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

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

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Lateral flow assay-based bacterial detection using engineered cell wall binding domains of a phage endolysin

Biosensors and Bioelectronics ◽

10.1016/j.bios.2017.05.010 ◽

2017 ◽

Vol 96 ◽

pp. 173-177 ◽

Cited By ~ 45

Author(s):

Minsuk Kong ◽

Joong Ho Shin ◽

Sunggi Heu ◽

Je-Kyun Park ◽

Sangryeol Ryu

Keyword(s):

Cell Wall ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Bacterial Detection ◽

Binding Domains ◽

Cell Wall Binding

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

Applied and Environmental Microbiology ◽

10.1128/aem.00801-10 ◽

2010 ◽

Vol 76 (17) ◽

pp. 5745-5756 ◽

Cited By ~ 108

Author(s):

Mathias Schmelcher ◽

Tatiana Shabarova ◽

Marcel R. Eugster ◽

Fritz Eichenseher ◽

Vincent S. Tchang ◽

...

Keyword(s):

Cell Wall ◽

Modular Organization ◽

Binding Assays ◽

Surface Plasmon Resonance Analysis ◽

Fluorescent Marker ◽

Binding Domains ◽

Cell Wall Binding ◽

Fluorescent Markers ◽

Severe Infections ◽

And Control

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

Applied Microbiology and Biotechnology ◽

10.1007/s00253-011-3637-0 ◽

2011 ◽

Vol 92 (5) ◽

pp. 921-928 ◽

Cited By ~ 25

Author(s):

Ganesh Ram R. Visweswaran ◽

Bauke W. Dijkstra ◽

Jan Kok

Keyword(s):

Cell Wall ◽

Binding Domains ◽

Cell Wall Binding

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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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LysPBC2, a Novel Endolysin Harboring aBacillus cereusSpore Binding Domain

Applied and Environmental Microbiology ◽

10.1128/aem.02462-18 ◽

2018 ◽

Vol 85 (5) ◽

Cited By ~ 3

Author(s):

Minsuk Kong ◽

Hongjun Na ◽

Nam-Chul Ha ◽

Sangryeol Ryu

Keyword(s):

Cell Wall ◽

Catalytic Domain ◽

Binding Activity ◽

Binding Domain ◽

Lytic Activity ◽

Content Type ◽

Cell Wall Binding ◽

A Cell ◽

Cell Wall Binding Domain

ABSTRACTTo control the spore-forming human pathogenBacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolatedB. cereusphage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against allBacillus,Listeria, andClostridiumspecies tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds toB. cereusspores but not to vegetative cells ofB. cereus. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells ofB. cereuscompared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCEBacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of aBacillus cereusphage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controllingB. cereus.

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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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