Chapter 18 : Listeria Phages: Basics and Applications

Relationships of different Listeria phage endolysins. The functional domains are shown as three-dimensional bars; amino acid sequence homologies (indicated by vertical lines between bars) identify specific relationships of individual domains. The similarities correspond to either the type of hydrolytic activity or the host cell serovar group infected by the phage specifying the endolysin. The exception is Ply511, encoded by the polyvalent phage A511, which can infect both serovar groups: its amidase domain is only weakly related to the N terminus of PlyPSA.

Listeria cells were immobilized on the even surface of a surface plasmon resonance biochip (BIAcore) and exposed to HGFP-CBD500.This hybrid protein consists of the CBD of the Ply500 murein hydrolase fused to GFP and targets a serovar-correlated carbohydrate ligand which is evenly distributed on the cell wall of Listeria. Cells were imaged by fluorescence microscopy; the decorated cell wall structure of the rodshaped bacteria is clearly visible by GFP-mediated fluorescence.

(A) Electron micrograph of negatively stained phage A118 particles. This temperate bacteriophage belongs to the Siphoviridae family and infects members of the serovar 1/2 subgroup of L. monocytogenes. The virion has a capsid diameter of approximately 60 nm and a long, rather flexible tail of roughly 300 nm. The baseplate at the distal end of the tail has a set of short tail fibers attached, which appear in a crown-like arrangement. These tail fibers mediate the specific recognition of, and binding to, teichoic acid sugar components present on the Listeria cell surface. (B) Schematic map of the L. monocytogenes temperate bacteriophage A118 genome. The virus features a terminally redundant, circularly permuted genome with a 40,834-bp unit length which displays a clearly defined life cycle-specific organization. The 72 ORFs are numbered consecutively and are indicated by arrows pointing in the direction of transcription. Black arrows indicate rightward transcription, and gray shaded ORFs point leftward. The attP site next to the integrase gene int is indicated by a thin black arrow. Upon infection and formation of lysogens, the viral DNA integrates into a comK homologue and disrupts its reading frame. The A118 DNA represents a terminally redundant and circularly permuted collection of molecules without cohesive ends. Therefore, both arms must recombine by homologous recombination within the terminally redundant ends before the circularized molecule can initiate replication or integrate into the host chromosome. As a result of recombination, all of the formerly permuted molecules are transformed into identical unit-length molecules, which is absolutely critical for maintaining genomic integrity. The genetic map is therefore drawn as a circle, which properly reflects the mixture of permuted molecules contained in an A118 phage population.

Listeria bacteriophage A511 belongs to the A1 subgroup of the Myoviridae and features a contractile, nonflexible tail and an isometric capsid. The approximate dimensions of the virion are indicated. This electron micrograph of a negatively stained A511 particle was digitally enhanced from the original image.

Plating phenotypes of a phage endolysin. (A) Plaques formed in a double-layer agar plate after infection of an L. monocytogenes host by phage A511. Note the distinct zones of secondary lysis around the core of the plaques.This is apparently due to the cell wall-hydrolyzing activity of the Ply511 endolysin, which is released from lysed cells and is able to diffuse into the soft agar layer. (B) Colonies of E. coli carrying a plasmid encoding the Ply511 endolysin. Cells were replica plated onto agar plates for the induction of enzyme synthesis, treated with chloroform to release cytoplasmic proteins, and overlaid with L. monocytogenes cells suspended in soft agar. Lysis of the Listeria cells is evident by the dark zones of clearing around the E. coli colonies. (C) Colonies of a recombinant L. lactis strain growing on an agar plate with a suitable medium, in which L. monocytogenes cells were directly incorporated. The cells carry a plasmid specifying Ply511 to which an N-terminal signal peptide was fused, resulting in the synthesis and secretion of a processed, fully functional enzyme into the surrounding medium. This leads to lysis of the Listeria cells in the medium around the colonies.

Schematic representation of the construction and function of A511::luxAB. An approximately 3-kb segment of the wild-type (wt) A511 genetic map is shown. The luxAB genetic fusion from V. harveyi was inserted immediately downstream of cps by homologous recombination from a plasmid carrying the luxAB fragment flanked by A511 DNA. Following infection of a Listeria host cell and transcription of this late gene region under control of the Pcps promoter, a bicistronic transcript is made, and Cps and bacterial luciferase are synthesized and accumulate within the cell, allowing for easy detection. T, transcription terminator; P, promoter.

Factors that increase the lytic activity of HPL118 on L. monocytogenes 10403S. For unknown reasons, this frequently used strain is slightly less sensitive to the action of the endolysin. (A) Detergents increase lytic action. Compared to the use of phosphate-buffered saline (PBS) alone, supplementing the buffer with Tween 20 and Triton X-100 can significantly enhance the lytic activity. Surprisingly, a mixture of nonionic, cationic, and anionic detergents containing a chelator and various protease inhibitors in RIPA buffer (used for the disruption of cell membranes for preparations of proteins) very strongly enhanced the lytic activity, indicating the insensitivity of the endolysins to these chemicals and enzymes. (B) Synergistic action of endolysins with different enzymatic specificities. The amount of enzyme added to each reaction was a total of 50 μl. When used alone, neither the HPL amidase nor the HPL118 peptidase achieved complete lysis in the measured time frame of 5 min. When used in combination, however, the different enzymatic activities complemented each other, and complete lysis was rapidly achieved.