The Auxiliary Role of the Amidase Domain in Cell Wall Binding and Exolytic Activity of Staphylococcal Phage Endolysins

Son, Bo‐Kyung; Kong, Minsuk; Ryu, Sangryeol

doi:10.3390/v10060284

Cited by 32 publications

(28 citation statements)

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“…Sequence analysis revealed that all 19 clones have one or two CHAP domains at the N-terminus and 13 out of the 19 selected clones harbored an amidase domain in the middle of the chimeric endolysins. Consistent with previous reports ( Becker et al, 2009a ; Schmelcher et al, 2012b ; Son et al, 2018 ), our results indicate that the N-terminal CHAP domain is essential for lysis of S. aureus cells and that the amidase domain at the central region may enhance the overall lytic activity of endolysins. Third, the use of heat-killed S. aureus cells allowed us to distinguish clearly active chimeric endolysins among clones in the screening process.…”

Section: Discussionsupporting

confidence: 93%

“…Then, how Lys109 has superior lytic activity to its parental endolysins? One possible reason is that the increased binding ability of the chimeric endolysin might have led to the improvement of antibacterial activity ( Son et al, 2018 ). Indeed, LysSA97 amidase plus CBD displayed higher binding affinity to the target bacteria than LysSA12 amidase plus CBD ( Supplementary Figure S3 ), suggesting that LysSA97 amidase plus CBD increases the lytic activity of LysSA12 CHAP by enhancing its accessibility to the target bacteria.…”

Section: Discussionmentioning

confidence: 99%

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Development of a Novel Chimeric Endolysin, Lys109 With Enhanced Lytic Activity Against Staphylococcus aureus

et al. 2021

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As the incidence of antibiotic-resistant bacteria has become increased, phage endolysins are believed as one of the promising alternatives to antibiotics. However, the discovery of potent endolysin is still challenging because it is labor intensive and difficult to obtain a soluble form with high lytic activity. In this respect, the modular structures of Gram-positive endolysins can provide an opportunity to develop novel endolysins by domain rearrangement. In this study, a random domain swapping library of four different endolysins from phages infecting Staphylococcus aureus was constructed and screened to obtain engineered endolysins. The novel chimeric endolysin, Lys109 was selected and characterized for its staphylolytic activity. Lys109 exhibited greater bacterial cell lytic activity than its parental endolysins against staphylococcal planktonic cells and biofilms, showing highly improved activity in eliminating S. aureus from milk and on the surface of stainless steel. These results demonstrate that a novel chimeric endolysin with higher activity and solubility can be developed by random domain swapping and that this chimeric endolysin has a great potential as an antimicrobial agent.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Development of a Novel Chimeric Endolysin, Lys109 With Enhanced Lytic Activity Against Staphylococcus aureus

et al. 2021

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“…The sequence analysis of the 481 amino acids inferred from the endolysin gene sequence of each isolated phage showed that these phage endolysins are also a modular enzyme. Three distint domains were identified on the endolysin sequences: cysteine; histidinedependent amidohydrolase/peptidase (CHAP); amidase 2 (N-acetylmuramyl-L-alanine amidase); and SH3b, involved in bacterial cell wall recognition according to Son et al (2018) (Figure 6 A). The endolysins expressed by the isolated phages potentially have domains for recognition and lysis of the S. aureus cell wall.…”

Section: Phagementioning

confidence: 99%

Lytic bacteriophages as a potential alternative to control Staphylococcus aureus

Leite¹,

Pereira

Borges

et al. 2019

Pesq. agropec. bras.

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The objective of this work was to characterize autochthonous bacteriophages and to determine their lytic activity on Staphylococcus aureus. Six phages were isolated from dairy barn flush water through enrichment cultures with three S. aureus strains. All phages were characterized by DNA digestion by restriction enzymes and sequencing of the DNA fragment encoding endolysin. Each phage was tested against 100 S. aureus strains isolated from bovine mastitis and from dairy products using the lysis-plate method. The sequences of the endolysin gene were highly conserved, with nucleotide similarity higher than 99% among the isolated phages. Three domains involved in the recognition and lysis of the bacterial cell wall were identified. Two bacteriophages isolated from the dairy barns present high lytic activity on S. aureus, on a wide range of host strains, indicating their potential for studies on phage therapy in dairy cattle or as a biological control agent for dairy products.

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“…Because endolysins are bactericidal enzymes with highly evolved specificities toward specific target bacteria, they have been considered novel antibacterial agents and have shown promising potential in therapy, disinfection, and diagnostics (10,11). While most endolysins from phages that infect Gram-negative bacteria adopt a single-domain globular structure, typical endolysins from Gram-positive phages have a modular structure consisting of one or more enzymatic active domains (EADs) and a C-terminal cell wall binding domain (CBD) (12,13). The EAD has conserved active sites and cleaves specific bonds within the peptidoglycan, whereas the CBD recognizes a highly specific ligand in the cell wall and targets the endolysin to its substrate (14).…”

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LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain

Kong

et al. 2019

Appl Environ Microbiol

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To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species 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 to B. cereus spores but not to vegetative cells of B. 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 of B. cereus compared 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. IMPORTANCE Bacteriophages 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 a Bacillus cereus phage 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 controlling B. cereus.

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The Auxiliary Role of the Amidase Domain in Cell Wall Binding and Exolytic Activity of Staphylococcal Phage Endolysins

Cited by 32 publications

References 39 publications

Development of a Novel Chimeric Endolysin, Lys109 With Enhanced Lytic Activity Against Staphylococcus aureus

Development of a Novel Chimeric Endolysin, Lys109 With Enhanced Lytic Activity Against Staphylococcus aureus

Lytic bacteriophages as a potential alternative to control Staphylococcus aureus

LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain

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