Phage-Derived Peptidoglycan Degrading Enzymes: Challenges and Future Prospects for In Vivo Therapy (original) (raw)
Related papers
Antibiotics
Due to the global emergence of antibiotic resistance, there has been an increase in research surrounding endolysins as an alternative therapeutic. Endolysins are phage-encoded enzymes, utilized by mature phage virions to hydrolyze the cell wall from within. There is significant evidence that proves the ability of endolysins to degrade the peptidoglycan externally without the assistance of phage. Thus, their incorporation in therapeutic strategies has opened new options for therapeutic application against bacterial infections in the human and veterinary sectors, as well as within the agricultural and biotechnology sectors. While endolysins show promising results within the laboratory, it is important to document their resistance, safety, and immunogenicity for in-vivo application. This review aims to provide new insights into the synergy between endolysins and antibiotics, as well as the formulation of endolysins. Thus, it provides crucial information for clinical trials involving en...
Phage Endolysins with Broad Antimicrobial Activity Against Enterococcus faecalis Clinical Strains
Microbial Drug Resistance, 2012
Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.
Phage lytic proteins: biotechnological applications beyond clinical antimicrobials
Critical Reviews in Biotechnology, 2015
Most bacteriophages encode two types of cell wall lytic proteins: endolysins (lysins) and virion-associated peptidoglycan hydrolases. Both enzymes have the ability to degrade the peptidoglycan of Gram positive bacteria resulting in cell lysis when they are applied externally. Bacteriophage lytic proteins have a demonstrated potential in treating animal models of infectious diseases. There has also been an increase in the study of these lytic proteins for their application in areas such as food safety, pathogen detection/diagnosis, surfaces disinfection, vaccine development and nanotechnology. This review summarizes the more recent developments, outlines the full potential of these proteins to develop new biotechnological tools and discusses the feasibility of these proposals.
Taking aim on bacterial pathogens: from phage therapy to enzybiotics
Current opinion in microbiology, 2007
The bactericidal activity of bacteriophages has been used to treat human infections for years as an alternative or a complement to antibiotic therapy. Nowadays, endolysins (phage-encoded enzymes that break down bacterial peptidoglycan at the terminal stage of the phage ...
Molecular Dissection of Phage Endolysin
Journal of Biological Chemistry, 2014
Background: Bacteriophages produce endolysins to hydrolyze host cell wall for their progeny release. Results: Mycobacterium phage endolysin is inactive in E. coli but is active in mycobacteria. Conclusion: An interdomain interaction makes Lysin A inactive in E. coli. Significance: A hydrolase with built-in mechanism attains host specificity. An in-depth study can help in developing strong anti-tuberculosis agents. Mycobacterium tuberculosis has always been recognized as one of the most successful pathogens. Bacteriophages that attack and kill mycobacteria offer an alternate mechanism for the curtailment of this bacterium. Upon infection, mycobacteriophages produce lysins that catalyze cell wall peptidoglycan hydrolysis and mycolic acid layer breakdown of the host resulting in bacterial cell rupture and virus release. The ability to lyse bacterial cells make lysins extremely significant. We report here a detailed molecular dissection of the function and regulation of mycobacteriophage D29 Lysin A. Several truncated versions of Lysin A were constructed, and their activities were analyzed by zymography and by expressing them in both Escherichia coli and Mycobacterium smegmatis. Our experiments establish that Lysin A harbors two catalytically active domains, both of which show E. coli cell lysis upon their expression exclusively in the periplasmic space. However, the expression of only one of these domains and the full-length Lysin A caused M. smegmatis cell lysis. Interestingly, full-length protein remained inactive in E. coli periplasm. Our data suggest that the inactivity is ensued by a C-terminal domain that interacts with the N-terminal domain. This interaction was affirmed by surface plasmon resonance. Our experiments also demonstrate that the C-terminal domain of Lysin A selectively binds to M. tuberculosis and M. smegmatis peptidoglycans. Our methodology of studying E. coli cell lysis by Lysin A and its truncations after expressing these proteins in the bacterial periplasm with the help of signal peptide paves the way for a large scale identification and analysis of such proteins obtained from other bacteriophages.
Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics
Critical Reviews in Microbiology, 2013
Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. In contrast to endolysins, PGHs that mediate lysis of the host bacteria at the end of the lytic cycle to release of phage progeny, the action of VAPGHs generates a small hole through which the phage tail tube crosses the cell envelope to eject the phage genetic material at the beginning to the infection cycle. The antimicrobial activity of VAPGHs was first discovered through the observation of the phenomenon of 'lysis from without' , in which the disruption of the bacterial cell wall occurs prior to phage production and is caused by a high number of phages adsorbed onto the cell surface. Based on a unique combination of properties of VAPGHs such as high specificity, remarkable thermostability, and a modular organization, these proteins are potential candidates as new antibacterial agents, e.g. against antibiotic-resistant bacteria in human therapy and veterinary as well as biopreservatives in food safety, and as biocontrol agents of harmful bacteria in agriculture. This review provides an overview of the different VAPGHs discovered to date and their potential as novel antimicrobials.
Journal of Biological Chemistry, 2013
Background: Peptidoglycan hydrolases, including bacterial autolysins and bacteriophage endolysins, contain generally a cell wall-binding domain (CWBD), responsible for their high affinity and specificity toward target cell walls. Results: Two Lactobacillus casei endolysins lyse only bacterial cells with a D-Asn cross-bridge in their peptidoglycan. Conclusion: The CWBD of these two endolysins recognizes specifically peptidoglycan with a D-Asn cross-bridge. Significance: This CWBD is a novel type of peptidoglycan-binding domain. Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a ␥-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala 4 3D-Asx-L-Lys 3 in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala 4 3L-Ala-(L-Ala/L-Ser)-L-Lys 3 ; moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala 4 3 D-Asp-L-Lys 3. In contrast, Lc-Lys could lyse the ampicillinresistant E. faecium mutant with 333 L-Lys 3-D-Asn-L-Lys 3 bridges replacing the wild-type 433 D-Ala 4-D-Asn-L-Lys 3 bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG. Peptidoglycan hydrolases (PGHs) 3 synthesized by Grampositive bacteria and their bacteriophages are able to degrade the protective cell wall peptidoglycan (PG) that surrounds the bacterial cytoplasmic membrane. PG is a macromolecule consisting of glycan chains made of alternating -1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) cross-linked by peptide chains, the composition of which varies between bacterial species. Bacterial PGHs or autolysins are required for cell wall remodeling during bacterial cell growth and division (1, 2). Bacteriophage PGHs, named endolysins, are synthesized in phage-infected cells at the end of the multiplication cycle leading to bacterial lysis and release of phage progeny (3, 4). Most PGHs have a modular structure with multiple domains, including a catalytic domain with PG hydrolyzing activity and a cell wall-binding domain (CWBD) that targets the enzyme specifically to the cell wall. The ligands of CWBDs may be structural motifs present either in PG or in secondary cell wall polymers, including polysaccharides or teichoic acids that decorate PG (3). Endolysins usually lack a signal peptide for their export and therefore rely on the synthesis of holins, which are able to insert into the cytoplasmic membrane and make pores (5). Most often, their catalytic domain is located at the N terminus and their CWBD at the C terminus (6). Generally, the catalytic domains found in endolysins belong to the same families as those encountered in bacterial PGHs (2). In contrast, certain endolysin CWBDs do not display any sequence similarity with well characterized CWBDs found in bacterial PGHs such as LysM or SH3b domains. CWBDs are considered to confer to endolysins high affinity and high specificity for their target bacteria (7). Endolysins from bacteriophages infecting Gram-positive bacteria can lyse bacteria from the outside and thus have been proposed as alternatives for preservatives and antibiotics used to destroy pathogens in food and medical applications (3, 4, 6, 8). Because of their high affinity for the cell wall, endolysin * This work was supported in part by INRA and Ré gion Ile de France and was an associated project from the Marie Curie FP7 Initial Training Network Cross Talk (Grant Agreement 21553-2). 1 Recipient of a fellowship from INRA.
The Journal of antimicrobial chemotherapy, 2015
In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs. PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model. Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by...