Research Collection Doctoral Thesis Specific receptor recognition and cell wall hydrolysis by bacteriophage structural proteins Author(s): Bielmann, Regula Publication Date: 2009 Permanent Link: https://doi.org/10.3929/ethz-a-005783673 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No 18255 Specific Receptor Recognition and Cell Wall Hydrolysis by Bacteriophage Structural Proteins A dissertation submitted to ETH Zurich for the degree of Doctor of Sciences presented by Regula Bielmann Dipl. Natw. ETH born September 29, 1978 from Rechthalten (FR) accepted on the recommendation of Prof. Dr. Martin Loessner, examiner Prof. Dr. Herbert Schmidt, co-examiner 2009 _________________________________________________________________________I Table of contents Table of contents ................................................................................................. I Abbreviations ..................................................................................................... III Summary..............................................................................................................V Zusammenfassung ...........................................................................................VII 1. Introduction .............................................................................................. 1 1.1. Listeria ................................................................................................................... 1 1.1.1. Listeria: History, taxonomy, ecology, and growth factors ...................... 1 1.1.2. Listeria monocytogenes – the causative agent of listeriosis.................. 3 1.1.2. Virulence of Listeria: Intracellular infection cycle ................................... 4 1.2. Bacteriophages...................................................................................................... 6 1.2.1. History, taxonomy, and morphology of bacteriophages......................... 6 1.2.2. Phage life cycle...................................................................................... 7 1.2.3. Listeria phages and their application ................................................... 12 1.2.4. Research objectives ............................................................................ 16 2. Material and Methods............................................................................. 17 2.1. Bacterial strains, growth conditions, phage propagation, and phage purification 17 2.2. Molecular cloning................................................................................................. 20 2.2.1. Constructs for recombinant protein expression ................................... 20 2.2.2. Construction of deletion mutant Listeria phages.................................. 20 2.3. Proteomics........................................................................................................... 23 2.3.1. Protein expression and purification...................................................... 23 2.3.2. Polyclonal rabbit-antibodies................................................................. 24 2.3.3. Mass spectrometry .............................................................................. 25 2.3.4. SDS-PAGE, Western blot analysis, visualization of lytic phage proteins by zymography, and 2D-gel electrophoresis......................... 25 2.4. Assays ................................................................................................................. 27 2.4.1. Binding assays..................................................................................... 27 2.4.2. “Pull-down” assay ................................................................................ 27 2.4.3. Transmission electron microscopy (TEM) ........................................... 28 2.5. Bioinformatics ...................................................................................................... 28 _________________________________________________________________________II 3. Results .................................................................................................... 29 3.1. Proteomics of different Listeria phages. .............................................................. 29 3.1.1. Profiles of three temperate phages A118, A500, A006 and three virulent phages P40, P35, and A511................................................... 29 3.1.2. Programmed translational frameshifting in A118 and A500 ................ 32 3.2. Identification of the lytic structural protein (LSP) ................................................. 40 3.2.1. LSP: a common element among Listeria phages ................................ 40 3.2.2. Identification of gp19 as the lytic structural protein (LSP) in A118 ...... 44 3.3. Topological model of the A118 tail tip.................................................................. 46 3.3.1. Antibodies against putative tail and baseplate proteins of A118 ......... 46 3.3.2. Gp18, gp19, and gp20 of A118 play an important role in the early steps of infection ................................................................................. 46 3.3.3. Transmission electron microscopy (TEM) analysis of Listeria phage A118.................................................................................................... 49 3.4. Identification of the receptor binding protein (RBP) ............................................. 53 3.4.1. Gp20 of A118 and A500 binds to Listeria cell walls............................. 53 3.4.2. The A118 RBP requires N-acetylglucosamine and rhamnose for binding................................................................................................. 56 4. Discussion .............................................................................................. 59 5. References.............................................................................................. 67 Publications....................................................................................................... 85 Danksagung ...................................................................................................... 87 Curriculum Vitae ............................................................................................... 89 _________________________________________________________________________III Abbreviations aa Amino acid ATCC American Type Culture Collection bp Base pairs CBD Cell wall binding domain cfu Colony forming units Cps/ Cps-L Major Capsid protein/ Major Capsid protein-Long CsCl Cesium Chloride DNA Deoxyribonucleic acid ds double stranded DTT Dithiothreitol E. coli Escherichia coli GFP Green fluorescence protein GlcNAc N-Acetylglucosamine HCCA hydroxy-alpha-cyanocinnamic acid ICTV International Committee on Taxonomy of Viruses IEF Isoelectric focusing InlA InternalinA InlB InternalinB IPTG Isopropyl-β-D-thiogalactopyranosid kDa kilo Dalton LB Luria Bertani LLO Listeriolysin-O L. monocytogenes Listeria monocytogenes LSP Lytic structural protein MW molecular weight NAM N-Acetylmuramic acid OD Optical density ORF Open reading frame pfu Plaque forming units RBP Receptor binding protein _________________________________________________________________________IV PCR Polymerase chain reaction PEG Polyethyleneglycol PlcA Phospholipase C Ply Phage lysin PVDF Polyvinylidenfluorid Rha Rhamnose RNA Ribonucleic acid SDS-PAGE Sodium-dodecylsulfate-polyacrylamide-gelelectrophoresis SLCC Special Listeria Culture Collection SV Serovar tal Tail associated lysin TB Tryptose broth TBS Tris buffered saline TEM Transmission electron microscopy TFA Trifluoro acetic acid Tmp Tape measure protein Tris Tris[hydromethyl]aminomethan Tsh/ Tsh-L Tail sheet protein/Tail sheet protein-Long WSLC Weihenstephan Listeria Collection Wt Wildtype _________________________________________________________________________V Summary Adsorption of a bacteriophage to the cell wall of the bacterial host requires recognition of a cell wall associated receptor by the phage receptor binding protein (RBP). This recognition event is extremely specific, and high affinity binding is important for rapid and efficient virus attachment. After adsorption, the phage-DNA is injected to the host cytoplasm, which requires penetration through the multilayered peptidoglycan of the Gram-positive Listeria cell wall. For this purpose, a lytic structural protein (LSP) locally digests the murein during the infection process. Little is known about the receptor binding and DNA delivery during the early steps of phage infection of Gram-positive bacteria. Listeria phage A118 was isolated from Listeria monocytogenes serovar (SV) 1/2. It features a non-contractile tail of approximately 300 nm in length, an isometric capsid with a diameter of 61 nm, and belongs to the Siphoviridae family of dsDNA bacterial viruses, in the order Caudovirales (B1 morphotype). The phage adsorbs to the SV-specific L-rhamnose and N-acetylglucosamine substituents in the cell- wall teichoic acids of host cell. Listeria phage A500 exhibits a non-overlapping and complementary host-range, infecting L. monocytogenes SV 4b. Although the host range of the two phages is different, they share significant sequence similarities in the predicted gene products of the late gene cluster. The identification of the RBP in phages A118 and A500 is reported here. Specific binding of GFP-RBP fusion proteins to the listerial cells