Rapid multiplex detection and differentiation of Listeria cells by use of fluorescent phage endolysin cell wall binding domains.
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.
Schmelcher
Mathias
M
Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse, 7, CH 8092 Zurich, Switzerland.
Shabarova
Tatiana
T
Eugster
Marcel R
MR
Eichenseher
Fritz
F
Tchang
Vincent S
VS
Banz
Manuel
M
Loessner
Martin J
MJ
D001431:Bacteriological Techniques / Q000379:methods*; D001435:Bacteriophages / Q000201:enzymology*; D002473:Cell Wall / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D008087:Listeria / Q000145:classification* / Q000302:isolation & purification*; D008164:Luminescent Proteins / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020349:Surface Plasmon Resonance
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C001143:endolysin