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PlyC: a multimeric bacteriophage lysin.

ABSTRACT: Lysins are murein hydrolases produced by bacteriophage that act on the bacterial host cell wall to release progeny phage. When added extrinsically in their purified form, these enzymes produce total lysis of susceptible Gram-positive bacteria within seconds, suggesting a unique antimicrobial strategy. All known Gram-positive lysins are produced as a single polypeptide containing a catalytic activity domain, which cleaves one of the four major peptidoglycan bonds, and a cell-wall-binding domain, which may bind a species-specific carbohydrate epitope in the cell wall. Here, we have cloned and expressed a unique lysin from the streptococcal bacteriophage C(1), termed PlyC. Molecular characterization of the plyC operon reveals that PlyC is, surprisingly, composed of two separate gene products, PlyCA and PlyCB. Based on biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of eight PlyCB subunits for each PlyCA. Inhibitor studies predicted the presence of an active-site cysteine, and bioinformatic analysis revealed a cysteine, histidine-dependent amidohydrolase/peptidase domain within PlyCA. Point mutagenesis confirmed that PlyCA is responsible for the observed catalytic activity, and Cys-333 and His-420 are the active-site residues. PlyCB was found to self-assemble into an octamer, and this complex alone was able to direct streptococcal cell-wall-specific binding. Similar to no other proteins in sequence databases, PlyC defines a previously uncharacterized structural family of cell-wall hydrolases.

SUBMITTER: Nelson D

PROVIDER: S-EPMC1487170 | biostudies-literature |

REPOSITORIES: biostudies-literature

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X-ray crystal structure of the streptococcal specific phage lysin PlyC.

Project description:Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria.

| S-EPMC3412044 | biostudies-literature

Dynamic Motion and Communication in the Streptococcal C1 Phage Lysin, PlyC.

Project description:The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency.

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Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent.

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Enzymatic characterization of a lysin encoded by bacteriophage EL.

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Thermosensitive Hydrogel Wound Dressing Loaded with Bacteriophage Lysin LysP53.

Project description:Wound infections are prone to attacks from infectious pathogens, including multidrug resistant bacteria that render conventional antimicrobials ineffective. Recently, lysins have been proposed as alternatives to conventional antimicrobials to tackle the menace of multidrug resistance pathogens. The coupling of lysins with a material that will cover the wound may prove beneficial in both protecting and treating wound infections. Hence, in this study, a Gram-negative lysin, LysP53, was coupled with a thermosensitive hydrogel, poloxamer P407, and its efficacy to treat wound infection was tested. In vitro, the addition of LysP53 to the poloxamer did not affect its thermosensitive characteristics, nor did it affect the hydrogel structure. Moreover, the lysin hydrogel could hydrolyze the peptidoglycan, demonstrating that it may have bactericidal activity. Up to 10.4% of LysP53 was released from the hydrogel gradually within 24 h, which led to a 4-log reduction of stationary phase Acinetobacter baumannii. Lastly, the lysin hydrogel was found safe with no cytotoxic effects observed in cells. Ex vivo, LysP53 hydrogel could inhibit bacterial growth on a pig skin decolonization model, with 3-log differences compared to non-treated groups. Overall, our results suggest that lysin-loaded hydrogels may provide a novel solution to treat wound infections caused by resistant bacteria.

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Enterococcal bacteriophage: A survey of the tail associated lysin landscape.

Project description:Bacteriophages are viruses that exclusively infect bacteria which require local degradation of cell barriers. This degradation is accomplished by various lysins located mainly within the phage tail structure. In this paper we surveyed and analysed the genomes of 506 isolated bacteriophage and prophage infecting or harboured within the genomes of the medically important Enterococcus faecalis and faecium. We highlight and characterise the major features of the genomes of phage in the morphological groups podovirus, siphovirus and myovirus, and explore their categorisation according to the new ICTV classifications, with a focus on putative extracellular lysins chiefly within tail modules. Our analysis reveals a range of potential cell-wall targeting enzyme domains that are part of tail, tape measure or other predicted base structures of these phages or prophages. These largely fall into protein domains targeting pentapeptide or glycosidic linkages within peptidoglycan but also potentially the enterococcal polysaccharide antigen (EPA) and wall teichoic acids of these species (i.e., Pectinesterases and Phosphodiesterases). Notably, there is a great variety of domain architectures that reveal the diversity of evolutionary solutions to attack the Enterococcus cell wall. Despite this variety, most phage and prophage possess a putative endopeptidase (70%), reflecting the ubiquity of this cell surface barrier. We also identified a predicted lytic transglycosylase domain belonging to the glycosyl hydrolase (GH) family 23 and present exclusively within tape measure proteins. Our data also reveal distinct features of the genomes of podo-, sipho- and myo-type viruses that most likely relate to their size and complexity. Overall, we lay a foundation for expression of recombinant TAL proteins and engineering of enterococcal and other phage that will be invaluable for researchers in this field.

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Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin.

Project description:Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively.

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Immunogenicity of Endolysin PlyC.

Project description:Endolysins are bacteriolytic enzymes derived from bacteriophages. They represent an alternative to antibiotics, since they are not susceptible to conventional antimicrobial resistance mechanisms. Since non-human proteins are efficient inducers of specific immune responses, including the IgG response or the development of an allergic response mediated by IgE, we evaluated the general immunogenicity of the highly active antibacterial enzyme, PlyC, in a human population and in a mouse model. The study includes the identification of molecular epitopes of PlyC. The overall assessment of potential hypersensitivity to this protein and PlyC-specific IgE testing was also conducted in mice. PlyC induced efficient IgG production in mice, and the molecular analysis revealed that PlyC-specific IgG interacted with four immunogenic regions identified within the PlyCA subunit. In humans, approximately 10% of the population demonstrated IgG reactivity to the PlyCB subunit only, which is attributed to cross-reactions since this was a naïve serum. Of note, in spite of being immunogenic, PlyC induced a normal immune response, without hypersensitivity, since both the animals challenged with PlyC and in the human population PlyC-specific IgE was not detected.

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