Functional characterization of the CXC chemokine-degrading cell envelope protease of Streptococcus pyogenes [Elektronische Ressource] / von Simran Jeet Kaur

Simran

Sujets

• Gesundheit

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2009
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Functional characterization of the CXC chemokine-
degrading cell envelope protease of Streptococcus pyogenes





Von Der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades einer

Doktorin der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n












von Simran Jeet Kaur
aus Jagraon/Indien


1. Referent: PD Dr. Manfred Rohde
2. Referent: Prof. Dr. Dieter Jahn
eingereicht am: 03.03.2009
mündliche Prüfung (Disputation) am: 19.05.2009

Druckjahr 2009 Vorveröffentlichungen der Dissertation


Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab
veröffentlicht:

Tagungsbeiträge

Kaur, S. J., Talay, S. R., Sastalla, I., Frank, R., Graham, R., Hanski, E. and Chhatwal, G. S.:
Functional studies on the IL-8 degrading surface protease of Streptococcus pyogenes (Poster)
MPP60, Annual Meeting of the German Society for Hygiene and Microbiology (DGHM)
Goettingen (2007).

Kaur, S. J., Talay, S. R., Rohde, M., Zähner, D., Graham, R., Hanski, E. and Chhatwal, G. S.:
Role of IL-8 degrading protease in the internalization of Streptococcus pyogenes by
endothelial cells (Poster), Joint Bilateral Seminar, Indian National Science Academy, New
Delhi (2007). Table of Content I
Table of Contents
 
1  Introduction ............................................................................................................... 1 
1.1  Epidemiology of S. pyogenes infections .......................................................................... 2 
1.2  Necrotizing Fasciitis ....................................................................................................... 2 
1.3  S. pyogenes virulence factors involved in impairing phagocytic defence mechanism of
the host ....................................................................................................................................... 4 
1.3.1  Hyaluronic acid capsule ................................................................................................................ 4 
1.3.2  M protein ...................................................................................................................................... 5 
1.3.3  C5a peptidase/ScpA ...................................................................................................................... 6 
1.3.4  Streptolysins .................................................................................................................................. 6 
1.3.5  Streptococcal chemokine protease C (ScpC/SpyCEP) ................................................................... 7 
1.3.5.1  Cleavage of chemokine interleukin-8 by ScpC ..................................................................... 7 
1.3.5.2  Role of ScpC in S. pyogenes pathogenicity in a murine model of soft-tissue infection ......... 8 
1.3.5.3  ScpC impairs the function of murine chemokines ................................................................ 9 
1.3.5.4  Reduced neutrophil priming via cleavage of host chemokines by ScpC ............................. 10 
1.3.5.5  Biological characteristics of ScpC ...................................................................................... 10 
1.3.5.6  Regulation of scpC expression ........................................................................................... 12 
1.3.5.6.1  Regulation of scpC expression by sil ............................................................................. 12 
1.3.5.6.2  Regulation of S. pyogenes virulence by two component system covRS .......................... 13 
1.4  S. pyogenes and the host innate immune response ....................................................... 14 
1.4.1  Neutrophils ................................................................................................................................. 14 
1.4.2  Neutrophil killing mechanism ..................................................................................................... 15 
1.4.3  Neutrophil Extracellular Traps .................................................................................................... 16 
1.4.4  Interleukin-8 ................................................................................................................................ 17 
1.4.4.1  Transendothelial migration of neutrophils .......................................................................... 17 
1.5  Intracellular invasion by S. pyogenes ........................................................................... 19 
1.5.1  Virulence factors involved in the invasion of S. pyogenes into host cells .................................... 20 
1.6  Objectives of the study ................................................................................................. 23 
2  Materials and Methods ............................................................................................ 24 
2.1  S. pyogenes strains ....................................................................................................... 24 
2.2  E. coli strains ............................................................................................................... 24 
2.3  Vectors ......................................................................................................................... 24 
2.4  Antibiotics .................................................................................................................... 24 
2.5  Chemical Reagents ...................................................................................................... 25 
2.6  Primers ........................................................................................................................ 27 
2.7  Antibodies .................................................................................................................... 28 
2.8  Cultivation of bacteria ................................................................................................. 28 
2.8.1  Cultivation of S. pyogenes ........................................................................................................... 28 
2.8.2  Cultivation of E. coli ................................................................................................................... 29 
2.9  DNA isolation and quantification ................................................................................ 29 
2.9.1  Isolation of genomic DNA from S. pyogenes ............................................................................... 29 
2.9.2  Quantification of DNA ................................................................................................................ 30 
2.10  Polymerase chain reaction (PCR) ................................................................................ 30 
2.11  Agarose gel electrophoresis ......................................................................................... 31 Table of Content II
2.12  Molecular cloning techniques ...................................................................................... 32 
2.12.1  Digestion of DNA with restriction endonucleases ................................................................... 32 
2.12.2  Dephosphorylation of vector ................................................................................................... 32 
2.12.3  Ligation of DNA fragments .................................................................................................... 32 
2.12.4  DNA precipitation .................................................................................................................. 33 
2.12.5  Preparation of chemically competent E. coli cells ................................................................... 33 
2.12.6  Transformation of E. coli cells ................................................................................................ 33 
2.12.7  Colony PCR ........................................................................................................................... 34 
2.12.8  Colony Hybridization ..................