Rapid multiplex detection and differentiation of Listeria cells by use of fluorescent phage endolysin cell wall binding domains - PubMed (original) (raw)

Rapid multiplex detection and differentiation of Listeria cells by use of fluorescent phage endolysin cell wall binding domains

Mathias Schmelcher et al. Appl Environ Microbiol. 2010 Sep.

Abstract

The genus Listeria comprises food-borne pathogens associated with severe infections and a high mortality rate. Endolysins from bacteriophages infecting Listeria are promising tools for both their detection and control. These proteins feature a modular organization, consisting of an N-terminal enzymatically active domain (EAD), which contributes lytic activity, and a C-terminal cell wall binding domain (CBD), which targets the lysin to its substrate. Sequence comparison among 12 different endolysins revealed high diversity among the enzyme's functional domains and allowed classification of their CBDs into two major groups and five subclasses. This diversity is reflected in various binding properties, as determined by cell wall binding assays using CBDs fused to fluorescent marker proteins. Although some proteins exhibited a broad binding range and recognize Listeria strains representing all serovars, others target specific serovars only. The CBDs also differed with respect to the number and distribution of ligands recognized on the cells, as well as their binding affinities. Surface plasmon resonance analysis revealed equilibrium affinities in the pico- to nanomolar ranges for all proteins except CBD006, which is due to an internal truncation. Rapid multiplexed detection and differentiation of Listeria strains in mixed bacterial cultures was possible by combining CBDs of different binding specificities with fluorescent markers of various colors. In addition, cells of different Listeria strains could be recovered from artificially contaminated milk or cheese by CBD-based magnetic separation by using broad-range CBDP40 and subsequently identified after incubation with two differently colored CBD fusion proteins of higher specificity.

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Figures

FIG. 1.

FIG. 1.

Listeria bacteriophage endolysin domain structures and amino acid sequence relatedness. (A) Schematic alignment of nine different Listeria phage endolysins. The N-terminal EADs and C-terminal CBDs are marked according to the different homology groups and classes, respectively. Regions of sequence homology are indicated by black lines. Ply006 features a deletion of the N-terminal portion of its CBD, compared to Ply118. Catalytic activities are indicated. (B) Phylogenetic tree indicating the relationships between CBDs examined in the present study. For calculation of the tree, the respective amino acid sequences, including the putative linker regions, were used. The lengths of the branches correspond to evolutionary distances between the CBDs. The tree was used as basis for the classification of CBDs.

FIG. 2.

FIG. 2.

Pairwise amino acid sequence alignments of the border regions between EADs and CBDs of Listeria phage endolysins characterized in the present study. Identical and similar residues are marked in yellow and green, respectively. Putative linker regions between EADs and CBDs are indicated by black boxes. The linker of PlyPSA is known from the crystal structure (21).

FIG. 3.

FIG. 3.

Listeria cells labeled with a set of 18 different fluorescent protein-CBD fusion proteins. Combinations of three different fluorescent reporters (CFP, cyan fluorescent protein; GFP, green fluorescent protein; RS, RedStar protein) and six different CBDs (one from each subclass) are shown. Listeria strains were selected according to the binding specificity of each CBD (see text). The samples were observed by epifluorescence microscopy using filter sets suitable for each fluorescent protein.

FIG. 4.

FIG. 4.

SPR analysis of the binding of CBD proteins to Listeria cell surfaces. The overlay plots show the binding kinetics of the indicated concentrations of HGFP-CBDP35 (A), HGFP-CBD006 (B), HGFP-CBD511 (C), and HGFP-CBDP40 (D) to L. monocytogenes cells immobilized on the sensor chip surface (see the text for explanation). The CBD association was monitored for 3 min; the subsequent dissociation was monitored for 12 min.

FIG. 5.

FIG. 5.

Differentiation of Listeria strains by multiplex fluorescent protein-CBD labeling (for details, see the text). (A) Cells of strains 1001 (sv. 1/2c) and 1042 (sv. 4b) incubated with HRS-CBD118 (red) and HCFP-CBD500 (blue), resulting in a serovar-specific cell wall decoration. (B) Strains 1066 (sv. 1/2b), 1020 (sv. 4a), and 1042 (sv. 4b) can be distinguished by using HRS-CBDP35 and HYFP-CBDPSA in a multiplex assay. Strain 1066 is labeled by CBD-P35 (red), strain 1042 is labeled by CBD-PSA (green), and strain WSLC 1020 is labeled by both CBDs (yellow). (C) Strains EGDe (sv. 1/2a), WSLC 1020 (sv. 4a), and WSLC 3010 (sv. 5) are distinguished by incubation with HRS-CBD511 and HBFP-CBD500. CBD511 strongly decorates WSLC 1020 and EGDe and only weakly binds to WSLC 3010, whereas CBD500 strongly binds to WSLC 1020 and 3010 and does not recognize EGDe cells. Therefore, strain EGDe appears red (square), WSLC 1020 is magenta (circle), and WSLC 3010 cells show up as purple (triangle). In panel D, detection and differentiation of Listeria strains CNL 103/2005 (1/2 a) and ScottA (4b) after recovery from contaminated milk and subsequent plating is shown, by a CBD binding assay. The green cells are strain ScottA, specifically tagged by HGFP-CBD500, while the red cells are CNL 103/2005 recognized by HRS-CBDP40. Note that deformation of the cells results from growth on drug-containing selective medium.

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