LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells

LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d -alanine of the stem peptide and glycine of the cross-bridge peptide, and N -acetylmuramoyl- l -alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK.     

Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci

A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.     

Lysis of staphylococcal mastitis pathogens by bacteriophage phi11 endolysin

The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative staphylococci (Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus), making it a strong candidate protein antimicrobial. This lytic activity is maintained at the pH (6.7), and the "free" calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins containing only the endopeptidase domain also lyse staphylococci in the absence of the SH3b-binding domain.     

The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis

Lysins are peptidoglycan hydrolases that are produced by bacteriophage and act to lyse the bacterial host cell wall during progeny phage release. Here, we describe the structure and function of a novel bacteriophage-derived lysin, PlyB, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342. This molecule comprises an N-terminal catalytic domain (PlyB(cat)) and a C-terminal bacterial SH3-like domain, SH3b. It is shown that both domains are required for effective catalytic activity against ATCC 4342. Further, PlyB has specific activity comparable to the phage lysin PlyG, an amidase being developed as a therapeutic against anthrax. In contrast to PlyG, however, the 1.6 A X-ray crystal structure of PlyB(cat) reveals that the catalytic domain adopts the glycosyl hydrolase (GH)-25, rather than phage T7 lysozyme-like fold. PlyB therefore represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.     

The Cell Lysis Activity of the Streptococcus agalactiae Bacteriophage B30 Endolysin Relies on the Cysteine, Histidine-Dependent Amidohydrolase/Peptidase Domain

The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity.     

Mycobacteriophage endolysins: diverse and modular enzymes with multiple catalytic activities

The mycobacterial cell wall presents significant challenges to mycobacteriophages--viruses that infect mycobacterial hosts--because of its unusual structure containing a mycolic acid-rich mycobacterial outer membrane attached to an arabinogalactan layer that is in turn linked to the peptidoglycan. Although little is known about how mycobacteriophages circumvent these barriers during the process of infection, destroying it for lysis at the end of their lytic cycles requires an unusual set of functions. These include Lysin B proteins that cleave the linkage of mycolic acids to the arabinogalactan layer, chaperones required for endolysin delivery to peptidoglycan, holins that regulate lysis timing, and the endolysins (Lysin As) that hydrolyze peptidoglycan. Because mycobacterial peptidoglycan contains atypical features including 3→3 interpeptide linkages, it is not surprising that the mycobacteriophage endolysins also have non-canonical features. We present here a bioinformatic dissection of these lysins and show that they are highly diverse and extensively modular, with an impressive number of domain organizations. Most contain three domains with a novel N-terminal predicted peptidase, a centrally located amidase, muramidase, or transglycosylase, and a C-terminal putative cell wall binding domain.     

Characterization of an endolysin, LysBPS13, from a Bacillus cereus bacteriophage

Use of bacteriophages as biocontrol agents is a promising tool for controlling pathogenic bacteria including antibiotic-resistant bacteria. Not only bacteriophages but also endolysins, the peptidoglycan hydrolyzing enzymes encoded by bacteriophages, have high potential for applications as biocontrol agents against food-borne pathogens. In this study, a putative endolysin gene was identified in the genome of the bacteriophage BPS13, which infects Bacillus cereus. In silico analysis of this endolysin, designated LysBPS13, showed that it consists of an N-terminal catalytic domain (PGRP domain) and a C-terminal cell wall binding domain (SH3_5 domain). Further characterization of the purified LysBPS13 revealed that this endolysin is an N-acetylmuramyl-l-alanine amidase, the activity of which was not influenced by addition of EDTA. In addition, LysBPS13 demonstrated remarkable thermostability in the presence of glycerol, and it retained its lytic activity even after incubation at 100 °C for 30 min. Taken together, these results indicate that LysBPS13 can be considered a favorable candidate for a new antimicrobial agent to control B. cereus.     

Structural insights into the binding and catalytic mechanisms of the Listeria monocytogenes bacteriophage glycosyl hydrolase PlyP40

Endolysins are bacteriophage‐encoded peptidoglycan hydrolases that specifically degrade the bacterial cell wall at the end of the phage lytic cycle. They feature a distinct modular architecture, consisting of enzymatically active domains (EADs) and cell wall‐binding domains (CBDs). Structural analysis of the complete enzymes or individual domains is required for better understanding the mechanisms of peptidoglycan degradation and provides guidelines for the rational design of chimeric enzymes. We here report the crystal structure of the EAD of PlyP40, a member of the GH‐25 family of glycosyl hydrolases, and the first muramidase reported for Listeria phages. Site‐directed mutagenesis confirmed key amino acids (Glu98 and Trp10) involved in catalysis and substrate stabilization. In addition, we found that PlyP40 contains two heterogeneous CBD modules with homology to SH3 and LysM domains. Truncation analysis revealed that both domains are required for full activity but contribute to cell wall recognition and lysis differently. Replacement of CBDP40 with a corresponding domain from a different Listeria phage endolysin yielded an enzyme with a significant shift in pH optimum. Finally, domain swapping between PlyP40 and the streptococcal endolysin Cpl‐1 produced an intergeneric chimera with activity against Listeria cells, indicating that structural similarity of individual domains determines enzyme function.     

LysGH15B,the SH3b Domain of Staphylococcal Phage Endolysin LysGH15, Retains High Affinity to Staphylococci

LysGH15, a phage endolysin, exhibits a particularly broad lytic spectrum against Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA). Sequence analysis reveals that this endolysin contains a C-terminal cell wall binding domain (SH3b), which causes the endolysin to bind to host strains. In this study, the substrate binding affinity of the SH3b domain (LysGH15B) was evaluated. A fusion protein of LysGH15B and green fluorescent protein (LysGH15B–GFP) were cloned and expressed in Escherichia coli. Laser scanning confocal microscopy was used to detect the fluorescence of the treated cells irradiated at different excitation wavelengths and to determine the binding activity of LysGH15B–GFP and GFP. We found that LysGH15B–GFP not only generated green fluorescence, but, more importantly, also displayed specific affinity to staphylococcal isolates, especially MRSA. In contrast, the single GFP did not display any binding activity. The high affinity was attributed to the portion of LysGH15B and the binding activity of the fusion protein was specific to staphylococci. This study provides an insight into the SH3b domain of LysGH15. The specific binding activity may cause LysGH15B to serve as an anchoring device, and offer an alternative approach for cell surface attachment onto staphylococci.     

Endolysin of bacteriophage BFK20: evidence of a catalytic and a cell wall binding domain

A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria.     

The cell lysis activity of the Streptococcus agalactiae bacteriophage B30 endolysin relies on the cysteine, histidine-dependent amidohydrolase/peptidase domain

The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity.     

Characterization of five novel endolysins from Gram-negative infecting bacteriophages

We here characterize five globular endolysins, encoded by a set of Gram-negative infecting bacteriophages: BcepC6gp22 (Burkholderia cepacia phage BcepC6B), P2gp09 (Escherichia coli phage P2), PsP3gp10 (Salmonella enterica phage PsP3), K11gp3.5 and KP32gp15 (Klebsiella pneumoniae phages K11 and KP32, respectively). In silico, BcepC6gp22, P2gp10 and PsP3gp10 are predicted to possess lytic transglycosylase activity, whereas K11gp3.5 and KP32gp15 have putative amidase activity. All five endolysins show muralytic activity on the peptidoglycan of several Gram-negative bacterial species. In vitro, Pseudomonas aeruginosa PAO1 is clearly sensitive for the antibacterial action of the five endolysins in the presence of the outer membrane permeabilizer EDTA: reductions are ranging from 1.89 to 3.08 log units dependent on the endolysin. The predicted transglycosylases BcepC6gp22, P2gp10 and PsP3gp10 have a substantially higher muralytic and in vitro antibacterial activity compared to the predicted amidases K11gp3.5 and KP32gp15, highlighting the impact of the catalytic specificity on endolysin activity. Furthermore, initial data exclude the synergistic lethal effect of a combination of the predicted transglycosylase PsP3gp10 and the predicted amidase K11gp3.5 on PAO1. As these globular endolysins show a lower enzymatic and antibacterial activity, in comparison to modular endolysins, we suggest that the latter should be favored for antibacterial applications.     

Novel Highly Thermostable Endolysin from Thermus scotoductus MAT2119 Bacteriophage Ph2119 with Amino Acid Sequence Similarity to Eukaryotic Peptidoglycan Recognition Proteins

In this study, we present the discovery and characterization of a highly thermostable endolysin from bacteriophage Ph2119 infecting Thermus strain MAT2119 isolated from geothermal areas in Iceland. Nucleotide sequence analysis of the 16S rRNA gene affiliated the strain with the species Thermus scotoductus. Bioinformatics analysis has allowed identification in the genome of phage 2119 of an open reading frame (468 bp in length) coding for a 155-amino-acid basic protein with an M_r of 17,555. Ph2119 endolysin does not resemble any known thermophilic phage lytic enzymes. Instead, it has conserved amino acid residues (His³⁰, Tyr⁵⁸, His¹³², and Cys¹⁴⁰) that form a Zn²⁺ binding site characteristic of T3 and T7 lysozymes, as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy, bacterial peptidoglycan. The purified enzyme shows high lytic activity toward thermophiles, i.e., T. scotoductus (100%), Thermus thermophilus (100%), and Thermus flavus (99%), and also, to a lesser extent, toward mesophilic Gram-negative bacteria, i.e., Escherichia coli (34%), Serratia marcescens (28%), Pseudomonas fluorescens (13%), and Salmonella enterica serovar Panama (10%). The enzyme has shown no activity against a number of Gram-positive bacteria analyzed, with the exception of Deinococcus radiodurans (25%) and Bacillus cereus (15%). Ph2119 endolysin was found to be highly thermostable: it retains approximately 87% of its lytic activity after 6 h of incubation at 95°C. The optimum temperature range for the enzyme activity is 50°C to 78°C. The enzyme exhibits lytic activity in the pH range of 6 to 10 (maximum at pH 7.5 to 8.0) and is also active in the presence of up to 500 mM NaCl.     

Structure and lytic activity of a Bacillus anthracis prophage endolysin

We report a structural and functional analysis of the lambda prophage Ba02 endolysin (PlyL) encoded by the Bacillus anthracis genome. We show that PlyL comprises two autonomously folded domains, an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the crystal structure of the catalytic domain; its three-dimensional fold is related to that of the cell wall amidase, T7 lysozyme, and contains a conserved zinc coordination site and other components of the catalytic machinery. We demonstrate that PlyL is an N-acetylmuramoyl-L-alanine amidase that cleaves the cell wall of several Bacillus species when applied exogenously. We show, unexpectedly, that the catalytic domain of PlyL cleaves more efficiently than the full-length protein, except in the case of Bacillus cereus, and using GFP-tagged cell wall-binding domain, we detected strong binding of the cell wall-binding domain to B. cereus but not to other species tested. We further show that a related endolysin (Ply21) from the B. cereus phage, TP21, shows a similar pattern of behavior. To explain these data, and the species specificity of PlyL, we propose that the C-terminal domain inhibits the activity of the catalytic domain through intramolecular interactions that are relieved upon binding of the C-terminal domain to the cell wall. Furthermore, our data show that (when applied exogenously) targeting of the enzyme to the cell wall is not a prerequisite of its lytic activity, which is inherently high. These results may have broad implications for the design of endolysins as therapeutic agents.     

Structure of bacteriophage SPN1S endolysin reveals an unusual two‐module fold for the peptidoglycan lytic and binding activity

Bacteriophage SPN1S infects the pathogenic Gram‐negative bacterium Salmonella typhimurium and expresses endolysin for the release of phage progeny by degrading peptidoglycan of the host cell walls. Bacteriophage SPN1S endolysin exhibits high glycosidase activity against peptidoglycans, resulting in antimicrobial activity against a broad range of outer membrane‐permeabilized Gram‐negative bacteria. Here, we report a crystal structure of SPN1S endolysin, indicating that unlike most endolysins from Gram‐negative bacteria background, the α‐helical protein consists of two modular domains, a large and a small domain, with a concave groove between them. Comparison with other structurally homologous glycoside hydrolases indicated a possible peptidoglycan binding site in the groove, and the presence of a catalytic dyad in the vicinity of the groove, one residue in a large domain and the other in a junction between the two domains. The catalytic dyad was further validated by antimicrobial activity assay against outer membrane‐permeabilized Escherichia coli. The three‐helix bundle in the small domain containing a novel class of sequence motif exhibited binding affinity against outer membrane‐permeabilized E. coli and was therefore proposed as the peptidoglycan‐binding domain. These structural and functional features suggest that endolysin from a Gram‐negative bacterial background has peptidoglycan‐binding activity and performs glycoside hydrolase activity through the catalytic dyad.     

Structural and Biochemical Characterization Reveals LysGH15 as an Unprecedented “EF-Hand-Like” Calcium-Binding Phage Lysin

The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.     

Peptidoglycan Hydrolase Fusions Maintain Their Parental Specificities

The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63°C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.     

Characterization of lytic enzyme open reading frame 9 (ORF9) derived from Enterococcus faecalis bacteriophage phiEF24C

In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage φEF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-l-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn(2+) ions for its activity, contrary to the bioinformatics prediction of a Zn(2+) ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-l-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.     

The novel Shewanella putrefaciens-infecting bacteriophage Spp001: genome sequence and lytic enzymes

Shewanella putrefaciens has been identified as a specific spoilage organism commonly found in chilled fresh fish, which contributes to the spoilage of fish products. Limiting S. putrefaciens growth can extend the shelf-life of chilled fish. Endolysins, which are lytic enzymes produced by bacteriophages, have been considered an alternative to control bacterial growth, and have been useful in various applications, including food preservation. We report here, for the first time, the complete genome sequence of a novel phage Spp001, which lyses S. putrefaciens Sp225. The Spp001 genome comprises a 54,789-bp DNA molecule with 67 open reading frames and an average total G + C content of 49.42 %. In silico analysis revealed that the Spp001 open reading frames encode various putative functional proteins, including an endolysin (ORF 62); however, no sequence for genes encoding the holin polypeptides, which work in concert with endolysins, was identified. To examine further the lytic activity of Spp001, we analyzed the lytic enzyme-containing fraction from phages released at the end of the phage lytic cycle in S. putrefaciens, using diffusion and turbidimetric assays. The results show that the partially purified extract contained endolysin, as indicated by a high hydrolytic activity towards bacterial peptidoglycan decrease in the OD₅₉₀ value by 0.160 in 15 min. The results will allow further investigation of the purification of natural Spp001 endolysin, the extension of Spp001 host range, and the applications of the phage-encoded enzymes.