Structural Investigations on the SH3b Domains of Clostridium perfringens Autolysin through NMR Spectroscopy and Structure Simulation Enlighten the Cell Wall Binding Function

Clostridium perfringens autolysin (CpAcp) is a peptidoglycan hydrolase associated with cell separation, division, and growth. It consists of a signal peptide, ten SH3b domains, and a catalytic domain. The structure and function mechanisms of the ten SH3bs related to cell wall peptidoglycan binding remain unclear. Here, the structures of CpAcp SH3bs were studied through NMR spectroscopy and structural simulation. The NMR structure of SH3b6 was determined at first, which adopts a typical β-barrel fold and has three potential ligand-binding pockets. The largest pocket containing eight conserved residues was suggested to bind with peptide ligand in a novel model. The structures of the other nine SH3bs were subsequently predicted to have a fold similar to SH3b6. Their ligand pockets are largely similar to those of SH3b6, although with varied size and morphology, except that SH3b1/2 display a third pocket markedly different from those in other SH3bs. Thus, it was supposed that SH3b3-10 possess similar ligand-binding ability, while SH3b1/2 have a different specificity and additional binding site for ligand. As an entirety, ten SH3bs confer a capacity for alternatively binding to various peptidoglycan sites in the cell wall. This study presents an initial insight into the structure and potential function of CpAcp SH3bs.

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A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm

Antibiotics ◽

10.3390/antibiotics10030245 ◽

2021 ◽

Vol 10 (3) ◽

pp. 245

Author(s):

Hiroshi Sekiya ◽

Maho Okada ◽

Eiji Tamai ◽

Toshi Shimamoto ◽

Tadashi Shimamoto ◽

...

Keyword(s):

Cell Wall ◽

Clostridium Perfringens ◽

Antimicrobial Agents ◽

Catalytic Domain ◽

Food Poisoning ◽

Binding Activity ◽

Lytic Activity ◽

Intestinal Bacterium ◽

Terminal Domain ◽

Cell Wall Binding

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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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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Recognition of the cell-wall binding site of the vancomycin-group antibiotics by unnatural structural motifs: 1H NMR studies of the effects of ligand binding on antibiotic dimerisation

Journal of the Chemical Society Perkin Transactions 1 ◽

10.1039/p19940000659 ◽

1994 ◽

pp. 659 ◽

Cited By ~ 8

Author(s):

Patrick Groves ◽

Mark S. Searle ◽

Ines Chicarelli-Robinson ◽

Dudley H. Williams

Keyword(s):

Cell Wall ◽

Ligand Binding ◽

Binding Site ◽

1H Nmr ◽

Nmr Studies ◽

Structural Motifs ◽

Cell Wall Binding ◽

Vancomycin Group

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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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