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

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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

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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

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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

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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

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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

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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 could be demonstrated. Binding of truncated versions of the putative RBPs suggested that the binding specificity of the RBP resides in the C-terminal part. Furthermore, the receptor on the host cell could be identified for the RBP of A118. It was shown by zymograms that lytic structural proteins are present in all tested Listeria phages. Whereas the tested temperate phages (A118, A500, and PSA) revealed a single lytically active band, located directly below the prominent major capsid protein (Cps), the virulent phage A511 demonstrated two lytically active bands of different sizes. Peptide fingerprinting and Western blot analysis of the zone responsible for lytic activity enabled assignment of a lytic activity to a baseplate protein (LSP) of A118.

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Finally, application of antibodies against several baseplate proteins and transmission electron micrography (TEM) enabled the proposition of a model of the A118 tail tip with the arrangement of both RBP and LSP within the baseplate. This thesis work provides answers to fundamental questions about the biology of Listeria bacteriophages and will also be useful to develop novel and effective tools for specific recognition and control of the foodborne pathogen L. monocytogenes.

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Zusammenfassung

Bakteriophagen benötigen zur Adsorption an die Zellwand einer Wirtszelle ein Protein, welches den Rezeptor erkennt und bindet. Es wird angenommen, dass dieses Rezeptorbindeprotein (RBP) hoch affin und extrem spezifisch bindet, denn genau diese Eigenschaften sind wichtig für ein effizientes und schnelles Anhaften des Virus an die Wirtszelle zu Beginn des Infektionsprozesses. Das Genom des Phagen wird nach Adsorption an die Zelle in das Cytosol des Wirts eingeschleust. Hierfür muss die Gram-positive Zellwand der Listerienzelle durchdrungen werden. Um dies zu bewerkstelligen, besitzt der Phage ein lytisches Strukturprotein (LSP), welches während des Infektionsprozesses lokal das Peptidoglykan lysiert. Bislang ist jedoch über die frühen Schritte der Phageninfektion in Gram-posiviten Bakterien bislang wenig bekannt. Der Phage A118 wurde aus Listeria monocytogenes Serovar (SV) 1/2 isoliert. Er hat einen nicht-kontraktilen, ca. 300 nm langen Schwanz und einen ikosaederförmigen Kopf mit einem Durchmesser von 61 nm und gehört zur Familie der Siphoviridae von dsDNA-Viren in der Ordnung der Caudovirales (B1 Morphotyp). Der Phage adsorbiert an SV-spezifische Kohlenhydrat-Reste (L- Rhamnose und N-Acetylglukosamin), die sich in den zellwandassoziierten Teichonsäuren der Wirtszelle befinden. Ein komplementäres und nicht überlappendes Wirtsspektrum zu A118 weist Phage A500 auf, der L. monocytogenes SV 4b infiziert. Obschon das Wirtsspektum von A118 und A500 unterschiedlich ist, sind sich die beiden Phagen in den späten Genen doch sehr ähnlich. Diese Arbeit beschreibt die Identifizierung der rezeporbindenden Proteine (RBP) der Phagen A118 und A500. Es konnte gezeigt werden, dass GFP-RBP Fusionsproteine spezifisch an Listerienzellen binden. Durch Bindung von verkürzten GFP-RBP an die Zellen konnte weiter gezeigt werden, dass die Spezifität der Bindung im C-Terminus des RBPs liegt. Zusätzlich konnte für das rezeptorbindende Protein von A118 der Zelloberflächen-Rezeptor identifiziert werden.

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Lytische Strukturproteine (LSP) wurden mit Hilfe von Zymogrammen in allen getesteten Listeria Phagen gefunden. Während drei der untersuchten temperenten Phagen (A118, A500 und PSA) nur eine lytisch aktive Bande zeigten, wurden im virulenten Phagen A511 zwei lytisch aktive Banden unterschiedlicher Grösse gefunden. Gelstücke, die das lytische Protein enthalten, wurden mittels Peptide Fingerprint und Western blot analysiert. Dies ermöglichte eine genaue Zuordnung des LSP zu einem Strukturprotein von A118. Um die Proteine im Phagenpartikel zu lokalisieren wurden Antikörper, die gegen verschiedene Proteine der Basalplatte aus A118 gerichtet waren, eingesetzt. Mit Hilfe von Transelektronenmikroskopiebildern gelang es schliesslich ein Modell für das Schwanzende und die Basalplatte von A118 zu erstellen, wo sowohl das RBP und das LSP darin zugeordnet werden konnten. In dieser Arbeit wurden fundamentale Aspekte der Biologie von Listeria- Bakteriophagen betrachtet. Sie liefert eine Grundlage für die Ausarbeitung von neuen und effizienten Werkzeugen für eine spezifische Erkennung und Kontrolle des Krankheitserregers L. monocytogenes.

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1. Introduction

1.1. Listeria

1.1.1. Listeria: History, taxonomy, ecology, and growth factors

Listeria was described more than 80 years ago by E.G.D. Murray and J. Pirie, independently of each other (93, 101). Both named the newly described bacterium differently. Due to the observation of a characteristic monocytosis in laboratory rabbits and guinea pigs, Murray named it “Bacterium monocytogenes”. Pirie on the other hand termed it “Listerella hepatolytica” as he isolated the organism from veld rodents from South Africa. Because of the identity of the organism, the current name Listeria monocytogenes was then given in 1940 (100). The genus Listeria belongs to the phylum Firmicutes in the order Bacilliales and is closely related to the genera Brochothrix, Bacillus, Staphylococcus, Streptococcus, Enterococcus and Clostridium (54, 105). They share the characteristic feature of a G+C-content less than 50% (105, 130). In recent years, the taxonomic position of Listeria species has been the subject of much work and debate. The ninth edition of Bergey`s Manual of Systematic Bacteriology (118) recognized five biochemically distinguishable species, namely L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri and L. ivanovii, whilst L. denitrificans, L. grayi and L. murrayi are listed as species incertae sedis. Recently, the genus Listeria was divided into 6 species: L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and L. grayi (105, 115). L. grayi and L. murrayi are considered not to be sufficiently different from each other and were merged in one species L. grayi with two subspecies L. grayi ssp. grayi and L. grayi ssp. murrayi (106). Further, the species L. ivanovii was divided into L. ivanovii ssp. ivanovii and L. ivanovii ssp. londoniensis (10). According to the pattern of somatic (O) and flagellar (H) antigens, a total of 17 serovars are known. L. monocytogenes is represented by 13 serovars (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e and 7) (37), some of which are shared by L. innocua and L. seeligeri (2).

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Bacteria of the genus Listeria are Gram-positive, short (0.4-0.5 x 0.5-2.0 µm), non-sporeforming rods that are motile at 20-25 °C by means of peritrichous flagella that provide a tumbling motility (103). On the other hand, at 37 °C they are not motile, as expression of the flagella is temperature dependent (98). Colonies of Listeria appear in a characteristic bluish color when illuminated by indirectly transmitted light (51). They are aerobic and facultative anaerobic. The temperature limits of growth are 1-2 °C to 45 °C (55), the optimum temperature is between 30-37 °C. Listeria can tolerate high salt concentrations (up to 10% NaCl) and survive low pH (pH 4.5); growth is optimally at pH 7 (118). These properties enable them to survive under extreme conditions. Therefore, it is not surprising that Listeria is widespread in nature. The bacteria have been isolated from many different environments, including soil, water, vegetation, sewage, animal feeds, farm environments and food-processing environments (38, 109, 133, 136-138).

Fig. 1. Transmission electron micrograph of L. monocytogenes (this work).

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1.1.2. Listeria monocytogenes – the causative agent of listeriosis

All of the different Listeria species are widespread in the environment, but only Listeria monocytogenes is considered to be a significant human pathogen. However, occasional human infections due to L. seeligeri or L ivanovii have also been reported (21, 53, 88, 108). L. ivanovii is responsible for some cases of abortion, and L. seeligeri is generally considered nonpathogenic (130). There are 13 serotypes of L. monocytogenes, but almost all clinical cases are due to types 4b, 1/2a, and 1/2b (115). They have been isolated from a broad variety of foods like milk, cheese (especially soft cheeses) and other diary products, meat and meat products, poultry and eggs, fish, fish products and seafood, raw vegetables, and salad (37). Transfer of the organism to the food occurs mostly by secondary contamination. At high risk are in general ready-to-eat products that are consumed without final heat treatment. The disease caused by L. monocytogenes is called listeriosis. Human listeriosis is typically acquired through ingestion of contaminated food, but other modes of transmission occur. These include transmission from mother to child transplacentally or through an infected birth canal and crossinfection in neonatal nurseries. Human-to-human infections have not been documented (18, 115). Human disease caused by L. monocytogenes occurs most frequently in women of childbearing age, infants, and the elderly. The risk of listeriosis is greatest among certain well defined high-risk groups, including pregnant women, neonates, and immunocompromised adults but may occasionally occur in persons who have no predisposing underlying conditions (37). Unlike infection with other common foodborne pathogens such as Salmonella, which rarely result in fatalities, listeriosis is associated with a mortality rate of approximately 30% (41). At least two different forms can appear, mainly depending on susceptibility of the patient: non-invasive, gastrointestinal infections usually occur in healthy, immunocompetent people, and are characterized by mild symptoms such as fever, vomiting, and diarrhea. Whereas invasive infections mainly affect persons belonging to one of the risk groups and are associated with severe symptoms such as meningitis or encephalitis, generalized bacteremia or septicemia, endocarditis, myocarditis or pneumonia (37, 40, 115, 130).

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Pregnant women infected with L. monocytogenes only develop fever-like symptoms, but the fetus can be infected via the placenta, which can result in abortion, stillbirth or generalized infection of the newborn (37, 115, 130).

1.1.2. Virulence of Listeria: Intracellular infection cycle

Internalization of the bacterium begins with the adhesion to the eukaryotic cell and subsequent penetration into the host cell. Invasion of nonphagocytic cells involves a zipper-type mechanism where the host cell surface surrounds the bacterium until it is complete engulfed. This internalization process differs to the membrane ruffles characteristic of invasion by Salmonella and Shigella (29, 56, 65, 124). The infection process has been studied extensively in cells in tissue culture (19). The entry of Listeria into mammalian cells is triggered by at least two surface proteins belonging to the internalin family: internalins InlA and InlB (20). The completion of the Listeria genome sequence revealed a large number of surface proteins, so that additional bacterial factors are probably involved in the uptake (13, 43). After phagocytosis, the bacterium is enclosed in a subcellular phagolysosom, a hostile and toxic environment for most bacteria. The low pH within this organelle activates listeriolysin-O (LLO), a pore-forming cytolysin that, together with phospholipase C (PlcA), lyses the membrane and allows L. monocytogenes to escape into the cytoplasm. The LLO is, in contrast to other members of the same family of toxins such as streptolysin-O or perfringolysin, optimally active at pH 5.5- 6.0 (corresponding to the internal pH of the vacuole) and less active at higher pHs (30). This lysis of the vacuole occurs about 30 min after infection. The bacterium is then able to multiply within the cytosol and through actin-based intracellular motility it is able to invade the neighboring cell. This spreading from cell to cell is also mediated by virulence factor ActA. During this process a two-membrane vacuole is formed, where the bacterium is again released into the cytosol, with help of LLO and another phospholipase C (PlcB) (130).

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Invasion (InlA, InlB)

Lysis of two- membrane vacuole Escape from (LLO, PlcB) phagolysosom (LLO & PlcA))

Actin recruitment and replication

Cell-to-cell spread Polymerized actin- (Listeriapods) Polymerization (ActA)

Fig. 2. Infection cycle of L. monocytogenes in host cells (Modified from Tilney et al. 1989 (126))

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1.2. Bacteriophages

1.2.1. History, taxonomy, and morphology of bacteriophages

Bacteriophages are viruses that infect bacteria. The name bacteriophage is derived from bacteria and Greek phagein “to eat”. The history of bacteriophages started in 1915 when F. W. Twort observed a “glassy transformation” within a layer of bacterial cocci, which could be induced in colonies of normal appearance after inoculating with substance of such “glassy” colonies (99). Two years later, F. d`Herelle described independently a similar phenomenon that was “antagonistic” to bacteria and that resulted in lysis in liquid culture and death in discrete patches, that he called plaques (22). He was interested in their biological nature and claimed the idea of an organized infection agent that is an obligate intracellular parasite. It was also d`Herelle who proposed this culture as a therapeutic agent in the preantibiotic era. Early studies dealt with the use of phages for the control of epidemics but the interest in phage therapy diminished after the invention of antibiotics in the forties. Nevertheless, phage therapy continued to be investigated extensively especially in the republic of former Soviet Union. Today, the use of phages as antimicrobial agent regained attention, as the increasing antibiotic resistances becomes a serious problem. Besides this, phages have become important model organisms for molecular biology and tools for application (see chapter 1.2.3. (123)).

The classification of bacteriophages goes back to Bradley (11, 12). Phages are divided into 6 groups based on the morphological differences and their differences in nucleic acids (single and double stranded DNA or RNA). These criteria are still the basis for the phage classification. According to the International Committee on Taxonomy of Viruses (ICTV), bacteriophages are classified in one order, 13 families, and 30 genera. At least 96% of all known bacteriophages are tailed and belong to the order Caudovirales. They are subdivided into 3 families, the Myoviridae (25%), phages with a contractile tail, the Siphoviridae (61%) with a non-contractile, flexible tail, and the Podoviridae (14%) with a non-contractile,

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short tail. Their genetic material is double stranded DNA (62). The remaining 4% of the bacteriophage virions are polyhedral, filamentous, or pleomorphic, and a few have lipid-containing envelopes (Fig. 3) (1).

Tailed (96%) Polyhedral (<4%)

Filamentous (<4%)

Pleomorphic (<4%)

Fig. 3. Basic bacteriophage morphotypes (Modified from Ackermann, 2003 (1))

1.2.2. Phage life cycle

Phages are obligate intracellular parasites and multiply within their host. At the end of the infection cycle they destroy their host cell, except for some filamentous phages which can cause chronic infections and are therefore constantly released from the host cell by forming protrusion without destroying the cell (1, 62). Among the remaining phages two phage life cycle types are distinguished, dividing the tailed phages into virulent phages and temperate phages. Both types are able to perform the lytic life cycle but temperate phages are further able to integrate their genome into the host at specific attachment sites and persist in a prophage stage.

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Under particular circumstances they reenter the lytic life cycle by excision of their genome and replicate within their host. In both cases, the initial step of phage infection is adsorption to and recognition of the host. This occurs after a random collision between the host and phage which is initially non-specific (91). This collision is followed by a specific recognition and attachment of specialized adsorption structures on the phage, for example tail fibers or spikes that recognize the receptor on the host cell surface. In Gram- negative bacteria any surface protein, oligosaccharides, and lipopolysaccharides can serve as receptor, whereas the situation in Gram-positive bacteria is slightly different. The more complex peptidoglycan layer, also known as murein, of Gram- positive bacteria offers a different set of potential binding sites. It consists of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (NAM) residues, linked to each other by peptide cross-bridges between NAM residues (26, 52). Many phages require additional cofactors for adsorption, such as Ca2+, Mg2+ or other divalent cations. This attachment of the virus particle requires a phage receptor binding protein (RBP). This recognition event of the RBP is extremely specific, and high affinity binding is important for rapid and efficient virus attachment. This interaction is the underlying principle of phage typing (76). Most of the information about this interaction in double-stranded DNA phages stems from research on T-even and lambdoid phages infecting E. coli (47, 48, 135). In contrast, very little is known about the infection process for phages infecting Gram-positive bacteria. Nevertheless some phages have been investigated more intensively. In recent years, the genes encoding for RBPs of Streptococcus thermophilus phages DT1 and MD4, Bacillus subtilis phage phi29, Lactococcus lactis phages bIL67 and CHL92 of the c2 species, sk1, bIL170, and p2 of the 936 species, and TP901-1 and Tuc2009 belonging to the P335 species, have been identified (23, 32, 33, 45, 117, 120, 122, 132).

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After the irreversible attachment, the phage genome passes through the tail into the host cell. Gram-negative bacteria have cell envelopes consisting of an inner and outer membrane and periplasmic space, in which a peptidoglycan layer is located. Peptidoglycan is considered to be responsible for the mechanical integrity of the cell and limits diffusion processes of macromolecules and hence, the major barrier for phage genome passage (25). The cell envelope of Gram-positive bacteria only consists of an inner membrane and a thick multilayered cover of peptidoglycan. These crucial differences of cell wall architecture between Gram- positive and Gram-negative bacteria must result in distinct infection strategies of phages. For phages infecting Gram-negative bacteria the infection process is well understood (4, 42, 68, 71, 92). For Gram-positive bacteriophages, similar structural proteins with cell wall degrading activity have been identified (61, 125). Lactococcal phage Tuc2009 was shown to have a tail associated structural component with cell wall-degrading activity (Tal2009 = tail associated lysin of

Fig. 4. Schematic representation of the lytic life cycle of tailed bacteriophages. Infection process starts with the specific recognition and the attachment to the host cell. The phage then ejects its DNA into the host cytoplasm where replication occurs. Phage progeny are then newly assembled before they are released by lysis of the host.

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phage Tuc2009) (61). The protein was identified as gp50, which exhibits high similarity (45% identity) to the known lytic protein Zoocin A from Streptococcus equi ssp. zooepidemicus. In addition the C-terminal portion of Tal2009 is a member of M37 peptidase family that includes endopeptidases that target the interpeptide bridge of the peptidoglycan layer. It was shown that gp50 undergoes autocatalytic processing at a glycine-rich domain after translation (87). Transelectron microscopy of immuno-gold-labeled Tuc2009 phages demonstrated that the lytic structural protein is located at the tail tip of the phage. Based on these results a putative model of the tail tip was proposed (Fig. 5) (87). TP-901, a phage related to Tuc2009, also features a tail-associated lytic protein. Likewise, a virion protein of Staphylococcus aureus phage P68 was shown to exhibit muralytic activity (61, 90). Such a virion enzyme that locally degrades the cell wall from the outside is believed to be common for most dsDNA phages (68, 90). The lytic activity of this protein is responsible for the “lysis from without” phenomenon, which is the phenotypic result of adsorption of many phage particles, leading to sudden lysis of the host cell, without infection of production of viral progeny (24). This lysis differ to the “lysis from within”, taking place at the end of the infection cycle in order to release the phage progenies (125). Upon peptidoglycan degradation, the phage genome is transferred to the host cell. Once the DNA is in the host cell, strong phage promoters lead to the transcription of the immediate early genes and the transition from host to phage- directed metabolism takes place. Products of these genes may protect the phage genome against modifications or degradation and inhibit host proteins. Further, a set of middle genes, involved in DNA-replication, is transcribed followed by the expression of the late genes that are responsible for the structural phage proteins. Phage particles are then assembled and the replicated genomes are packed into preassembled icosahedral protein shells that are called procapsids. In most phages the assembly involves the interaction between specific scaffolding proteins and the major head structural proteins (Cps). The head expands during packaging and becomes more stable. At one vertex a portal complex serves as starting point for head assembly, the docking site for the DNA packaging

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enzymes, a conduit for the passage of DNA, and, for Myoviruses and Siphoviruses, a binding site for the phage tail. The tail on the other side is assembled separately. The Siphoviridae build up an initiator complex to which one or more fibers may be attached. The tail’s precisely defined length is determined

Tsh Tmp

BppU

Dit

RBP (BppL) BppA

Tal2009

Fig. 5. Detail of proposed protein architecture of bacteriophage Tuc2009 tail adsorption apparatus. Identified proteins are indicated by arrows.Tal2009: tail associated lysozyme; RBP: Receptor binding protein; Tsh: Tail sheet protein; Tmp: Tape measure protein; Dit, BppA, BppU: other baseplate proteins. (Modified from Mc Grath et al. (87)).

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by the tail tape measure protein and the completed tail is stabilized by a terminator protein, which interacts with the completed head. For Myoviridae, the many components of the contractile tail and the baseplate are also assembled in a highly ordered fashion (9, 49, 58, 59). As the final step after assembly, the host cell is lysed in order to release the phage progeny. This lysis is highly controlled and timed. Tailed phages almost always use two components: a muralytic enzyme, called lysin or endolysin, which is capable to cleave the peptidoglycan and a holin, a protein which form pores into the plasma membrane. These lesions in the membrane allow the endolysins to access their substrate in order to digest the peptidoglycan, followed by lysis of the host cell (134, 142).

1.2.3. Listeria phages and their application

Bacteriophages have been applied as therapeutic agents against bacterial infections in humans and animals. F. d`Herelle realized as first the promising potential of phage therapy in medicine (31). Phages specifically destroy the pathogenic agents in human diseases and may contribute to healing. Especially in recent times when multi-antibiotic resistance is emerging, phages may become a valuable alternative for treating infectious bacterial diseases (94). Bacteriophages infecting the genus Listeria were first reported by Schultz in 1945 (116). Until today more than 400 phages were isolated and partly characterized. These phages infect all different Listeria species except L. grayi, where currently no infecting phage could be found. Examination of more than 120 Listeria phages by transelectron microscopy demonstrated a relatively limited diversity. Most of the phages infecting Listeria were shown to belong to the Siphoviridae family morphotype B1 (isometric capsid, long non-contractile tail). The remaining phages were classified as Myoviridae of morphotype A1 (isometric capsid, long, contractile tail) (77). The genome size of Listeria phages range from 36 to more than 100 kb. The G+C-content is 34.7 – 40.8 mol%, which corresponds to the G+C-content of Listeria (63, 81, 107, 144). Further, they are well adapted to their host and complete lytic cycles at temperatures from 10 °C to 37 °C (77).

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Fig. 6. Transmission electron micrograph of bacteriophage A118 infecting a listerial host cell (This study).

All known Listeria phages are strictly genus specific. The temperate phages display a narrow host range, infecting bacteria of individual serovar groups, while the virulent ones can attack strains of all species and serovars, displaying a broad host range (5, 6, 76, 78, 102, 104). It has been demonstrated that the teichoic acid substituents N-acetylglucosamine (GlcNAc) and rhamnose are major determinants of phage adsorption in serovar (SV) 1/2 strains, while GlcNAc and galactose are important in SV 4 strains (17, 127, 139). In contrast it is assumed that the virulent phage A511, which is able to infect about 80% of all Listeria strains, recognizes the peptidoglycan itself as receptor (139). Listeria phage A118 was isolated from L. monocytogenes SV 1/2 (76). It features a non-contractile tail of approximately 300 nm in length, an isometric capsid with

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diameter of 61 nm (145), and belongs to the Siphoviridae family of dsDNA bacterial viruses, in the order Caudovirales (B1 morphotype). A118 was the first Listeria phage which was completely sequenced and analyzed in molecular detail (80); and it represents the prototype of a temperate Listeria phage. The A118 genome contains 72 ORFs, organized in three major, life-cycle specific gene clusters. Listeria phage A500 exhibits a non-overlapping and complementary host-range, infecting L. monocytogenes SV 4b (82). Although the host range of the two phages is different, they share significant sequence similarities in the predicted gene products with respect to the late gene cluster. Listeria phages and their components have found many practical applications, not only as tools in the research laboratories. With respect to foods, the biological specificity of these viruses can be used to detect and control Listeria. Virulent Listeria phages were studied for control of L. monocytogenes during food processing and storage. The use of lytic bacteriophages applied on food showed significant reduction of bacterial populations by two to five log units of viable bacteria cells (14, 44, 46, 72). Due to their specificity, Listeria phages are useful for subtyping of Listeria strains in epidemiological investigations concerning outbreaks of listeriosis. The application benefits from the different host spectra of a set of bacteriophages, resulting in distinct lysis patterns of the strains investigated (69, 76, 78, 146). Evaluation of an improved phage set for Listeria typing revealed that about 90% of all the strains tested are typable (128). This specificity also makes bacteriophages appropriate agents for the detection of viable Listeria contaminants in food. A prominent example is the construction of a genetically engineered reporter phage A511::luxAB that expresses a bacterial luciferase gene during infection and facilitates the detection of the infected bacteria via measurement of emitted bioluminescence (76, 83, 84). This reporter phage represents an appropriate agent for the detection of Listeria in foods. (83). The utilization of this reporter phage in a reliable assay was proven for large scale screening of L. monocytogenes in foods and environmental samples (84). Single phage components that are recombinantly expressed can also be used to improve control and detection of pathogenic host cells. In this context, purified

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endolysins can be used for rapid lysis of listerial cells. The C-terminal cell wall binding domains (CBDs) of these phage endolysins can be fused to green fluorescent protein (GFP) for specific detection of Listeria cells in mixed bacterial populations (81, 114). Additional application of these CBDs includes also the immobilization of host cells to solid surfaces. For example magnetic beads coated with CBDs offer the opportunity to develop useful applications, such as recovering Listeria cells from food samples (64). Phage endolysins and virulent phages against Listeria exhibit a broad field of possible applications in food science, in microbial diagnostics or for treatment of experimental infections. They may also be applied in bio-disinfection of solid surfaces and equipment in combination with common disinfectants as well as in biocontrol of pathogenic organisms (73, 75).

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1.2.4. Research objectives

The major aim of this work is to obtain information on different virion associated proteins. Specifically, the focus will be on the receptor binding proteins (RBP), which are involved in attachment and host recognition, and the lytic structural proteins (LSP) that possess cell wall hydrolytic activity.

Phage-encoded lytic proteins able to digest cell wall peptidoglycan recently received much attention because of their possible uses as antimicrobial agents against diverse pathogens. Investigations for the identification of the LSP include activity-based zymogram assays with subsequent allocation to a putative gene product.

Host recognition is a very specific event and high affinity binding is needed for efficient phage attachment. The RBP is believed to bind specifically to listerial host cells. The identification of putative RBPs will be investigated by binding assays. For this, the putative RBPs are first fused to GFP, and the fusion proteins are then analyzed for specific decoration of the host cells.

The RBP and the LSP are believed to represent parts of the baseplate. Analysis of the A118 tail tip will be carried out with help of polyclonal antibodies directed against putative baseplate proteins. Finally, transelectron microscopy will be applied to gain more information on the prototype temperate Listeria phage A118.

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2. Material and Methods

2.1. Bacterial strains, growth conditions, phage propagation, and phage purification

E. coli strain XL1-Blue MRF’ (Stratagene, La Jolla, USA) was used for molecular cloning and expression of recombinant proteins. E. coli strains were cultured in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 0.8% NaCl, pH 7.4) at 37 °C. For overexpression of recombinant proteins the strains were grown at 30 °C, while for overexpression of GFP-fusion proteins, LB medium containing 0.5% NaCl was used. Plasmid selection was accomplished by addition of ampicillin (100 μg/ml). Listeria monocytogenes strain WSLC 1001, WSLC 1042, and WSLC 3009 (SV 1/2c, SV 4b, and SV 5) were used as strains for phage propagation, substrate cells for zymograms, pull down, and binding assays. Strains 1/2a3 (SV 1/2) and HLT 2 (SV 1/2) (127) and HLT 2/2 (SV 1/2) (S. Kathariou, personal communication) were used for binding assays. Concentration of 10 µg/ml for erythromycin and 1 mg/ml for streptomycin were used for the selection of Listeria mutants. Listeria strains were grown in Tryptose Broth (TB) (Biolife) at 30 °C. Bacterial strains used are listed in Table 2.1. Phages A118 (80), A500 (27), P35 (27), P40 (28), and A006 (27) were propagated in softagar overlay plates and purified by PEG precipitation and CsCl- gradient centrifugation as described earlier (80, 145). Isolation and purification of phage structural proteins was performed as described in Loessner et al. (82). Phage A511 was propagated using liquid culture method and purified by PEG precipitation and CsCl-gradient centrifugation as described previously (85, 111). For protein analysis phages A118, A500, A006, P40, P35, and A511 were used. Zymogram analysis were carried out with phages A118, A500, A511, and PSA (144). Phages are listed in Table 2.2.

______18 GlcNAcRha 1999 al. et Tran communication personal Kathariou, S. Δ Δ deletion mutant in WSLC 1001 mutant deletion in WSLC 1001 mutant deletion this study in WSLC 1001 mutant deletion this study this study orf18 orf18 orf19 orf20 18 A118 19 A118 20 A118 Δ Δ Δ Collection, D Collection, Listeria Listeria HLT 2/2 WSLC 1001::A118 1001::A118 WSLC A118 for lysogenic WSLC 1001 1/2a3 L.monocytogenes stock laboratory WSLC WSLC 1001WSLC 10421/2a3 wildtype 1/2c, SV HLT 2 wildtype4b, SV 5764 of SLCC derivative Streptomycin-resistant 1001::A118 WSLC Tran1999 et al. 1/2a3 L.monocytogenes 1001::A118 WSLC ATCC 19112 ATCC23074 Culture Collection, Collection, D Culture XL1-Blue MRF' MRF' XL1-Blue cells Electroporation-competent USA La Jolla, Stratagene, Listeria WSLC 3009 SV 5, wildtype SLCC 4769 Listeria monocytogenes monocytogenes Listeria monocytogenes Listeria Listeria monocytogenes monocytogenes Listeria ATCC = American Type Culture Collection, USA Collection, Culture Type ATCC= American SLCC = Special WSLC = Weihenstephan Weihenstephan = WSLC Table 2.1. Bacterial strains. Bacterial 2.1. Table Strain monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria Remarks coli Escherichia or reference Source Listeria monocytogenes monocytogenes Listeria ivanovii Listeria monocytogenes Listeria Listeria monocytogenes monocytogenes Listeria

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> 80% of strains of all SV Klumpp et Klumpp al. 2008 phoviridae Myoviridae ~75 % of SV 1/2 strains Dorscht Dorscht 2007 most strains strains most of SV 4, 5, 6; ~50 % of SV 1/2 strains al., in preparation iridae Siphoviridae Si strains Dorscht 2007Dorscht et Dorscht only SV 4only SV 1/2 mainly Zimmer et Zimmer al. 2003 mainly SV mainly 4b, 6 strains Dorscht 2007 B1 B1 B1 B1 B1 B1 A1 61 nm 62 nm 61 nm 62 nm 56 nm 58 nm 80 nm 300 nm 274 nm 180 nm 280 nm 110 nm 110 nm 180 nm 40834 bp 38867 bp 37618 bp 38124 bp 35638 bp 35822 bp 134494 bp temperate temperate temperate temperate virulent virulent virulent Siphoviridae Siphoviridae Siphoviridae Siphov A118 A500 PSA A006 P40 P35 A511 AJ242593 DQ003637 AJ312240 DQ003642 EU855793 DQ003641 DQ003638 mainly SV mainly 1/2 strains Loessner et al. 2000 r r y e e h g g cle yp y Pha Famil hot numbe p Database Life c Reference accession Tail lent Tail Host range Mor sid diamete Genome size p Ca Table 2.2. Bacteriophages. Table

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2.2. Molecular cloning

2.2.1. Constructs for recombinant protein expression

The genes encoding gp16 (C-terminus), gp17 to gp21 of A118, and gp20 of A500 were amplified by PCR using purified phage DNA as template and a proofreading polymerase (ProofStart, Qiagen). Primers contained suitable restriction sites for cloning (Table 2.3). PCR products were digested with the appropriate restriction enzyme BamHI, PstI, or SacI (Fermentas) and cloned into pQE-30 (Qiagen) or its derivative pHGFP (81). After successful transformation of E. coli XL1-Blue MRF’ plasmids were reisolated (GenElute Plasmid Miniprep Kit, Sigma) and sequenced. Same procedure was done for cloning of orf97 and orf102 of A511. Primers used are listed in Table 2.3.

2.2.2. Construction of deletion mutant Listeria phages

In order to test the functionalities of gp18, gp19, and gp20 in A118, several deletion mutant phages were created. Temperature dependent integration vector pKSV7 (119) was used for the deletion of the corresponding genes within the prophage genome of A118. Flanking regions of orf18, orf19, and orf20 of A118, respectively were PCR amplified using splicing by overlap extension PCR (143). The primers contained suitable restriction sites (Table 2.3). PCR products were digested with the appropriate restriction enzyme BamHI or SacI (Fermentas) and cloned into pKSV7. After successful transformation of E. coli XL1-Blue MRF’, plasmids were reisolated (GenElute Plasmid Miniprep Kit, Sigma), sequenced, and transformed into L. monocytogenes WSLC 1001::A118 carrying the prophage A118. The plasmid was then forced to integrate into the genome by a temperature shift to 41 °C. Excision of the plasmid was obtained by a temperature downshift. The excision of the plasmid can lead to wt-situation but also to the allelic exchange. Strains were UV induced for 3-4 min (254 nm, 220 µW/cm2) and after 4 h of incubation in the dark plated in serial dilutions on Listeria WSLC 1001 for plaque formation. Phage lysates were further screened by PCR for deletion of the corresponding genes. Primers X, Y, and Z listed in Table 2.3 were used.

______21 GCT CGT TAT AAA AAA CACGCT CGT TAT AAA G ATG ACA AAT CAAAATACAATC TTT AAA TCA GCT ATT ACTATTAAC GAAAAA GTT CAT AAT GGT ATT GCC AAC TTC GTA TAA TAT CGT TGA GCC ATT ATA AAT TTT AGT TAA TGT CAC CTC TGT AC AGG TTC GAA AAA C AG AGC ATT ATC ACT GC TAC TAT AG AA TAG CGA AAA TCT TGA TAA ATG TT AC AAA TCA AAT CTT TAA ATC AGC TAT T AA CTA TAA ACA GTT TTA CGC ATA TGA T GA AAA CACCTATAA TCG TA GCC CCT TTC CGT AAA ATG CCT TTC CGT AAA TA GCC CT TAT ACA AAA AGG AGA AAT CC TT TCG CTC CTT TC CC CTC GAC TCT ATA GA A CTTAT TGC CAA CGT ATA TCG TTG ATG CCG C ACA CGA TAC TTT CC CTA AAT TAG AG ATT GTT TCT TGT TTC TG AT TAT CTAAAA GCAAT TAT TAA ATG TTA ATG TTA A AT G TT A AT G TT A AT G TT A AT G TT A AT G TT A AAT TG AAA TGA GT GGA AAT TCT TCA AT G TT A TT CGA GAG ATT AAA CAC TAA ATT AG TT G TT A GGA GAA ATT ACT GA TT TAT CAA CTG CAG TAT CAA CTG CAG AAC TGA GCT C TAT CAA GAG CTC AAC TGA GCT C C ATC AGG ATC ATC AGG ATC C ATC AGG ATC C ATC AGG ATC TAT CAA GAG CTC C ATC AGG ATC C CGA AGT ATC C ATC AGG ATC ATC AGA GCT C ATC AGA GCT C ATC AGG ATC C ATC AGG ATC R_A118_gp20 R_A118_gp20 R_A500_gp20 R_A118_gp16C-term R_A118_gp16C-term AGA GCT C ATC R_A118_gp17 R_A118_gp18 AGA GCT C ATC R_A118_gp19 AGA GCT C ATC R_A118_gp20 AGA GCT C ATC R_A118_gp21 F_A118_gp17 F_A118_gp17 F_A118_gp18 ATC C AGG ATC F_A118_gp19 ATC C AGG ATC ATC C AGG ATC F_A118_gp20 Fwd_BamHI_102 Rev_SalI_97 Rev_BamHI_102 F_A118_gp20C-term F_A500_gp20C-term A511 Fwd_BamHI_97 A511 A511 Table 2.3. Oligonucleotide Table usedprimers für amplification of the different gene products. Restrictionsites are underlined; boldare start/stop sites ORF17 A118 ORF17 A118 ORF18 A118 ORF19 A118ORF20 A118ORF21 F_A118_gp20 A500ORF20 full orf97 length A500 orf102 F_A118_gp21 F_A500_gp20 Amplification C-termORF16 A118 Template A118 Primer F_A118_gp16C-term (5`-3`) Sequence

______22 GC GAT CTA AAC AGG AC CTA AAC AGG GC GAT CT CCC GCT TGT TTC CGC GGG CT TGT CCG TTC CA CCG CGG TGG AGC GA GG GAC TTA GC CAC TCA AGG ATC C ATC TCA AGG TCA AGA GCT C C ATC TCA AGG TCA AGA GCT C C ATC TCA AGG TCA AGA GCT C Fwd X Fwd 17504-1752118461-18479 Rev Y GCA AGG TGC TGG TAC GGC Rev Z 19053-19068G GAT CCC TTT GCT TGT GCT TCC AAC GCG G CCC CAT Fwd X Fwd 16427-1644717282-17300 Rev Y CCA GTC ACA TAC ACT AGC CCG Rev Z 17973-17989C GCT ATT GCC CCG TAT CGT TTA CCT CTG CCG CG GCG Fwd X 15373-15388Fwd 16119-16136 Y Rev GCC AAG G TCG AGT GGC Rev Z 16922-16941 GC CCT GCC AAA TGA CCA AAA CCGTCT CCAAC GCT ACA Rev_BFwd_CRev_D_SacI Rev_BFwd_CTTC ACC TAC CAT GTA ACC ATT TCC GGT CAT CTA TTT CGC Rev_D_SacI ATG TAG AAA GGA GCG ACC ATG AAT GGT GAA TAC GTA GGT Rev_BFwd_C GAACTG ACC TCC TAT GGT ACC TAA CAT CTA TTT CGC T Rev_D_SacI ATT C GAT AGG TCA CGG AGG TAC AGC GAA ATA GAT GTT TGA CC ACC CAT TAG AAT ATC AAC TTC GGT GCC CTA ATT ATT AG TTG GCA AAG TGG GTA CCG TAA TTC ATA GGT CAG Fwd_A_BamHI Fwd_A_BamHI 20 19 18 Δ Δ Δ A118 Fwd_A_BamHI A118 A118 A118 A118 A118 A118 Table 2.3. (Continued) 2.3. Table Restrictionsites are underlined; boldare start/stop sites orf20 A118 Deletion mutant orf18 orf19 A118 orf19 orf20 Primers forscreeningof used deletion mutantstrains orf18A118

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2.3. Proteomics

2.3.1. Protein expression and purification

In order to produce recombinant proteins the expression was induced in E. coli at

OD600 0.5 with 0.1 mM IPTG for 4 h. Then cells were harvested by centrifugation directly after the 4 h induction or in the case of GFP-fusion-proteins after additional overnight incubation at 4 °C, resuspended in buffer A (500 mM NaCl,

50 mM NaHPO4, 5 mM Imidazole, 0.1% Tween 20, pH 8.0) and frozen at -20 °C. After thawing, cells were disrupted by a French cell press (SLC Aminco), centrifuged (30’000 x g, 1 h) and the supernatant was sterilized by filtration (0.2 µM PES membrane, Millipore). Raw extracts of gp97 and gp102 (A511) were directly assayed. Raw extracts of His-tagged proteins were loaded on a Ni-NTA column (1 ml His-Trap HP, GE Healthcare) using an ÄKTA Purifier (Amersham). GFP-fusion proteins were purified on 1 ml Ni-NTA sepharose (High Density Ni- NTA-Affarose, Interchim, France) in gravity flow columns (BioRad). Elution of proteins was carried out in buffer B (500 mM NaCl, 50 mM NaHPO4, 250 mM

Table 2.4. Proteins used in this study. Name MW [kDa] RE PP ABBA LA A118 gp16 (C-term) 52 - + + - + A118 gp17 32.4 - + + - + A118 gp18 40.8 - + + - + A118 gp19 38.7 - + + - + A118 gp20 41 - + + - + A118 gp21 14.3 - + + - + GFP-RBP A118 67.7 - + - + - GFP-RBP A500 67.5 - + - + - GFP-RBP A118 (C-term) 50 - + - + - GFP-RBP A500 (C-term) 49.6 - + - + - A511 gp97 131.1 + - - - + A511 gp102 26.4 + - - - + RE: Raw extracts were directly tested for activity PP: Purification of proteins on Ni-NTA AB: Used for immunization (Antibody-production) BA: Binding assays LA: Tested for lytic activity

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Imidazole, 0.1% Tween 20, pH 8.0). The eluted proteins were dialyzed against buffer containing 100 mM NaCl, 50 mM NaHPO4, and 0.005% Tween 20, pH 8.0 and stored at -20 °C in 50% glycerol. Proteins produced and used in this study are listed in Table 2.4.

2.3.2. Polyclonal rabbit-antibodies

Polyclonal rabbit-antibodies were generated at the Institut für Labortierkunde, University of Zurich, Switzerland. Proteins gp16 C-term, gp17, gp18, gp19, gp20, and gp21 of A118 were purified on Ni-NTA columns and dialyzed against PBST

(120 mM NaCl, 50 mM NaHPO4, 0.1% Tween 20, pH 8.0). For each antigen one rabbit was used for immunization (six rabbits in total). Aliquots of 200 µg antigen, diluted in 500 µl PBST, were used for each immunization and booster injection. Immunization followed a standard immunization protocol (Table 2.5.). Obtained sera (α-gp16 C-term; α-gp17; α-gp18; α-gp19; α-gp20; α-gp21) were analyzed by Western blot for their immune reaction against the corresponding antigens. Each tested immune antisera showed reactivity after 14 weeks. Sera were further directly used for Western blots or were ProteinA purified (ProteinA-antibody purification Kit, Sigma) for TEM.

Table 2.5. Standard immunization protocol for rabbits. Day 0 Pre-immune serum; 1st immunization with Freund's Complete Adjuvant Week 4 1st booster injection (with Freund's Incomplete Adjuvant) Week 6 Control bleed (10 ml) Week 8 2nd booster injection with Freund's Incomplete Adjuvant) Week 10 Standard bleed (50 ml) Week 12 3rd booster injection Week 14 Final bleed (antisera)

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2.3.3. Mass spectrometry

Phage structural proteins were separated by a horizontal discontinuous SDS- PAGE (ExcelGel gradient 8-18% PAA-gels, GE Healthcare, Germany). Samples were diluted in reducing sample loading buffer (4% SDS, 100 mM Tris-HCl, 0.02% Servablue G-250, 0.02% Bromophenolblue, 1% DTT stock solution 6.3 M). Gels were run with 200 V - 600 V, 25 mA, and 15 W at 12 °C and stained for 30 min in Coomassie blue R-350 (GE-Healthcare). Unstained protein marker (Fermentas) was used as a molecular weight marker. Protein bands were excised, cut in small pieces and washed twice with 100 µl of 100 mM NH4HCO3/50% acetonitrile, and with 50 µl acetonitrile. Supernatants were discarded. The individual protein species were proteolytically digested (“in gel” digestion) with 15 µl trypsin (10 ng/µl trypsin (Promega, sequencing grade modified) in 10 mM TrisCl, 2 mM

CaCl2, pH 8.2). Supernatant was removed after incubation overnight at 37 °C and gel pieces were extracted twice with 100 µl in 0.1% TFA/50% acetonitrile. Eluted supernatants were pooled and vacuum dried. Peptides were dissolved in 15 μl 0.1% TFA. 10 μl of the samples were desalted by using a ZipTip C18 and mixed 1:1 with matrix solution (5 mg/ml 4-HCCA in 0.1% TFA, 50% acetonitrile). Remaining sample after ZipTip was dried, dissolved in 0.1% formic acid and transferred to autosampler vials for LC/MS/MS. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.1.04). Scaffold (version Scaffold-01_06_17, Proteome Software Inc., Portland, USA) was used to validate MS/MS based peptide and protein identifications (60). Probabilities were assigned by the Protein Prophet algorithm (96).

2.3.4. SDS-PAGE, Western blot analysis, visualization of lytic phage proteins by zymography, and 2D-gel electrophoresis

Sodium-dodecylsulfate-polyacrylamide-gelelectrophoresis (SDS-PAGE) was performed as described previously (66, 111). 14% Tris/Tricin SDS-PAGE were performed according to Schagger and Jagow (113), run for about 4-5 h at 100 V in a Mighty Small II SE250/SE260 chamber (Hoefer), and Coomassie stained. Molecular mass of the proteins was determined using prestained molecular mass

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marker (Fermentas). Protein samples for electrophoresis were diluted in SDS- loading buffer and boiled for 10 min. For Western blot analysis proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Immobilon-P Transfer Membranes 0.45 μm, Millipore) with transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3). Membranes were blocked with 10% skimmed milk in TBST (10 mM TrisCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 1 h, and incubated with serum containing polyclonal rabbit antibodies (α-gp16 C-term; α- gp17; α-gp18; α-gp19; α-gp20; α-gp21, and α-Ply118 (39) at a dilution of 1:5000 in 3% skimmed milk) 1 h at room temperature, or alternatively overnight at 4 °C. Membranes were washed with TBST and incubated for 30 min with HRP-labelled secondary goat α-rabbit IgG antibody (Calbiochem, VWR, Switzerland) and washed with TBST. Chemiluminescent signals (Chemiluminescence Blotting Substrate, Roche) of bound antibodies were detected using a Kodak Image Station IS2000R (Carestream Health, New Haven, USA). Zymogram analysis was performed as described previously (70, 125). Briefly, protein samples were separated on a 12% SDS-PAGE containing ~1012 heat inactivated Listeria substrate cells per 5 ml resolving gel for detection of bacteriolytic activity. Substrate cells were prepared as follows: TB medium was inoculated with an overnight culture of Listeria, cells were grown until late exponential phase (OD600 0.6-0.8) and inactivated by steaming (100 °C, 10 min). The zymogram was incubated for 30 min in distilled water at room temperature, then transferred into regeneration buffer containing 25 mM Tris (pH 7.3) and 0.1% Triton X-100 and further incubated overnight at 15 °C. Peptidoglycan hydrolase activity was detected as a clear zone. Molecular weight of active proteins in zymogram was estimated using prestained protein marker (Fermentas). For 2D-gel electrophoresis 20 μl of diluted phage sample was boiled for 10 min with 0.2% SDS, treated with 1 U/µl BenzonNuclease (Merck) for 30 min at room temperature and mixed 1:10 with buffer containing 8-10 M urea, 2% CHAPS, 50 mM DTT, 0.2% ampholytes (Bio-Lyte 3-10; BioRad) and 0.001% Bromophenol. IEF-strips (pH 4-7, 7 cm) (BioRad) were passively rehydrated with the sample overnight at 20 °C and then focused using BioRad IEF system under recommended standard conditions (50 mA/Gel). Strips were stored at -70° C until

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use. After thawing for 15 min the strips were equilibrated twice for 10 min in equilibration buffer (6 M urea, 2% SDS, 2% DTT, 0.375 M TrisCl, 20% glycerol, pH 8.8) and casted into a SDS-PAGE for the second dimension. Gels were either silverstained (95) or in case of 2D-zymograms renaturated overnight in buffer containing 25 mM Tris (pH 7.3) and 0.1% Triton X-100. For identification of bands with muralytic activity, gel pieces (Tris/Tricin-gel) of phage protein profiling were excised, cut into small pieces, equilibrated with 4 μl SDS-loading buffer and applied in a slot of the stacking gel on top of a zymogram. Treatment of the zymogram after the run was carried out as described above.

2.4. Assays

2.4.1. Binding assays

The binding of GFP-RBP fusion proteins was tested similar to the procedures as described by Loessner et. al. (2002). Before incubation with Listeria cells, the purified GFP-RBP fusion proteins were centrifuged for 1 h with 30’000 x g at 4 °C. 0.5 ml of exponentially growing Listeria cells were centrifuged and resuspended in

120 µl SM buffer (50 mM TrisCl, 0.55% NaCl, 0.2% MgSO4 7H2O pH 7.4), supplemented with 1 mM CaCl2 and 20 µl GFP-RBP and incubated for 1 h at room temperature. Known SV-specific cell wall binding proteins (CBD-118 and CBD-500) served as positive and negative controls (81). Cells were washed twice with SM buffer and binding to the listerial cell wall was tested and observed by fluorescence microscopy.

2.4.2. “Pull-down” assay

100 μl of phage suspensions (107 pfu/ml) were pre-incubated with 5 μl of the different antisera (α-gp16 to α-gp21) and the corresponding pre-immune sera for 1 h at 30 °C. Samples were incubated for 10-15 min with 0.5 ml of an overnight culture Listeria WSLC 1001 (SV 1/2). As controls, samples without preliminary antisera incubation, were incubated with either Listeria WSLC 1001 (SV 1/2) or Listeria WSLC 1042 (SV 4b). Cells were then centrifuged with 12’000 x g and

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washed twice in PBST (pH 8.0). After dilution of the pellet in 1 ml SM Puffer, 10 μl of a 10-2 dilution was plated out with new host cells, incubated overnight at 30 °C and plaques were counted.

2.4.3. Transmission electron microscopy (TEM)

CsCl-purified phage particles (10 µl of 1012 pfu/ml) were mixed with 60 μl TBT

(20 mM Tris, 50 mM NaCl, 10 mM MgCl2) incubated with either 10 μl (for α-gp16, α-gp18, and α-gp20) or 20 μl (for α-gp17, α-gp19, and α-gp21) of ProteinA purified antisera and filled up with MQ-water to a total volume of 120 μl. Sample without antisera was used as control. After incubation overnight at 4 °C the samples were centrifuged for 15 min at 100’000 x g (Beckman, Airfuge Air-Driven ultracentrifuge), 100 μl of the supernatant were carefully removed and the remaining pellet was washed with 100 μl ½ TBT. These steps were repeated twice. The phages present in the pellet were then either directly prepared for TEM or further incubated with the secondary 5 nm gold conjugate goat α-rabbit IgG antibody (British Biocell, Plano). Negative stain and sample preparation were done with 2% uranyl acetate or 2% ammoniummolybdate solution and adsorption on carbon-coated G400 Hex-C3 grids (Science Services, Munich, Germany) (121). The samples were observed in a Philips CM100 at 100 kV acceleration voltage (FEI Company, Hillsboro, USA) equipped with a TVIPS Fastscan CCD camera (Tietz Systems, Gauting, Germany) or Tecnai G2 Spirit at 120 kV, equipped with an EAGLE CCD camera (FEI).

2.5. Bioinformatics

Nucleotide and amino acid sequence analysis and interpretation were performed using VectorNTI (Version 10.3, Invitrogen). Pairwise sequence alignments were done using the BLASTn and BLASTp programs available at the NCBI website (3). Multiple sequence alignments were conducted by ClustalW (http://www.ebi.ac.uk/Toolx/clustalw) (67). Sequences of A118 (AJ242593), A500 (DQ003637), PSA (AJ312240), and A511 (DQ003638) were retrieved from Genbank.

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3. Results

3.1. Proteomics of different Listeria phages.

3.1.1. Profiles of three temperate phages A118, A500, A006 and three virulent phages P40, P35, and A511

Structural proteins of the six viruses were separated by horizontal SDS-PAGE (Fig. 7). All phages exhibited relatively specific protein profiles. The individual bands were analyzed by mass spectrometry. Peptide fingerprints permitted allocation of many of the bands to predicted phage gene products. The portal protein, major capsid protein (Cps) and tail sheet protein (Tsh) were identified in all analyzed phages. Within the different profiles, the most abundant proteins were identified as Cps and Tsh. All detected gene products of analyzed members of the Siphoviridae family, namely A118, A500, A006, P40, and P35, were assigned to the late gene cluster encoding for structural proteins. The Myovirus A511 displays homologies to staphylococcal phage 812 (36, 63). We observed similar degradation products of predominant structural components. For example, Tsh and Cps were found in several bands. Furthermore, the identified gp145 of A511 is not located in the late gene cluster. Correspondingly, the homologous protein of bacteriophage 812 was detected as well. Therefore, the presence of gp145 is not unexpected in the mature virion. Although the protein profile of phage A118 has been studied before (145), additional structural proteins could be identified. Other identified gene products, such as putative head associated proteins, include gp8 (14.6 kDa), gp9 (13.8 kDa), gp11 (15.1 kDa), and the portal protein (55.3 and 56.5 kDa). The tail tape measure protein (Tmp), with a calculated molecular weight of 186 kDa, was found in a band of lower molecular weight (Fig. 7). Furthermore two proteins (gp17 and gp20), with predicted molecular mass of 30.9 kDa and 39.2 kDa, respectively, were assigned as putative tail or baseplate proteins (80). Gp18 and gp19, two additional putative baseplate proteins were not directly identified in the

______30 Tmp gp17 gp16 portal Cps Cps Tsh (Cps) Tsh A006 25 66 35 45 18.4 116 14.4 gp19 Tsh gp18/Cps-L gp19 portal Cps Tsh-L gp8/11/(9) (Cps) Tmp A500 25 45 35 18.4 14.4 66 116 gp17 Tsh, Cps Tsh, Tsh-L Cps gp20/Cps-L portal Tsh gp11 gp8, gp9 Tmp A118 25 18.4 14.4 45 35 66 116

Fig. 7. Protein profiles of different Listeria phages. SDS-PAGE analysis of different phages structural protein contents. Based on MALDI-MS peptide fingerprints, assignment of gel bands to predicted gene products is shown. Abbreviations: Tmp: Tape measure protein, Tsh/Tsh-L: Tail sheet protein/Tail sheet protein long; Cps/Cps-L: Major capsid protein/Major capsid protein long. Unstained protein marker (Fermentas) indicates the molecular weight in kDa (Continued next page).

______31 gp97, gp106 gp104 portal gp108 gp88 gp106 Tsh Cps gp105 gp145 gp94 Cps, Tsh gp106, gp103 Cps, gp106 gp102, gp145 A511 25 45 35 18.4 14.4 66 116 Tmp portal gp15 gp16 Cps/Tsh gp17 P35 25 45 35 18.4 14.4 66 116 Tmp portal gp15 gp16 Tsh Cps gp17 gp7 gp10 P40 45 66 25 35 116 18.4 14.4

Fig. 7. Protein profiles of different Listeria phages. (Continued).

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A118 protein profile by mass spectrometry, but could be identified in the A500 profile (145). In this band, one can assume the presence of A118 gp18 (39.4 kDa) and gp19 (37.2 kDa) at the same designated bands compared to A500, since A118 and A500 share high homologies within the late gene cluster and show other similarities such as their morphology and protein profile (145). Regarding the protein profiles of the recently identified phage P40 and P35, the relatedness between these two became apparent. Only the Tsh protein of P40 (MW: 34.7 kDa) migrates faster than the Cps (MW: 32.3 kDa), whereas in P35 Tsh (35 kDa) and Cps (32.9 kDa) form one single band. Comparison of their genomes revealed not only similar organization of the late gene cluster but also high identities among individual gene products, as e.g. the Cps, Tmp, and the portal protein.

3.1.2. Programmed translational frameshifting in A118 and A500

Both the major capsid protein (Cps) and the major tail protein (Tsh) are represented by two proteins of different size in phage A118 and A500. Bioinformatical analysis indicated that in both proteins a programmed translational (ribosomal) frameshift (-1 in Cps and +1 in Tsh) at the 3’ end of the analogous genes could result in the synthesis of a larger polypeptide species. Such recoding events may result in two products of different sizes, sharing the same N-termini but varying in the length of their C-termini. The calculated molecular weight of these proteins corresponds to the observed bands on the gels. To provide evidence for the actual existence of the elongated products, and to determine the location and type of frameshift involved, mass spectrometry was employed. MALDI-MS peptide fingerprints of Cps-L and Tsh-L were generated, and the determined masses of the individual tryptic polypeptide fragments were compared with the deduced masses for Cps-L and Tsh-L in both phages (Table 3.1). The analysis yielded total fragment coverage of 73% and 56.5% for Cps-L, 77.5% and 68% for Tsh-L in A118 and A500, respectively. Protein coverage is shown in Fig. 9.

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MALDI-MS enabled identification of the peptides spanning the potential frameshifting sites, and therefore also permitted determination of the location and modus of the shift. Fig. 8 shows that in both phages the frameshift in cps occurs at a location close to the 3’ end of the gene, at the mRNA sequence GCG GGA AAC (corresponding to coordinates 6071-6079 and 6048-6056 of the A118 and A500 genomes, respectively). The ribosome apparently slips from the GGA glycine codon one nucleotide into the 5’ direction (underlined) and continues from the overlapping glycine codeon GGG in the -1 frame until it reaches the stop codon at position 6248 in A118 and 6214 in A500. Thus, Cps-L contains in both phages most of the sequence of Cps (299 (A118) and 278 (A500) residues), with 53 extra amino acids (aa) from the alternate frame added onto the C-terminus. With respect to tsh mRNA, the slippery sequence AAA CCC UGA (corresponding to coordinates 8173-8181, and 8153-8161 of the A118 and A500 genomes, respectively) is also located at the end of the gene. In contrast to the frameshift in cps, the ribosome slips one nucleotide position in the 3’ end (underlined), and continues translation in the +1 frame ending at position 8440 (A118) and 8420 (A500) respectively. The shift in Tsh-L results to an addition of 87 aa in A118 and A500, whereas the Tsh consist of 144 aa (A118) and 145 aa (A500).

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A -1 frameshift

Cps 289 A F S A V Q P K A G N * 865 GCG TTC TCT GCT GTT CAA CCA AAA GCG GGA AAC TAA

298 G K L M A A R S G Cps-L 891 GGG AAA CTA ATG GCG GCG CGG TCG GGT

307 K T D S A P I K D F S V M T V A E L 918 AAA ACT GAT AGC GCG CCG ATT AAA GAC TTT TCA GTT ATG ACA GTA GCA GAA TTG

325 K E E L A N R N I E F A S N A K K A 972 AAA GAA GAG CTT GCG AAT AGA AAT ATC GAA TTT GCA AGT AAT GCG AAA AAA GCG

343 E L V A L L E G S E * 1026 GAG TTA GTT GCG CTG TTG GAA GGT AGT GAG TGA

B

+1 frameshift

136 D E T P T V T K P * Tsh 406 GAT GAA ACA CCT ACG GTT ACA AAA CCC TGA

144 P E E S P S S V E Tsh-L 431 CCT GAG GAG AGC CCG TCC AGC GTC GAA 153 V G H N T I T V K V G E T F T I N A 458 GTG GGC CAC AAT ACA ATT ACC GTT AAA GTA GGA GAA ACA TTT ACT ATT AAT GCT

171 S V L P V G A S Q E V T Y T S S N P 512 TCT GTA TTG CCA GTG GGA GCT AGT CAA GAA GTA ACT TAC ACT TCA TCT AAT CCA

189 P K A K I N S V G T G E G V A E G T 566 CCG AAG GCA AAA ATC AAT AGC GTG GGT ACA GGT GAA GGC GTA GCA GAA GGA ACA

207 A N I T V A S K E S T S I N K V V Q 620 GCA AAC ATA ACA GTT GCA TCT AAA GAA AGT ACT TCT ATC AAC AAA GTA GTA CAA

225 V T V E A A D * * 674 GTA ACA GTA GAA GCA GCA GAT TAA TAA

Fig. 8. Programmed translational frameshift near the 3’ ends of the genes results in synthesis of two different length products for Cps and Tsh major structural proteins in the two Listeria phages A118 and A500. A) The -1 frameshift in cps of A118 is shown, leading to an extended version of the Cps. B) The +1 frameshift is shown in the sequence encoding for the Tsh of phage A118 (Continued next page).

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C

-1 frameshift

Cps 272 V V P V A G N * 814 GTA GTT CCA GTT GCG GGA AAC TAA

277 G K L M A A R S V E T D S Cps-L 828 GGG AAA CTA ATG GCG GCG CGG TCG GTT GAA ACT GAT AGC

290 A P I Q D F S T M T V A E L K E E L 867 GCG CCG ATT CAA GAC TTT TCA ACT ATG ACA GTA GCA GAA TTG AAA GAA GAG CTT

308 V T R N I E F A S N A K K A E L V A 921 GTG ACT AGA AAT ATC GAA TTT GCA AGT AAT GCG AAA AAA GCG GAG TTA GTG GCG

326 L L E G S D * 975 CTG TTG GAA GGT AGT GAT TGA

D

+1 frameshift

137 D E T P K V T K P * Tsh 409 GAT GAA ACG CCT AAG GTA ACA AAA CCC TGA

145 P E E S P S S V T Tsh-L 434 CCT GAG GAG AGC CCG TCC AGC GTT ACA

154 V D H D T I T V K V G E T F T I N A 461 GTG GAC CAC GAT ACA ATT ACC GTT AAA GTA GGA GAA ACA TTT ACT ATT AAT GCT

172 S V L P A G A S Q E V T Y T S S N P 515 TCT GTA TTG CCA GCG GGA GCT AGT CAA GAA GTA ACT TAC ACT TCA TCT AAT CCA

190 P K A K I N S V G T G E G V A E G T 569 CCG AAG GCA AAA ATC AAT AGC GTG GGT ACA GGT GAA GGC GTA GCA GAA GGA ACA

208 A N I T V A S K E S P S I N K V V Q 623 GCA AAC ATA ACT GTC GCA TCT AAA GAA AGT CCT TCT ATC AAC AAA GTA GTG CAA

226 V T V E A A D * * 677 GTA ACA GTA GAA GCA GCA GAC TAA TAA

Fig. 8. (Continued). (C) and (D) Similar to A118 the Cps-L of phage A500 results through a -1 frameshift whereas the Tsh-L of phage A500 is produced through a +1 frameshift.

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10 20 30 40 50 60 MGFNPDTTTM QSAKTGSIPI NISEQIITGV KNGSAAMKLA KAVPMTKPEE EFTFMSGVGA A 70 80 90 100 110 120 FWVDEAERIQ TSKPTFTKAK MRSKKMGVII PTTKENLNYS VTNFFSLMQA EIVEAFYKKF

130 140 150 160 170 180 DQAVFTGVES PYNWNILKSA TDASNLVEET ANKYDDLNEA IGLIEAEDLE PNGIATIRKQ

190 200 210 220 230 240 RVKYRSTKDG NGMPIFNTAT SNGVDDVLGL PIAYTPKYTF GDKDISELVG DWNQAYYGIL

250 260 270 280 290 300 RGVEYEILTE ATLTTVADET GKPLNLAERD MAAIKATFEV GFMVVKDEAF SAVQPKAG KL

310 320 330 340 350 MAARSGKTDS APIKDFSVMT VAELKEELAN RNIEFASNAK KAELVALLEG SE

10 20 30 40 50 60 MRIKNAKTKY SVAEIVAGAG EPDWKRLSKW ITNVSDDGSD NTEEQGDYDG DGNEKTVVLG B 70 80 90 100 110 120 YSEAYTFEGT HDREDEAQNL IVAKRRTPEN RSIMFKIEIP DTETAIGKAT VSEIKGSAGG

130 140 150 160 170 180 GDATEFPAFG CRIAYDETPT VTKP EESPSS VEVGHNTITV KVGETFTINA SVLPVGASQE

190 200 210 220 230 VTYTSSNPPK AKINSVGTGE GVAEGTANIT VASKESTSIN KVVQVTVEAA D

Fig. 9. Complete amino acid sequences of Cps-L and Tsh-L polypeptides of phages A118 and A500. Fragments found by peptide mass fingerprinting (MALDI-MS) (Table 3.1) are indicated in bold letters. Amino acid residues resulting from the frameshift are underlined. A) Amino acid sequence of A118 Cps-L. B) Amino acid sequence of A118 Tsh-L. (Continued next page).

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10 20 30 40 50 60 C MADLTTKLAN LIDPEVMGPM ISAKLPKAIK FGKIAPIDNS LEGQPGSEIT VPKYKYIGDA

70 80 90 100 110 120 QDVAEGAAID YSALETESVK HGIKKAGKGV KLTDESVLSG YGDPVEEAQK QIRMAIASKV

130 140 150 160 170 180 DNDILEEALT TTLEVKGAIN IGLIDKIENT FTDAPDAIED ESITTTGVLF LNYKDTAKLR

190 200 210 220 230 240 EEAAGSWTKA SQLGDDLLVK GAFGELLGWE IVRTKKLADG NALAVKAGAL KTFLKRNLLA

250 260 270 280 290 300 ESGRDMDHKL TKFNADQHYA VALVDETKAV KVVPVAG KLM AARSVETDSA PIQDFSTMTV

310 320 330 AELKEELVTR NIEFASNAKK AELVALLEGS D

10 20 30 40 50 60 MARIKNAKTK YFVAEIVDGV GEPVWKRLSK WITNVSDDGS DNTEEQGDYD GDGNEKTVVL D 70 80 90 100 110 120 GYSEAYTFEG THDREDEAQN LIVAKRRTPE NRGIMFKIEI PDTETAVGKA TVSEIKGSAG

130 140 150 160 170 180 GGDATEFPAF ACRIAYDETP KVTKP EESPS SVTVDHDTIT VKVGETFTIN ASVLPAGASQ

190 200 210 220 230 EVTYTSSNPP KAKINSVGTG EGVAEGTANI TVASKESPSI NKVVQVTVEA AD

Fig. 9. (Continued). C) Amino acid sequence of A500 Cps-L. D) Amino acid sequence of A500 Tsh-L.

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Table 3.1: Peptide mass fingerprinting (MALDI-MS) of tryptic fragments Cps-L of A118 Fragment MW (exp) MW (calc) Δ MW [aa] [Da]a [Da]b [%]c Corresponding aa-sequence d 15-31 1769.91 1770.00 0.00 TGSIPINISEQIITGVK 69-78 1150.59 1150.65 0.00 IQTSKPTFTK 86-94 959.47 959.56 0.01 MGVIIPTTK 119-138 2356.08 2356.20 0.00 KFDQAVFTGVESPYNWNILK 120-138 2228.00 2228.10 0.00 FDQAVFTGVESPYNWNILK 139-153 1549.65 1549.73 0.01 SATDASNLVEETANK 154-178 2744.17 2744.36 0.01 YDDLNEAIGLIEAEDLEPNGIATIR 218-241 2823.16 2823.36 0.01 YTFGDKDISELVGDWNQAYYGILR 224-241 2111.94 2112.04 0.00 DISELVGDWNQAYYGILR 242-269 3033.30 3033.56 0.01 GVEYEILTEATLTTVADETGKPLNLAER 276-286 1227.59 1227.64 0.00 ATFEVGFMVVK 287-299 1347.56 1347.69 0.01 DEAFSAVQPKAGK 305-314 1003.48 1003.54 0.01 SGKTDSAPIK 308-325 1951.90 1952.00 0.01 TDSAPIKDFSVMTVAELK 332-340 993.46 993.50 0.00 NIEFASNAK 341-352 1258.62 1258.69 0.01 KAELVALLEGSE

Peptide mass fingerprinting (MALDI-MS) of tryptic fragments of Tsh-L of A118 Fragment MW (exp) MW (calc) Δ MW [aa] [Da]a [Da]b [%]c Corresponding aa-sequence d 10-25 1692.05 1691.83 0.01 YSVAEIVAGAGEPDWK 30-55 2860.46 2860.13 0.01 WITNVSDDGSDNTEEQGDYDGDGNEK 56-73 2045.25 2044.96 0.01 TVVLGYSEAYTFEGTHDR 97-108 1286.87 1286.68 0.01 IEIPDTETAIGK 116-132 1599.91 1599.69 0.01 GSAGGGDATEFPAFGCR 133-161 3128.91 3128.56 0.01 IAYDETPTVTKPEESPSSVEVGHNTITVK 162-190 2993.87 2993.51 0.01 VGETFTINASVLPVGASQEVTYTSSNPPK 193-214 2075.34 2075.06 0.01 INSVGTGEGVAEGTANITVASK

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Table 3.1. (Continued).

Peptide mass fingerprinting (MALDI-MS) of tryptic fragments of Csp-L of A500 Fragment MW (exp) MW (calc) Δ MW [aa] [Da]a [Da]b [%]c Corresponding aa-sequence d 8-24 1799.05 1798.94 0.01 LANLIDPEVMGPMISAK 34-53 2065.28 2065.08 0.01 IAPIDNSLEGQPGSEITVPK 56-80 2615.48 2615.24 0.01 YIGDAQDVAEGAAIDYSALETESVK 92-110 2037.13 2036.97 0.01 LTDESVLSGYGDPVEEAQK 120-136 1903.13 1902.99 0.01 VDNDILEEALTTTLEVK 147-174 3117.75 3117.52 0.01 IENTFTDAPDAIEDESITTTGVLFLNYK 179-189 1247.71 1247.64 0.01 LREEAAGSWTK 190-200 1158.70 1158.64 0.01 ASQLGDDLLVK 201-213 1446.86 1446.77 0.01 GAFGELLGWEIVR 217-226 971.61 971.55 0.01 LADGNALAVK 237-244 859.52 859.46 0.01 NLLAESGR 284-304 2269.09 2269.25 0.01 SVETDSAPIQDFSTMTVAELKEELVTR

Peptide mass fingerprinting (MALDI-MS) of tryptic fragments of Tsh-L of A500 Fragment MW (exp) MW (calc) Δ MW [aa] [Da]a [Da]b [%]c Corresponding aa-sequence d 11-26 1807.89 1807.93 0.00 YFVAEIVDGVGEPVWK 31-56 2859.99 2860.13 0.00 WITNVSDDGSDNTEEQGDYDGDGNEK 57-85 3255.33 3255.58 0.01 TVVLGYSEAYTFEGTHDREDEAQNLIVAK 98-109 1272.62 1272.67 0.00 IEIPDTETAVGK 117-133 1613.74 1613.70 0.00 GSAGGGDATEFPAFACR 134-141 936.43 936.47 0.00 IAYDETPK 142-162 2269.13 2269.16 0.00 VTKPEESPSSVTVDHDTITVK 163-191 2965.32 2965.48 0.01 VGETFTINASVLPAGASQEVTYTSSNPPK

a. Expected molecular w eight determined from the observed molecular mass of the protonated ions. b. Molecular w eight of the corresponding fragment calculated from the deduced aa sequence. c. Difference betw een the expected and the calculated molecular w eights. d. Amino acid residues resulting from the frameshift are indicated in bold letters

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3.2. Identification of the lytic structural protein (LSP)

3.2.1. LSP: a common element among Listeria phages

To assess whether different Listeria phages posses a structural component with muralytic activity, zymogram assays were performed with heat inactivated substrate cells of SV 1/2 (WSLC 1001) or SV 4b (WSLC 1042), according to the host range of the analyzed phage. For that purpose the temperate phages A118, A500, and PSA and the virulent phage A511 were propagated and purified. Phage proteins were separated under denaturing conditions. Upon renaturation a single, transparent ~30-kDa band appeared in the case of A118, A500, and PSA, whereas for A511 two bands (~24 kDa, ~36 kDa) with muralytic activity appeared (Fig. 10 B). The same phage proteins were simultaneously separated on a classic SDS-PAGE (Fig. 10 A) and compared to the corresponding zymograms. The LSP in A118, A500, and PSA is directly located below the major capsid protein (Cps). Because the Cps overlaid the LSP, a direct allocation to a designated band was not possible for these phages. 2D-gel electrophoresis with protein sample of phage A500 was performed to improve separation of the zone responsible for the muralytic activity (Fig. 11) (82). The combination of a high-resolution IEF in immobilized pH gradients with molecular sizing in either SDS gel or zymogram rendered it possible to correlate these two gels. As control served the same phage protein sample separated by molecular sizing. After renaturation of the zymogram, a muralytic band appeared only in control lane (Fig. 11-2). In further experiments we were able to show that the urea used in 2D-separation irreversibly affected the ability of the LSP to renature, excluding this alternative strategy (data not shown). Therefore, we opted for another alternative method in which the renaturation capacity is conserved (no urea) and thus activity based detection remains possible. Phage proteins were separated by SDS-PAGE on 14% Tris/Tricin gels. The protein profile of PSA showed an improved separation within the 30 kDa size region including the major capsid protein (Cps) and the frameshifted tail protein (Tsh-L) (144). This led to the

______41 e

. Tsh Cps re renaturated overnight. Zones with muralytic activity wer re renaturated overnight. Zones Tsh Cps Phage proteins in denaturating SDS buffer were subjected to SDS buffer were subjected Phage proteins in denaturating Coomassie stained corresponding protein profiles of the phages (A) the phages of profiles protein Coomassie stained corresponding A B A B A B A Tsh Cps Tsh-L Cps-L phages A118, A500, PSA, and A511. phages Listeria A B A A118 A500 PSA A511 Tsh Cps

Fig. 10. LSP of zymography on SDS-PAGE gels with embedded substrate cells (B) and we cells (B) and zymography on SDS-PAGE gels with embedded substrate visible as clear lysis zones, and could be correlated to the and could be correlated as clear lysis zones, visible

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IEF (pH 4-7) IEF (pH 4-7) + - + - - A500 A500 12% SDS-PAGE 12%

+ 2 1

Fig. 11. Improved separation of A500 phage proteins by 2D-gel electrophoresis. Two dimensional separation of phage proteins using IEF in immobilized pH gradient gels pH 4-7 in the first dimension (left to right) and molecular sizing in SDS-PAGE (1) or zymogram (2) in the second dimension (top to bottom). Phage A500 proteins served as control. detection of new protein species in this region (Fig. 12 A). The bands were excised from the gel and these gel pieces reloaded onto a zymogram gel for confirmation of lytic activity. The band responsible for lytic activity in phage PSA was then subjected to mass spectrometry. However, due to the low abundance of the protein from the gel piece and high contamination with peptides of Cps, a direct assignment of the LSP to a structural protein was not yet possible. As shown in Fig. 10, zymograms of A511 proteins showed two distinct lytically active bands. In contrast to the temperate phages A118, A500, and PSA, the corresponding proteins are not located immediately next to any major protein band, which simplified identification and separation. As performed for the LSP of PSA, the band(s) suspected to harbor the lytic activity were excised from SDS- PAGE gels and reloaded onto a zymogram. Protein samples from gels were then analyzed by MS-based peptide fingerprinting. The results indicated that the lower

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SDS-PAGE zymogram PSA SDS-PAGE 1 2 3 C

Cps 1 2 Tsh-L 3

SDS-PAGE zymogram A511 SDS-PAGE 1 2 3 4 5 C Tsh

Cps 5 4 gp106 3

2 1 gp102 gp145

Fig. 12. Zymogram based detection of LSP from phage PSA (A) and A511 (B). A) The band excised from first dimension SDS-PAGE was loaded onto a zymogram gel. Only the protein from the band loaded in lane 1 showed a zone of lytic activity, whereas neither the second identified small band between Cps and Tsh-L (lane 2), nor the band corresponding to Tsh-L (lane 3) showed muralytic activity. PSA phage proteins loaded in lane C served as positive control. B) The A511 virion contains two cell wall hydrolases. Phage proteins were first separated by 12% SDS-PAGE and Coomassie stained. Gel pieces corresponding to the indicated bands (left panel; 1-5) were excised and reloaded onto a 12% zymogram gel containing Listeria host cells as substrate (right panel; lanes 1- 5). A511 proteins served as positive control (lane C). After renaturation, the proteins from bands loaded in lanes 1 and 4 showed lytic activity, and were identified by mass spectrometry.

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band contains a mixture of gp102 and gp145, and the upper band consists of truncated form of gp106. Gp145 is unlikely to represent a putative LSP, because orf145 is not located in the late genes cluster encoding the structural proteins, but putatively encodes an “early protein” of unknown function. Gp106 was found in several bands of different mass, possibly due to proteolytic cleavage or degradation and was therefore not further analyzed. In agreement with the activity-based zymogram results, BLAST analysis suggested a possible lysozyme activity for gp102. Gp102 was recombinantly produced but did not reveal any muralytic activity (data not shown). Interestingly, a strong homology to conserved lysozyme domains was also revealed for gp97 (MW: 131 kDa), which, however could not be identified among the lytic bands from zymogram. To test whether this gp97 displays a lytic activity it was recombinantly produced and tested in lysis assays and zymogram. No activity could be detected (data not shown).

3.2.2. Identification of gp19 as the lytic structural protein (LSP) in A118

Phage A118 was analyzed for lytic structural proteins by zymogram which revealed a lytic protein of about 30 kDa in mass as shown in Fig. 10. To correlate the LSP to specific phage proteins, the zymogram was compared to Coomassie stained SDS-PAGE of A118 (Fig. 13 A / B). The putative LSP is located immediately below the band comprising the major capsid protein (Cps). It was found that the lytic principle of a single protein species was conserved. Therefore, fractions out of several 14% Tris/Tricin gels were removed, combined in a single tube, and reloaded on a second gel. Content of 8 gel pieces per slot were reloaded on a 12% SDS-PAGE and analyzed by Western blot using the antisera against the baseplate proteins (α-gp16 (C-term Tmp), α-gp17, α-gp18, α-gp19, α- gp20, and α-gp-21) and α-ply118 antiserum as negative control (39). α-gp19 revealed a weak signal in the Western blot analysis (Fig. 13 C). The same sample was analyzed by zymogram to ensure the presence of this lytic protein and we found its lytic activity to be conserved (data not shown). In addition the same equivalents were subjected to mass spectrometry where gp19 was identified in homogenized gel pieces (Fig. 13 D), indicating that gp19 is responsible for the

______45

lytic band in zymograms. However, to test whether recombinantly produced gp19 displays lytic activity against listerial host cell, the protein was tested in zymogram and lysis assays but no activity could be observed.

A BC

~30 kDa

16 17 18 19 20 21 Ply118

1 MLNLDKWGNT LFDSNKYQQF NANMEKLEKD SLAKDVDINA D 41 TNNRIDNVVL EAGGNNITEV VDARTSKNGQ VYSTLNSRLN 81 GDYSAIASDL AESNALLQTV NEENKVLKSK LDELYGNSAS 121 NIEYYVSSTN GNDVTGTGAI DAPFKTIQKA VNMVPKVKVG 161 GFIYIFCEPG QYNEDVVVQS FSGAECFYIQ PTNLATIDPT 201 TGQTGFFVKS ILFSGIMFQC VVQGLNSMST AVNNNSTVIQ 241 FARCWYGTVT KCRFDTNLKA TNITTVQYNQ SRGNCYSNYF 281 KNQNIIMSSE YMGHALFAST NTCEATSNVG LKAASGGILV 321 KSGTPVLNAT TAELKQAGGQ IF

Fig. 13. Identification of gp19 as the LSP in A118. Phage A118 proteins in denaturating SDS buffer were subjected to zymography on SDS-PAGE gels with embedded substrate cells (B). The region with muralytic activity was visible as clear lysis zone, and could be compared to the Coomassie stained protein profile of A118 (A). C) Western analysis with different antisera (indicated by numbers) of gel pieces displaying lytic activity in zymograms. Fragments found by peptide mass fingerprinting of gp19 are indicated in bold letters (D).

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3.3. Topological model of the A118 tail tip

3.3.1. Antibodies against putative tail and baseplate proteins of A118

Both the RBP and the LSP are believed to be integral parts of the baseplate, whose structural proteins are encoded by the late gene cluster. Polyclonal rabbit- antibodies were raised against six distinct gene products encoded by these late genes. Specifically the C-terminal part of the tape measure protein (Tmp; gp16), and gp17 to gp21. These six antisera were tested for specific binding to phage structural proteins. Purified phage particles were separated by SDS-PAGE and Western blotted using the different antisera. Individual protein bands were recognized and labeled by specific antibodies, which correlated well to the results obtained by peptide fingerprinting (Fig. 14). Apart from the α-gp16 (C-terminal part of Tmp) that bound to a protein with lower molecular mass than the full-length Tmp with a calculated molecular mass of about 186 kDa. The faint band generated by α-gp21 did, however, not correlate to the calculated molecular mass of gp21 (MW: 12.4 kDa).

3.3.2. Gp18, gp19, and gp20 of A118 play an important role in the early steps of infection

Besides the binding to the protein profile the antisera generated were tested for binding to specific structural proteins that are important in phage attachment and/or infection. The infectivity of phage particles after pre-incubation with serum was tested using a pull down assay. Phages pre-incubated with antiserum were mixed with Listeria host cells SV 1/2 (WSLC 1001). Adsorbed phages in the pellet were plated on suitable host cells. Phages incubated with the antisera were compared to controls challenged with the corresponding pre-immune sera. All counts were normalized to 100%. As controls, pull down of untreated phages on either SV 1/2 (WSLC 1001) or SV 4b (WSLC 1042), to which A118 cannot adsorb, were performed (Fig. 15). Antibodies α-gp16, α-gp17, and α-gp21 had no effect

______47 gp18 (A500) portal Cps Tsh-L gp17 Tsh, Cps Tsh gp8, gp11 gp9 gp20/Cps-L gp19 (A500) 25 45 35 18.4 14.4 66 20 21 19 MW (calc) [kDa](calc) MW gene of products 30.9 39.4 37.2 39.2 12.4 Western blot analysis 186 16 17 18 AB

Fig. 14. Western blot analysis of the A118 protein profile using antibodies generated against several baseplate proteins. Numbers indicate the antisera used for immunoprobing (A). Position of Cps is marked by blue lines. Calculated molecular weight of the different gene products are indicated in table below the lanes. Protein profile of A118 is shown on in panel B.

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200

150 t

100 % pfu in pelle

50

0 anti-Tmp anti-gp17 anti-gp18 anti-gp19 anti-gp20 anti-gp21 w/o Ab w/o Ab (C-term)

SV 1/2 SV 1/2 SV 1/2 SV 1/2 SV 1/2 SV 1/2 SV 1/2 SV 4b

Fig. 15. Gp18, gp19, and gp20 of A118 play an important role in the early steps of infection. A118 phage particles, pre-incubated with either antisera against gp16 to gp21 and corresponding pre-immune sera were tested for their ability to attach and infect Listeria SV 1/2 host cells in a pull down assay. Plaque forming units (pfu) of adsorbed phages were determined in%. Pre-immune sera were normalized to 100%. Adsorbed phages were counted as plaques. Pull down of untreated A118 with SV 1/2 and SV 4b cells served as negative control. on phage infectivity. In contrast, α-gp18, α-gp19, and α-gp20 completely inhibited infectivity, indicating that their binding partners gp18, gp19, and gp20 play vital roles in the early recognition and infection process. To support this finding, different A118 deletion mutants were constructed. The genes encoding for gp18, gp19, and gp20 were deleted in the WSLC 1001::A118 strain. Each of the prophage was then induced by U.V. light and corresponding lysates were tested for presence of infective phage particles. Compared to

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induced wildtyp WSLC 1001::A118, none of the three tested induced lysates of 1001::A118Δ18, 1001::A118Δ19, or 1001::A118Δ20 showed infective phage particles. Detection of the mature phage virions in the lysates was only possible by PCR and not by Western blot analysis or by transelectron microscopy.

3.3.3. Transmission electron microscopy (TEM) analysis of Listeria phage A118

To determine whether the antibodies generated against the putative baseplate proteins bind to the phage particles, transmission electron micrography (TEM) was performed (Fig. 16). For all tested primary antibodies, the gold conjugated secondary antibodies located to the baseplate (Fig. 16 A). Since antibodies have two antigen-binding sites, these antibodies were able to crosslink phages with each other. This property could be used to better locate the antibody binding site (Fig. 16 B / C). Antibody α-gp16 (C-terminus Tmp) bound in the interconnection of tail tube and tail tip. Binding of α-gp19 (LSP) was restricted to the lower baseplate ring. Antibody α-gp20 crosslinked phages at the upper baseplate ring. It appeared that α-gp17 linked phages at two different positions within the phage baseplate: one directly located below gp16 at the interconnection of tail to tail tip and the other between the upper and lower baseplate. This suggested that gp17 may form an inner core of the tail tip and is accessible from different sites. α-gp18 was able to bind at the center of the lower baseplate ring. Crosslinkage by α-gp21 demonstrated that the connection of upper and lower baseplate ring is at least partly made up of gp21. Since each of the different antibodies bound in a characteristic pattern, it was possible to allocate all of the tested putative baseplate proteins. Based on the data obtained by TEM, a model of the A118 tail tip could be generated. The proposed model is presented in Fig. 17 and is compared to detail TEM micrographs of the tail tip in side and bottom view.

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Fig. 16. Transmission electron micrographs of A118. A) TEM of immunogold-labeled A118 baseplate proteins. (Continued next page).

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B 50 nm

α-gp19 (LSP)

α-gp16 (C-term) α-gp20 (RBP)

50 nm 50 nm

α-gp17 α-gp18 α-gp21

50 nm 50 nm 50 nm

α-gp16 α-gp17 α-gp18 α-gp19 α-gp20 α-gp21 C (C-term) (LSP) (RBP)

Fig. 16. Transmission electron micrographs of A118. (Continued). B) TEM of A118 particles following incubation with α-gp16 to α-gp21 antibodies. C) Proposed crosslink is indicated with an arrow. Scale bars correspond to 50 nm.

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11 nm

A C

Tsh / Tsh-L

gp16 (C-term; Tmp) gp17 300 nm (tail300 nm tohead junction) gp20 (RBP) gp21

gp19 (LSP) 15 nm 15

gp18

gp20 (RBP) gp19 (LSP) B

20 nm

Fig. 17. TEM analysis and proposed protein architecture of the A118 tail tip. Results of the antibody-bindings were summarized by a schematic model of the phage tail tip showing anatomical features and dimensions (C). Proposed model of the tail tip apparatus is compared to TEM pictures in side view (A) and bottom view (B).

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3.4. Identification of the receptor binding protein (RBP)

3.4.1. Gp20 of A118 and A500 binds to Listeria cell walls

Due to the non-overlapping and complementary host range of Listeria phages A118 and A500, putative RBP genes were assumed to show no or only partial sequence homology with each other. Furthermore, based on the genomic location and on comparison to other known RBPs in Gram-positive bacteria, the putative RBP in phage A118 and A500 is located in the late gene cluster (33, 132). Therefore, the putative baseplate and tail fiber proteins of both A118 and A500 (gp17 to gp22 in both phages) were compared to each other and analyzed. The gp17 (50% identity 138/271; e-value = 2e-68), gp18 (60% identity; e-value = 8e- 118), and gp19 (34% identity 117/344; e-value = 6e-41) proteins revealed high sequence similarities and were therefore unlikely to represent candidates for the putative RBP. However, bioinformatic analyses suggested that gp20, gp21, or gp22 represent the RBP. While there was no significant similarity found for gp21 and gp22, the similarity between gp20 of A118 and A500 was restricted to the N- terminal part. The amino acid sequences of gp20 were also compared to gene products of PSA, another phage displaying the same host range as A500. The various homologies of the putative RBP proteins from A118, A500, and PSA were indicated in Fig. 18. The alignments revealed that gp15 of PSA is 96% identical in the C-terminal part over 121 amino acids (e-value 4e-42) to gp20 of A500. All three proteins showed an identity of 50-62% over 56-59 amino acids (e-values: 9e-11 and 2e-11) in the core part of the proteins. To determine the specificity of the putative receptor binding proteins of A118 and A500, labeling and localization studies were performed. For this, purified GFP-tagged proteins were mixed with exponentially growing Listeria cells of SV 1/2 (WSLC 1001) and SV 4b (WSLC 1042) and after incubation, specific binding was analyzed under the fluorescence microscope (Fig. 19 A / B). Known SV-specific cell wall binding proteins (CBD-118 and CBD-500) served as positive and negative controls. GFP-RBP A118 (gp20 of A118) bound to SV 1/2 but not to SV 4b cells, whereas GFP-RBP A500 (gp20 of A500) bound to cells of SV 4b but not to SV 1/2 (Fig. 19 B1 / B3). Interestingly, the two truncated versions of the putative RBP of phages A118 and A500

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displayed the same SV restricted binding pattern as observed for the full length proteins (Fig. 19 B2 / B4). Nevertheless the fluorescence microscope images showed that compared to full-length versions, the labeling of the truncated proteins fused to GFP occurred at discrete spots only, and the overall intensity of the decoration by the truncated proteins was lower.

Φ infects SV proposed RBP

4b PSA gp15 N C

4b A500 gp20 N C

1/2 A118 gp20 N C

0 160 220 357 aa

Fig. 18. Alignments of 3 putative RBPs of Listeria phages A118, A500, and PSA. Color bars and vertical lines indicate homologies. Red: identity of 54% (86/157; e-value = 7e-40)/ orange: A118 to A500: identity of 50% (28/56; e-value = 9e-11), PSA to A500: 62% (37/59, e-value = 2e-11)/ yellow: identity of 96% (117/121, e-value = 4e-42)/ white: no similarity.

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) FP G /o w /2 b a, 1 4 (a V V h S S gt to to n g g le in in P d d B in in A Protein R B B 0 156 220 357 aa

GFP C GFP-RBP A118 357 ++ (1) -

GFP C GFP-RBP A118 (C-term) 201 + (2) -

0 157 220 355 aa (3) GFP C GFP-RBP A500 355 - ++ (4) GFP C GFP-RBP A500 (C-term) 198 - +

Φ A118 ++ - Φ A500 - ++

B SV 1/2 SV 4b

1 3

2 4

Fig. 19. Identification of gp20 as the receptor binding protein (RBP) in A118 and A500. A) Putative RBPs of A118 and A500 (Full length and N-terminally truncated version) were fused to GFP and tested for binding to Listeria cells of SV 1/2 and SV 4b. Lengths of the different RBPs (w/o Gfp) are indicated in aa (Gfp not in scale). B) SV specific binding by the putative RBP of A118 and A500. Fluorescence microscopy images of SV 1/2 or 4b cells labeled with full-length GFP-RBP A118 (1) or GFP-RBP A500 (3). Binding of the N-terminally truncated versions GFP-RBP A118 (C-term) (2) and GFP-RBP A500 (4) to either SV 1/2 or 4b cells.

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3.4.2. The A118 RBP requires N-acetylglucosamine and rhamnose for binding

Listeria strains resistant to A118 plaque formation were used to study the capacity of binding of the putative RBP protein in the absence of bacteriophages. In a previous study, the receptor molecules for A118, N-acetylglucosamine (GlcNAc) and rhamnose (Rha), were identified (139). Further it was shown that A118 was unable to attach and to infect SV 1/2 ΔGlcNAc (HLT 2/2, (127)) or SV 1/2 ΔRha (HLT 2, S. Kathariou, personal communication) compared to SV 1/2 parental strain (1/2a3 (57)) (127). In order to confirm that phage A118 and its putative RBP use the same cell wall ligand, the binding of GFP-RBP was studied. The ability of GFP-RBP A118 to bind to SV 1/2 ΔGlcNAc and SV 1/2 ΔRha Listeria strains was analyzed (Fig. 20). The two mutant strains, ΔGlcNAc and ΔRha, were not labeled by GFP-RBP A118 (Fig. 20 B / C). GFP-RBP A118 displays the same binding pattern as A118 to the tested strains, confirming the role of gp20 as the RBP in Listeria phage A118.

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/2 1 V S n ai tr s c al A nt N e lc ha ar G R Δ Δ ) P ) ) Φ A118 (A (B (C

Attachement + - - Infection + - -

GFP-RBP A118 + - -

A

B C

Fig. 20. Binding of A118 RBP to phage resistant strains of SV 1/2. GFP-RBP A118 was incubated with SV 1/2 parental strain (A), SV 1/2 ΔGlcNAc (B), and SV 1/2 ΔRha (C).

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4. Discussion

In this study, the lytic structural protein (LSP) and the receptor binding protein (RBP) of Listeria phage A118 have been identified and localized. The data allowed the proposal of a topological model of the phage A118 tail tip. Gp19 most likely represents the LSP in phage A118, as shown by zymogram data. The protein profile of A118 showed no signal when immunoprobed with α- gp19, compared to the corresponding lytic band on the zymogram (about 30 kDa). However, a band was observed at the predicted full-length gp19. Only when the protein concentration was increased eightfold, gp19 could be identified in the region responsible for the lytic zone in zymograms by mass spectrometry and Western blot analyses. This finding suggests that mainly the full-length gp19 is incorporated in the mature phage particle, and that gp19 is rare in assembled virion particles. The LSP might be present only in low copy numbers within the phage tail or baseplate, or may be activated only upon infection. The latter is supported by the finding that recombinantly expressed full length LSP (gp19) showed no lytic activity against Listeria host cells (data not shown). However, judging from the size of the zymogram lysis zone, the active form of gp19 possesses a strong lytic activity. Processing of phage structural proteins is a common phenomenon (50). It was demonstrated that Tal2009 of L. lactis phage Tuc2009 can undergo auto-proteolytic cleavage at a glycine-rich region, which detaches this lytic activity from the rest of the protein. Both processed and unprocessed forms of Tal2009 are present in the mature phage particle and were shown to be located at the tip of the tail (87, 131). Incorporation of an inactive precursor protein or only few copies of the active protein might be essential as a self-protection strategy of the phage. Indeed, carrying highly muralytic proteins may have negative effects on the interaction of phage with host cells. Consequently, the initial steps of the phage infection cycle would be impaired by formation of uncoordinated lesions into host cell wall to which the phage is adsorbed. The data obtained by mass spectrometry, however, exclude a C- terminal truncation of gp19. Thus, cleavage of the N-terminal part might be the activation mechanism for the LSP in A118. The exact cleavage site could not be

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determined by N-terminal sequencing, due to the low abundance of the active protein. The copy number and location of the LSP within the tail tip in A118 differs compared to the lactococcal phage Tuc2009 (61, 87). Whereas in the case of Tuc2009 the LSP (Tal2009) form a fiber structure at the tip of the phage tail (87), gp19 of A118 was shown to form the entire lower baseplate ring (Fig. 17). However, the activation of gp19 (A118) remains to be elucidated. It could be shown that the presence of a LSP is a common feature among the Listeria phages tested. It was previously described that all Siphoviridae infecting Gram-positive hosts tested, contain murein hydrolases in their virions (86, 90, 129). This was observed not only for the described Listeria phages, but also for other phages investigated during this study, such as P35, B054, B025, A006 (data not shown). Nevertheless, zymograms of these phages displayed only weak bands. Interestingly, in SDS-PAGE the LSP was often located closely to the dominant band, corresponding to the major capsid protein (Cps). Why the molecular mass of Cps and LSP are so similar is still unknown, but this seems to be common in many phages (I. Molineux, personal communication). However, this finding might be coincidental. In case of A511, two lytically active proteins of different sizes (~26 kDa and ~36 kDa) were identified by zymogram analysis. The size of the upper band is in perfect agreement to the molecular weight of the native endolysin Ply511 (MW: 36.5 kDa). It was previously shown that lysozyme e of phage T4, the endolysin responsible for “lysis from within”, can also be found associated to the phage particle (average 0.5 molecule/phage particle), but actually plays no role in the infection process (35). Another example where the association of the endolysin to the mature phage particle was also demonstrated is Pseudomonas phage ФKZ (89). PRD1 is a lipid-containing virus and its morphology differs from the other described phages, but it also carries the protein responsible for host cell lysis and liberation of progeny phages (110). T4, ФKZ, and PRD1 are infecting Gram- negative bacteria. However, Western blot analysis of the A511 proteins, using a Ply511-crossreacting antibody (39), indicated that Ply511 is probably not associated to the phage (data not shown). Although it was possible to identify

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proteins within the bands allocated to the size of the lytic bands, it is believed that another protein might be responsible for this effect. Gp97 displays strong homology to conserved lysozyme domains, and is therefore likely the protein responsible for the activity observed within zymograms. In the protein profile of A511, gp97 (131 kDa) was found to be allocated to a protein band, together with gp106 (128 kDa), with a molecular weight of around 100 kDa. Zymogram analyses revealed no muralytic band at this position. This suggests that a full length and inactive gp97 is incorporated in the mature virion. In both A118 and A511, the band comprising the full length gp19 (A118) or gp97 (A511) did not correlate to the smaller lytic band found in zymograms. Processing to a smaller and active form, as it is likely for gp19 of A118, might also be possible for gp97 of A511. Unfortunately, the identification of gp97 was not possible on the zymograms. This might be again due to the low concentration of the LSP, as suspected for gp19 in A118. Further, the in vitro expression of full length A511 gp97 in E. coli failed, suggesting that gp97 might have detrimental effects on the bacterial cell. However, the actual role of gp97 in A511 remains to be elucidated. Throughout the process of identifying gp19 as the LSP in A118, it was noticed that urea, used in the first dimension isoelectric focusing of 2D-gel electrophoresis, irreversibly affected the ability of the lytic active protein to refold. The actual mechanism how the activity is inhibited remains unclear. However, it was shown to exhibit residual activity when the zone with the assumed LSP was cut out of a gel and subsequently reloaded onto a zymogram. SDS-treatments remained reversible even after several sequential denaturation and staining steps. This characteristic was essential for the isolation and identification of gp19 as the LSP of A118.

In this study, gp20 in A118 was identified as the RBP. Moreover, comparison to A118 related phages, such as A500 and PSA, enabled the identification of a RBP in these phages. The GFP-RBP-fusion proteins of A118 and A500 were able to bind SV-specific Listeria cells and moreover display the same binding pattern, as the phage host range (Fig. 19). The genes encoding the putative RBPs are

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modularly organized and the binding specificity resides in the C-terminal domain. This finding is supported by the fact that N-terminally truncated GFP-fusion proteins bound to host cells as well, although binding was less pronounced and occurred in a spot-like, localized fashion, compared to the full-length proteins. However, lack of the N-terminus may cause improper folding of the truncated protein. Further, the N-terminal domain of the RBPs of A118 and A500 could be responsible for proper phage assembling, stabilization of the baseplate, and may be involved in a strong protein-protein interaction with other phage tail proteins as shown for the T-even phages (47). Such a modular organization in genes encoding RBPs was previously described for phage DT1 and MD4 (32). The A118 phage receptors are sugar residues in the teichoic acid of the cell wall, namely N-acetylglucosamine (GlcNAc) and rhamnose (Rha) (139). The data from this study confirms that GFP-RBP A118 (gp20) binds to the same substituents. The binding was restricted to the parental strain of SV 1/2 but not to ΔGlcNAc or ΔRha mutant strains. Resistance to A118 was previously shown by lack of either of the two sugar components within the listerial cell wall teichoic acids (127). As a result, it can be concluded that Rha and GlcNAc are necessary for phage attachment. Taking into consideration the binding specificity of gp20 of both, A118 and A500, and the binding to the phage receptor substituents of gp20 of A118, these proteins are believed to be responsible for attachment and host range determination in these Listeria phages.

The LSP and the RBP are believed to represent structural components of the phage baseplate in mature viruses. The baseplate proteins are encoded in a region located between ORF16 (Tmp) and ORF 23/24 (lysis cassette Ply118/Hol118) in the late gene cluster of the A118 genome. The use of polyclonal antibodies against these gene products and subsequent TEM analysis allowed us to propose a model of the A118 tail tip. Gp16 to gp21 were allocated to a designated location within the tail tip. Gp16 is believed to be the Tail tape measure protein (Tmp) (80), which determines the tail length and is located in the

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tail tube (49, 58). It is localized by α-gp16, (directed against the C-terminal Tmp part). The fact that the C-terminal domain is so easily accessible for the antibodies suggests that it might also be involved in tail sheet to baseplate connection. Labeling by Tmp-antibodies was also found in a mutant lactococcal phage, lacking the double baseplate structure and therefore allowing access of the antibodies to the Tmp (131). The precise role of the Tmp at this interconnection between tail and baseplate remains to be elucidated. No evidence was found for Tmp processing, as shown to be the case for other phages (49, 97, 144).

As demonstrated by phage pull down using antisera, not only α-gp19 (LSP) and α-gp20 (RBP) were able to neutralize A118 phages, but also α-gp18. Since attachment and receptor recognition are often described as a two-step process (91), it cannot be excluded that gp18 is also involved in receptor recognition. Furthermore, the ability of α-gp18 to neutralize phage adsorption may be due to a sterical obstruction, disabling recognition and binding to the major cell wall receptor by the RBP (gp20). No putative function could be assigned to gp18 based on BLAST analyses. The importance of the three proteins gp18, gp19, and gp20 in the mature virion has also been suggested by introducing deletions in any of the three protein genes. Lysates of the mutant prophages harbored no infective phage particles. Unfortunately, it was not possible to obtain evidence for the actual presence of mutant phages in the lysates (via transmission electron microscopy) because of concentration effects. Interestingly, induced numbers of wildtype A118 prophage were also too low to be detected. The inability of the mutant phages to infect did not allow propagation to higher titers. Therefore, it remains unclear whether the inability to infect is due to a phage assembly defect, or due to the lack of a crucial element that is important in host recognition. Further investigations are needed to assign functionalities to all of the baseplate proteins.

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Finally, the protein profiles of six Listeria phages were compared and analyzed by mass spectrometry in order to allocate additional protein bands consisting of minor proteins to predicted gene products (27, 79). The major proteins of five of the analyzed phages (A511, P35, A500, A118, and A006) have already been characterized (27, 63, 145). The protein profile of phage P40 was newly characterized. Related phages display a similar protein profile, therefore A118 and A500, or P35 and P40 were observed as being similar. Interestingly, programmed translational frameshifts were identified in A118 and A500. Both utilize +1 as well as -1 programmed translational frameshifting for generating Cps and Tsh proteins with different length C-termini. The obtained data showed that the mode of the translational frameshift in both phages is identical (28). Considering the icosahedral symmetry of a phage capsid, the possible role of the C-terminally modified Cps was explained for PSA (144). PSA, similar to the newly tested phages A118 and A500, features a capsid structure with a triangulation number T = 7 (16). These capsids consist of a total of 420 protein subunits, which are organized in 12 pentameric and 60 hexameric ring structures, the capsomeres (140). The ratio of Cps and Cps-L in PSA is explained by the different ratio of these subunits and is in perfect agreement with the experimentally determined ratio. Ribosomal frameshift within Tsh might also play a role in correct assembly of the tail. For B. subtilis phage SPP1, it was shown that the tail morphology was altered, when only one of the two tail proteins was expressed (7). In bacteriophage λ, a frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly (74). The correct relative amounts of proteins for virion assembly seems to be crucial; the head and tail proteins of phage λ are produced in very different amounts as a result of different translation efficiencies (112). It has been shown for phage T4 that a correct ratio of different structural proteins is crucial to achieve efficient phage production (34). Consequently, programmed translational frameshifts seem to be important for the biological role of the products, since they are widespread among phages and strongly conserved (8, 141). The use of such ribosomal frameshifts was described as a recoding event specified in the sequences of phages and insertion sequence (IS) elements (8) and might refer to

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a common ancestor or lateral transfer of genes (8, 15). Generation of N-terminal identical proteins could also ensure best fit for developed phage assembly strategies. Nevertheless, these translational frameshifts in the structural proteins were identified in representative members of several hundreds known Listeria phages, suggesting a universal mechanism rather than an unusual finding.

Control of L. monocytogenes has become an important issue in the food industry in recent years. The use of bacteriophages for specific recognition and elimination of this human pathogen offers powerful tools and alternative approaches. So far, research mainly focused on the application of whole phages or phage endolysins. These methods have been shown to be very effective in reduction or elimination of Listeria in food (44, 46, 64, 81). The results presented here describe virion associated components, namely the LSP and RBP in Listeria phage A118. Both RBP and LSP appear to harbor significant potential for a number of applications. For example, it is believed that the RBP, similar to the CBDs of endolysins (64), can be elegantly used for detection and immobilization of Listeria. The binding of RBPs is strictly restricted to the corresponding phage sensitive strains, therefore displaying a higher specificity as CBDs do. This correlates well with a finding that the CBD-ligands differ to the phage receptors on the surface of the listerial cell (81). These are the first identifications of baseplate components with designated functions in Listeria phages. The morphology and distinct functional assignments identified in representing members of Listeria phages suggests universal horizontal exchange of such genetic elements, as they are universally located at similar positions. Therefore, the results of this report may be extrapolated to other phages infecting Firmicutes. Evaluation on the application possibilities and characterization of these functional proteins will provide for interesting future studies.

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Publications

Bielmann R., R. Lurz, R. Calendar, M.J. Loessner. 2009. Identification and Localization of the Lytic Structural Protein (LSP) Receptor Binding Protein (RBP) in Listeria monocytogenes Bacteriophage A118. (In preparation).

Dorscht, J., R. Bielmann, M. Schmelcher, Y. Born, M. Zimmer, R. Calendar, J. Klumpp, and M. J. Loessner. 2009. Comparative genomics and proteomics of Listeria bacteriophages reveals an extensive mosaicism and programmed translational frameshifting as common elements. (In preparation).

Klumpp, J., J. Dorscht, R. Lurz, R. Bielmann, M. Wieland, M. Zimmer, R. Calendar, and M. J. Loessner. 2008. The terminally redundant, nonpermuted genome of Listeria bacteriophage A511: a model for the SPO1-like myoviruses of gram-positive bacteria. J Bacteriol 190:5753-65.

Szathmary, R., R. Bielmann, M. Nita-Lazar, P. Burda, and C. A. Jakob. 2005. Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol Cell 19:765-75.

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Danksagung

Ein herzlicher Dank geht als erstes an Prof. Martin Loessner, der mir die Möglichkeit gegeben hat, meine Forschungsarbeit in seiner Gruppe zu absolvieren. Sein stets offenes Ohr, die zahlreichen Besprechungen und Anregungen und sein Vertrauen in mich und diese Arbeit haben massgeblich zum Gelingen dieser Dissertation beigetragen.

Bedanken möchte ich mich auch bei Prof. Herbert Schmidt für das Übernehmen des Korreferats.

Vielen Dank an Rudi Lurz für die phänomenalen Bilder am Elektronenmikroskop und die interessante und sehr erfolgreiche Zeit, die ich am Max-Planck Institut in Berlin verbringen durfte. Richard Calendar gebührt ein Dank für das perfekte Aufreinigen der verschiedenen Phagen, die für die Protein-analysen eingesetzt wurden.

Ein spezieller Dank geht an meine Kollegen Yannick Born, Yves Briers, Simone Dell`Era, Jeannette de Vries, Dominik Doyscher, Fritz Eichenseher, Marcel Eugster, Lars Fieseler, Susanne Günther, Steven Hagens, Monique Herensperger, Thomas Huber, Jochen Klumpp, Kwang-Pyo Kim, Rainer Lehmann, Miluse Mares, Patricia Romero, Barbara Schnell, Uschi Schuler- Schmid, Markus Schuppler, Timo Takala und Markus Zimmer. Die stete Unterstützung bei „Pipettier-Problemen“ und das gute Arbeitsklima waren von unschätzbarem Wert. Auch die unzähligen Znüni-Kuchen, die den Laboralltag massgeblich versüsst haben, und die besondere Atmosphäre bei Laborausflügen und -events, BQM/DVD/Grill-Feierabenden sind positiv zu erwähnen. Vielen Dank!

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Weiter geht ein grosser Dank an meine Studenten Thomas Luchsinger und Manuel Kradolfer und meine Studentin Anna-Maria Gabryjonczyk für ihre motivierte und tolle Arbeit während den absolvierten Forschungsprojekten.

Danke auch allen anderen Mitarbeitern, Freunden und Kollegen am Institut für Lebensmittelwissenschaften für ihre Hilfsbereitschaft.

Erwähnen möchte ich ausserdem die Personen, die ausserhalb der direkten Forschungstätigkeit für mich wichtig waren und mich massgeblich beeinflusst haben. Der wohl grösste Dank geht an meine Freunde und Freundinnen, die mich während der ganzen Zeit begleitet, gestützt und gestärkt haben. Neben den Aufmunterungen und wichtigen Entscheidungshilfen in allen Lebenslagen, auch danke für sportliche Badmintonabende, abkühlende Tauchevents, plaudernde Joggingrunden, gemütliches Beisammensein und eine spannende und unvergessliche Studienzeit. An dieser Stelle möchte ich mich auch bei Patrice Tscherrig bedanken. Seine grosse Unterstützung, nicht nur im Hinblick auf diese Arbeit, war und ist für mich von grösster Bedeutung.

Mein herzlichster Dank gebührt meinen Eltern Lilly und Paul Bielmann. Nicht nur, dass ich ohne sie nicht da wäre wo ich jetzt bin, sie haben mich stets unterstützt und es mir immer ermöglicht mein Leben nach meinen Vorstellungen zu gestalten und zu leben.

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Curriculum Vitae

Regula Bielmann

Born, 29th of September, 1978 in Freiburg, Switzerland Citizen of Rechthalten (FR), Switzerland

2004 – 2009 Ph.D. student, Food Microbiology Laboratory Institute of Food Science and Nutrition, Swiss Federal Institute of Technology (ETH), Zürich

2004 Internship in the Laboratory of Dr. E. Chevet, Department of Surgery, McGill University, Montreal, Canada

2001 – 2004 Studies in Biology at ETH Zürich Diploma thesis in the Institute of Microbiology ETH Zürich

1999 – 2001 Basic studies in Biology at the University of Freiburg (CH)

1994 – 1999 Degree in Elementary Education, Primarlehrerseminar Freiburg (CH)

1991 – 1994 Secondary school, Plaffeien (FR)

1985 – 1991 Elementary school, St. Silvester (FR)