Potential Role of the Host-Derived Cell-Wall Binding Domain of Endolysin CD16/50L as a Molecular Anchor in Preservation of Uninfected Clostridioides difficile for New Rounds of Phage Infection (original) (raw)
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Microorganisms
Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacillus and is a major cause of healthcare-associated infections. Whereas the vegetative form of the pathogen is susceptible to treatment with antibiotics, its ability to persist in the gut as antibiotic-resistant spores means that reinfection can occur in cases were the individual fails to re-establish a protective microflora. Bacteriophages and their lysins are currently being explored as treatment options due to their specificity, which minimizes the disruption to the other members of the gut microflora that are protective. The feasibility of employing recombinant endolysins to target the vegetative form of C. difficile has been demonstrated in animal models. In this study, we cloned and expressed the enzyme active domain of LysCD6356 and confirmed its ability to lyse the vegetative forms of a diverse range of clinical isolates of C. difficile, which included members of the hypervirulent 027 ribotype. Lytic ac...
Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291
Environmental Microbiology, 2021
Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.
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.
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...
Using a novel lysin to help control Clostridium difficile infections
Antimicrobial agents and chemotherapy, 2015
As a consequence of excessive antibiotic therapies in hospitalized patients, Clostridium difficile, a Gram-positive anaerobic spore-forming intestinal pathogen, is the leading cause of hospital-acquired diarrhea and colitis. Drug treatments for these diseases are often complicated by antibiotic resistant strains and a high frequency of treatment failures and relapse; therefore, novel non-antibiotic approaches may prove to be more effective. In this study, we recombinantly expressed a prophage lysin initially identified from a C. difficile strain, CD630, which we named PlyCD. PlyCD was found to have lytic activity against specific C. difficile strains. Moreover, the recombinantly expressed catalytic domain of this protein, PlyCD1-174, displayed significantly greater lytic activity (>4-log kill) and a broader lytic spectrum against C. difficile strains, while still retaining a high degree of specificity towards C. difficile versus commensal Clostridia and other bacterial species. O...
The CD27L and CTP1L Endolysins Targeting Clostridia Contain a Built-in Trigger and Release Factor
PLoS Pathogens, 2014
The bacteriophage WCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-Lphenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.
Invisible steps for a global endemy: molecular strategies adopted by Clostridioides difficile
Therapeutic Advances in Gastroenterology
Clostridioides difficile infection (CDI) is on the rise worldwide and is associated with an increase in deaths and socio-health burden. C. difficile has become ubiquitous in anthropized environments because of the extreme resistance of its spores. Based on the epidemiological data and knowledge of molecular pathogenesis of C. difficile, it is possible to predict its progressive colonization of the human population for the following reasons: first, its global spread is unstoppable; second, the toxins (Tcds) produced by C. difficile, TcdA and TcdB, mainly cause cell death by apoptosis, but the surviving cells acquire a senescence state that favours persistence of C. difficile in the intestine; third, proinflammatory cytokines, tumour necrosis factor-α and interferon-γ, induced during CDI, enhance the cytotoxicity of Tcds and can increase the survival of senescent cells; fourth, Tcds block mobility and induce apoptosis in immune cells recruited at the infection site; and finally, after...
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.
Molecular characterization of clostridium difficile isolated in Hong Kong SAR
International Journal of Infectious Diseases, 2012
Clostridium difficile infection is increasing in both frequency and severity, with the emergence of new highly virulent strains highlighting the need for more rapid and effective methods of control. Here, we show that bacteriophage endolysin can be used to inhibit and kill C. difficile. The genome sequence of a novel bacteriophage that is active against C. difficile was determined, and the bacteriophage endolysin gene was subcloned and expressed in Escherichia coli. The partially purified endolysin was active against 30 diverse strains of C. difficile, and importantly, this group included strains of the major epidemic ribotype 027 (B1/NAP1). In contrast, a range of commensal species that inhabit the gastrointestinal tract, including several representatives of the clostridium-like Firmicutes, were insensitive to the endolysin. This endolysin provides a platform for the generation of both therapeutic and detection systems to combat the C. difficile problem. To investigate a method for the protected delivery and production of the lysin in the gastrointestinal tract, we demonstrated the expression of active CD27L endolysin in the lactic acid bacterium Lactococcus lactis MG1363.