Rapid Multiplex Detection and Differentiation of Listeria Cells by Use of Fluorescent Phage Endolysin Cell Wall Binding Domains

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 broadrange CBDP40 and subsequently identified after incubation with two differently colored CBD fusion proteins of higher specificity.Listeria belong to the low GϩC Gram-positive bacteria; are ubiquitously present in nature; and can be isolated from many sources such as soil, water, sewage effluents, and the feces of humans and animals (52). Within the genus Listeria, six species are recognized (Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi), in addition to the recently proposed new species L. marthii (12) and L. rocourtiae (24). Five major serovar (sv.) groups exist (1/2, 3, 4, 6, and 7), and at least 16 subserovars can be distinguished. Variations are mainly due to the structure and composition of cell wall-associated carbohydrates, wall teichoic acid (WTA), and lipoteichoic acid (LTA). However, serotyping does not necessarily correlate with the species (7, 8). Listeria monocytogenes is an opportunistic, intracellular pathogen causing an infection termed listeriosis and is exclusively transmitted via contaminated food such as raw meat, milk products, fish products, and vegetables. Listeriosis is a serious infection primarily affecting immunocompromised patients, pregnant women, the elderly, and newborns and is characterized by a high mortality rate up to Ͼ40% (5). Strains belonging to sv. groups 1/2 and 4b have been responsible for the majority of Listeria infections in hu...

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

“…The use of affinity-based magnetic separation can simultaneously tackle two critical parameters to reduce detection assay time and increase sensitivity, by (i) enriching viable cell numbers to a detectable level and (ii) isolating cells from contaminants and debris found in food samples for more accurate downstream identification and characterization (51). Toward our aim to develop a sensitive and specific assay for bacterial enrichment that can outcompete the conventional application of antibodies for biomolecular recognition, the bacteriophage S16 LTF joins other approaches employing either whole phages (25,32,33,58,59) or phage-encoded proteins, such as endolysin cell wall-binding domains (CBDs) (36)(37)(38) and tail spike RBPs (39)(40)(41).…”

“…A simplified alternative to applying whole phages for bacterial detection is to utilize phage-encoded host interaction proteins as affinity molecules. Cell wall binding domains (CBDs) of phage endolysins (peptidoglycan hydrolases) have proven effective for detecting and enriching Gram-positive bacteria, such as Listeria (36,37), Bacillus cereus, and Clostridium perfringens (38). Alternatively, phage receptor binding proteins (RBPs), which exist as long tail fibers (LTFs) or short tail spikes attached to the baseplate of tailed phages, have been applied for bacterial detection.…”

Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

“…GFP-tagged CBD proteins derived from phage endolysins Ply118, Ply511, PlyP40, and PlyP35 were produced in E. coli and purified by affinity chromatography as described earlier (33,43). Protein concentration was determined using a NanoDrop ND-1000 analyzer, and purity was checked by SDS-PAGE analysis.…”

“…In contrast, the CBDs of Listeria phage endolysins Ply118, Ply511, and PlyP40 (34,43) investigated here are assumed to recognize a more common, serovar-independent motif in the peptidoglycan structure (43). Green fluorescent protein (GFP)-tagged CBD118, CBD511, and CBDP40 molecules mainly attach to poles and septal regions of Listeria cells (33,43), while the nature of the binding ligands remains unclear.…”

“…In contrast, the CBDs of Listeria phage endolysins Ply118, Ply511, and PlyP40 (34, 43) investigated here are assumed to recognize a more common, serovar-independent motif in the peptidoglycan structure (43). Green fluorescent protein (GFP)-tagged CBD118, CBD511, and CBDP40 molecules mainly attach to poles and septal regions of Listeria cells (33,43), while the nature of the binding ligands remains unclear. The specific spatial distribution of binding ligands on the Listeria cell surface could also suggest that CBD targeting might be regulated and/or affected by the presence of other components in the cell envelope, such as the extremely abundant WTA polymers, which were reported to constitute up to 70% of the Listeria cell wall dry weight (13,14).…”