Murein and pseudomurein cell wall binding domains of bacteria and archaea—a comparative view

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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Insight into the Lytic Functions of the Lactococcal Prophage TP712

Viruses ◽

10.3390/v11100881 ◽

2019 ◽

Vol 11 (10) ◽

pp. 881 ◽

Cited By ~ 3

Author(s):

Escobedo ◽

Campelo ◽

Wegmann ◽

García ◽

Rodríguez ◽

...

Keyword(s):

Cell Wall ◽

Signal Peptide ◽

Catalytic Domain ◽

Experimental Conditions ◽

Binding Domains ◽

Cell Wall Binding ◽

Additional Cell ◽

Translational Start ◽

Bacterial Peptidoglycan

The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria.

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Molecular dissection of phage lysin PlySs2: integrity of the catalytic and cell wall binding domains is essential for its broad lytic activity

Virologica Sinica ◽

10.1007/s12250-014-3535-6 ◽

2015 ◽

Vol 30 (1) ◽

pp. 45-51 ◽

Cited By ~ 13

Author(s):

Yanling Huang ◽

Hang Yang ◽

Junping Yu ◽

Hongping Wei

Keyword(s):

Cell Wall ◽

Lytic Activity ◽

Molecular Dissection ◽

Binding Domains ◽

Cell Wall Binding ◽

Phage Lysin

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Thermophile Lytic Enzyme Fusion Proteins that Target Clostridium perfringens

Antibiotics ◽

10.3390/antibiotics8040214 ◽

2019 ◽

Vol 8 (4) ◽

pp. 214 ◽

Cited By ~ 1

Author(s):

Steven M. Swift ◽

Kevin P. Reid ◽

David M. Donovan ◽

Timothy G. Ramsay

Keyword(s):

Cell Wall ◽

Clostridium Perfringens ◽

Animal Feed ◽

Lytic Enzyme ◽

Necrotic Enteritis ◽

Lytic Enzymes ◽

Thermostable Enzymes ◽

Catalytic Domains ◽

Binding Domains ◽

Cell Wall Binding

Clostridium perfringens is a bacterial pathogen that causes necrotic enteritis in poultry and livestock, and is a source of food poisoning and gas gangrene in humans. As the agriculture industry eliminates the use of antibiotics in animal feed, alternatives to antibiotics will be needed. Bacteriophage endolysins are enzymes used by the virus to burst their bacterial host, releasing bacteriophage particles. This type of enzyme represents a potential replacement for antibiotics controlling C. perfringens. As animal feed is often heat-treated during production of feed pellets, thermostable enzymes would be preferred for use in feed. To create thermostable endolysins that target C. perfringens, thermophile endolysin catalytic domains were fused to cell wall binding domains from different C. perfringens prophage endolysins. Three thermostable catalytic domains were used, two from prophage endolysins from two Geobacillus strains, and a third endolysin from the deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2). These domains harbor predicted L-alanine-amidase, glucosaminidase, and L-alanine-amidase activities, respectively and degrade the peptidoglycan of the bacterial cell wall. The cell wall binding domains were from C. perfringens prophage endolysins (Phage LYtic enzymes; Ply): PlyCP18, PlyCP10, PlyCP33, PlyCP41, and PlyCP26F. The resulting fifteen chimeric proteins were more thermostable than the native C. perfringens endolysins, and killed swine and poultry disease-associated strains of C. perfringens.

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

Journal of Bacteriology ◽

10.1128/jb.00808-12 ◽

2012 ◽

Vol 194 (23) ◽

pp. 6498-6506 ◽

Cited By ~ 35

Author(s):

Marcel R. Eugster ◽

Martin J. Loessner

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Teichoic Acid ◽

Single Copy ◽

Wall Teichoic Acid ◽

Content Type ◽

Binding Domains ◽

Cell Wall Binding ◽

Wall Teichoic Acids

ABSTRACTThe C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. TheListeria monocytogenesPly118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure ofListeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding usingL. monocytogenesserovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundanttagOhomologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of eithertagO1(EGDelmo0959) ortagO2(EGDelmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficientListeriacells. Substitution oftagOfrom an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from otherListeriaphage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.

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