Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies

• 1 Introduction

  • 1.1  Shigella

    • 1.1.1  Epidemiology

    • 1.1.2 Properties

    • 1.1.3 Pathogenicity

    • 1.1.4 Virulence Factors

  • 1.2 Innate immune host cells: neutrophils

    • 1.2.1  Neutrophil recruitment

    • 1.2.2 Bacterial recognition by neutrophils

    • 1.2.3 Neutrophil killing mechanisms

  • 1.3 Interaction of neutrophils with Shigella

  • 1.4 Serine Proteases: NE and CG

    • 1.4.1  Serine Proteases

    • 1.4.2 Chymotrypsin-like serine proteases

    • 1.4.3 Human neutrophil cathepsin G (CG) and neutrophil elastase (NE)

  • 1.5  Aim of study

• 2 Materials and Methods

  • 2.1  Bacteria – Escherichia coli

    • 2.1.1  Strains

    • 2.1.2 Growth conditions and media

    • 2.1.3 Protein expression

    • 2.1.4 Protein purification

  • 2.2 Bacteria – Shigella flexneri

    • 2.2.1  Strains

    • 2.2.2 Growth conditions and media

    • 2.2.3 Cleavage of IpaB

    • 2.2.4 Cleavage of IcsA and OmpA

  • 2.3 Cells

    • 2.3.1  Cell line

    • 2.3.2 Growth conditions and media

    • 2.3.3 Transient transfection

    • 2.3.4 Stable transfection and single-cell dilution

    • 2.3.5 Cell lysis

  • 2.4 Chemicals

  • 2.5 Enzymatic Assays

    • 2.5.1  β-galactosidase activity

    • 2.5.2 Reagents to measure NE and CG activity

    • 2.5.3 NE and CG activity of cell lysates

    • 2.5.4 NE and CG kinetics / activity units

  • 2.6 Molecular cloning techniques

    • 2.6.1  Cloning of NE for expression in E. coli

    • 2.6.2 Cloning of NE for expression in RBL-1 cells

    • 2.6.3 Mutagenesis of ELA2 (NE gene)

  • 2.7 Protein analysis

    • 2.7.1  Determination of protein concentrations

    • 2.7.2 TCA precipitation

    • 2.7.3 SDS polyacrylamide gel electrophoresis (SDS-PAGE)

    • 2.7.4 Immunoblotting

    • 2.7.5 Coomassie staining

  • 2.8 Structure analysis

• 3 Results

  • 3.1  The Specificity of NE for virulence factors is not encoded in NE substrates

    • 3.1.1  A recognition motif for NE was not detected in the primary sequence of IpaB

    • 3.1.2 The secondary and tertiary structures of the substrate do not affect the specificity of NE for virulence factors

  • 3.2  The Specificity of NE for virulence factors is encoded in NE

    • 3.2.1  Expression of recombinant wildtype NE in mammalian cells

    • 3.2.2 Kinetics of recombinant wildtype NE

    • 3.2.3 Specificity of recombinant wildtype NE

    • 3.2.4  Design of NE mutants

    • 3.2.5 Analysis of NE mutants – Cleavage of peptide substrates

    • 3.2.6 Analysis of NE mutants – Cleavage of Shigella virulence factors

• 4 Discussion

  • 4.1  Do virulence factors contain a recognition motif for NE?

    • 4.1.1  A recognition motif for NE was not detected in the primary sequence of IpaB

    • 4.1.2 The secondary and tertiary structures of the substrate do not affect the specificity of NE for virulence factors

  • 4.2 The specificity of NE for virulence factors is encoded in NE

    • 4.2.1  Recombinant expression of NE

    • 4.2.2 The specificity of NE for virulence factors is encoded in NE

      • NE mutants 35-41 and 58A-61

      • NE mutants F192A, F192K and A213V

      • NE mutants F215Y and F215A

      • NE mutants 216-218 and 216-224

      • NE mutant N98L and N98A

    • 4.2.3 Conclusion

• 5 Appendix

  • 5.1 Expression of recombinant wt NE in bacteria

  • 5.2 Analysis of single-cell derived cell lines of the different NE mutants

  • 5.3  Sequence alignment of NE, CG, trypsin and chymotrypsin

  • 5.4  Amino acids – abbreviations and structural formula

• Abbreviations

• References

• Curriculum Vitae

• Publications

• Acknowledgements

• Selbständigkeitserklärung

• Table 3.1: Plasmids and Primers used in this study.

• Table 3.2: Primary and secondary antibodies used in this study.

• Fig. 1.1: Shigella infection.

• Fig. 1.2: Schematic presentation of the oxygen-dependent and oxygen-independent mechanisms during neutrophil phagocytosis of bacteria.

• Fig. 1.3: Structure of bovine chymotrypsin.

• Fig. 1.4: Nomenclature of substrate residues at the scissile bond.

• Fig. 1.5: Representation of protease-substrate interactions.

• Fig. 1.6: Specificity pocket of trypsin.

• Fig. 1.7: Schematic model of S1 pockets from different chymotrypsin-like serine proteases.

• Fig. 1.8: Superimposition of the NE and CG crystal structures.

• Fig. 3.1: Schematic representation of IpaB mutants.

• Fig. 3.2: The primary sequence of IpaB does not encode for NE specificity.

• Fig. 3.3: NE but not CG degrades native and denatured IpaB.

• Fig. 3.4: Expression of active recombinant NE in transiently transfected cells.

• Fig. 3.5: Expression of active recombinant wildtype NE in stably transfected RBL-1 cells.

• Fig. 3.6: Kinetics of recombinant wildtype NE.

• Fig. 3.7: Degradation of IpaB by lysates from RBL-1 cells.

• Fig. 3.8: NE activity units of wt lysate and pNE in the presence or absence of the inhibitor cocktail (IC).

• Fig. 3.9: The inhibitor cocktail (IC) can block degradation of IpaB by endogenous proteases without inhibiting NE specificity.

• Fig. 3.10 Schematic representation of the NE substrate binding pockets (SBP).

• Fig. 3.11: NE mutants and localization within the NE structure.

• Fig. 3.12: NE and CG activity of different cell lines expressing NE mutant N98A

• Fig. 3.13: NE and CG activity of the different NE mutants.

• Fig. 3.14: NE activity of wt and NE-like mutants in the presence or absence of NE or CG inhibitors.

• Fig. 3.15: CG activity of CG-like mutants in the presence or absence of NE or CG inhibitors.

• Fig. 3.16: NE mutants with NE activity cleave IpaB.

• Fig. 3.17: IpaB is cleaved by NE-like but not by CG-like mutants.

• Fig. 3.18: IcsA is cleaved by NE-like but not by CG-like mutants.

• Fig. 4.1: Schematic representation of the NE residues 35-41 and 58A-61.

• Fig. 4.2: Schematic representation of the S1 pocket of NE.

• Fig. 4.3: Schematic representation of the S2 pocket of NE.

• Fig. 4.4: Schematic representation of the NE segment 216-224.

• Fig. 4.5: Schematic representation of NE.

• Fig. 5.1: Mature NE expressed in bacteria is not active.

• Fig. 5.2: NE activity of different cell lines of the 11 NE mutants.

• Fig. 5.3 : CG activity of NE mutants 58A-61, N98A, 216-218, and 216-224.

• Fig. 5.1: Sequence alignment

• Fig. 5.2: Amino acids with non-polar side chains.

• Fig. 5.3: Amino Acids with uncharged-polar side chains.

• Fig. 5.4: Amino Acids with acidic side chains.

• Fig. 5.5: Amino acids with basic side chains.