
  • 2.1.7. Cell culture reagents ...................................................................................................... 26
  • 2.2. Methods ................................................................................................................................. 27
  • 2.2.1. Isolation and maintenance of human umbilical vein endothelial cell culture (HUVEC) ............................................................................................................................ 27
  • 2.2.2. Coating cell culture plates with or without glass coverslips .................................... 28
  • 2.2.3. Preparation of bacteria inoculum ................................................................................ 28
  • 2.2.4. Maintaining a HUVEC culture ....................................................................................... 29
  • 2.2.5. Infection of HUVEC with Staphylococcus aureus ...................................................... 29
  • 2.2.6. Immunostaining and fluorescence microscopy during caspase-3 assay ................ 29
  • 2.2.7. Annexin V/PI apoptotic assay ....................................................................................... 30
  • 2.2.8. Staphylococcus aureus intracellular survival ............................................................. 30
  • 2.2.9. Infected HUVEC phase contrast microscopy .............................................................. 30
  • 2.2.10. Protein kinases inhibition assay ................................................................................... 31
  • 2.2.11. Gene expression study .................................................................................................. 31
  • 2.2.11.1. The mRNA isolation and reverse transcription .................................................... 31
  • 2.2.11.2. Real-time qPCR ......................................................................................................... 32
  • 2.2.11.3. Quantification of RT-qPCR results .......................................................................... 35
  • 2.2.12. Microarray performance ............................................................................................... 36
  • 2.2.13. Statistics ........................................................................................................................... 36
  • 3. Results ..................................................................................................................................... 37
  • 3.1. Staphylococcus aureus strains that actively translocate prevent TNF-α-induced ICAM-1 upregulation in human endothelial cells ....................................................................... 37
  • 3.2. S. aureus display strain-related pathophysiological features in EC ............................... 39
  • 3.2.1. Expression of virulence factors is strain related ........................................................ 40
  • 3.3. Morphodynamics of cytotoxic effect of S. aureus strains in infected endothelial cell cultures .................................................................................................................................. 41
  • 3.3.1. Strain-specific S. aureus release from infected endothelial cells by
  • “outbursts” ..................................................................................................................... 45
  • 3.4. Cytotoxic staphylococcus aureus strains are able to survive and grow in the endothelium .......................................................................................................................... 46
  • 3.5. S. aureus induced endothelial cell death .......................................................................... 48
  • 3.5.1. Cytotoxic strains of S. aureus induce early apoptosis in endothelial cells ............. 48
  • 3.6. Global transcriptome analysis of HUVEC infected with different isolates of S. aureus ................................................................................................................................ 52
  • 3.6.1. The study of gene expression and experimental design .......................................... 52
  • 3.7. Microarray controls .............................................................................................................. 54
  • 3.7.1. Microarray internal controls ........................................................................................ 54
  • 3.7.2. Determining sample similarity by scatter plotting .................................................... 57
  • 3.7.3. Validation of microarray data by RT-qPCR ................................................................. 60
  • 3.7.4. Principal component analysis ...................................................................................... 63
  • 3.8. Microarray results ................................................................................................................ 64
  • 3.8.1. Differential expression of HUVEC infected with different S. aureus
  • strains .............................................................................................................................. 64
  • 3.8.2. Differential expression of HUVEC innate immunity genes ....................................... 66
  • 3.8.3. Differential expression of cell death-related genes in endothelium ..................... 72
  • 3.8.4. Differential expression of HUVEC cytoskeleton and developmental genes .......... 76
  • 3.8.4.1. Strain-specific expression of structural and developmental genes ........................ 80
  • 3.8.5. Differential expression of HUVEC transcription factors and signaling molecules
  • .......................................................................................................................................... 81
  • 3.9. S. aureus strain related induction of genes involved in endothelial
  • cell death ............................................................................................................................... 85
  • 3.9.1. Cytotoxic strains of S. aureus induce cell death related genes in endothelial cells
  • .......................................................................................................................................... 85
  • 3.9.2. Different transcription factors involved in S. aureus-induced cell death gene expression ....................................................................................................................... 89
  • 3.10. Influence of kinases signaling on survival of endothelium upon S. aureus infection ................................................................................................................................................. 91
  • 3.10.1. Inhibition of protein kinases affects HUVEC survival upon S. aureus infection .... 91
  • 4. Discussion ............................................................................................................................ 100
  • 4.1. HUVEC expression study and microarray data analysis ................................................ 102
  • 4.2. HUVEC infected with the Staphylococcus aureus isolates demonstrate the strain-specific gene expression response ................................................................................... 105
  • 4.3. Strain-specific mechanisms of HUVEC cell death induction .......................................... 108
  • 4.4. Verification of the role of protein kinases phosphorylation in S. aureus-induced endothelial apoptosis ........................................................................................................ 110
  • Summary ....................................................................................................................................... 114
  • References ..................................................................................................................................... 116.

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