Staphylococcal Phage 2638A endolysin is lytic for Staphylococcus aureus and harbors an inter-lytic-domain secondary translational start site

Abaev, Igor; Foster‐Frey, Juli; Korobova, Olga; Shishkova, N.A.; Киселева, Н. В.; Копылов, П. Х.; Pryamchuk, S. D.; Schmelcher, Mathias; Becker, Stephen C.; Donovan, David M.

doi:10.1007/s00253-012-4252-4

Cited by 53 publications

(68 citation statements)

References 22 publications

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“…1A). The 9 candidates are listed in Table 1 and include (besides lysostaphin) the constructs CHAPK_CWT-LST, a fusion protein consisting of the CHAP endopeptidase domain of the phage endolysin LysK (23,24) and the SH3b-type cell wall targeting domain (CWT) of lysostaphin; Ami2638A, a truncation construct harboring the amidase domain of the 2638A endolysin (25); CHAPK_CBD2638A, a fusion of the LysK CHAP domain with the SH3b CBD of the 2638A endolysin; M23LST_CBD2638A, a fusion of the M23 endopeptidase domain of lysostaphin and the CBD of 2638A; CHAPK_CHAPK_CWT-LST, a construct containing the duplicated CHAP domain of LysK fused to the CWT of lysostaphin; M23LST_CHAPH5_CWT-LST, a fusion protein featuring an N-terminal M23 domain of lysostaphin, a centrally located CHAP domain from LysH5 (an endolysin reportedly active in cow's milk) (26), and a C-terminal CWT of lysostaphin; CHAPH5_LST, which includes the CHAP domain of LysH5 fused to lysostaphin; and CHAPTwort, a truncation construct consisting of the CHAP domain of the phage Twort endolysin (27). Of note, this selection of 9 PGH constructs comprises enzymes with diverse domain architectures, ranging from single EADs without a cell wall binding module to chimeric multidomain fusion proteins, and includes EADs targeting multiple PG cleavage sites (amidase, CHAP endopeptidases, and lysostaphin M23 endopeptidase).…”

Section: Resultsmentioning

confidence: 99%

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Carolin

Dätwyler

Meile

et al. 2017

Appl Environ Microbiol

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Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of Ͼ170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of Ͼ9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by Ͼ3.5 log units within 3 h. IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.KEYWORDS antibiotic resistance, antimicrobial agents, bovine mastitis, peptidoglycan hydrolases

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Section: Resultsmentioning

confidence: 99%

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Carolin

Dätwyler

Meile

et al. 2017

Appl Environ Microbiol

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“…One exception seems to be the endolysin of the staphylococcal phage 2638A, which has been described as having an Nterminal M23 peptidase domain, a central amidase CD and a C-terminal SH3b CWB domain. Interestingly, in the assayed conditions, the amidase CD of this endolysin was shown to contribute to most of the enzyme's lytic activity (Abaev et al 2013).…”

Section: Discussionmentioning

confidence: 99%

EC300: a phage-based, bacteriolysin-like protein with enhanced antibacterial activity against Enterococcus faecalis

Proença

Leandro

García

et al. 2015

Appl Microbiol Biotechnol

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Bacteriophage lytic enzymes, either endolysins or virion-associated lysins, have been receiving considerable attention as potential antibacterial agents, particularly for the combat of antibiotic-resistant Gram-positive pathogens. A conclusion that easily emerges from the careful analysis of a great number of reports on the field is that the activity of phage lytic enzymes is rarely studied in conditions that support robust growth of the target bacteria. Here, we report the construction and study of a chimerical lysin, EC300, which was designed to target and kill Enterococcus faecalis in conditions supporting vigorous bacterial growth. EC300 resulted from the fusion of a predicted M23 endopeptidase domain of a virion-associated lysin to the putative cell wall binding domain of a previously characterized amidase endolysin, both produced by the E. faecalis phage F170/08. This bacteriolysin-like protein exhibited a clear enhanced lytic activity over the parental endolysin when both were assayed in a rich bacterial growth medium. We demonstrate the killing efficacy of EC300 against growing cells of a panel of typed E. faecalis clinical strains with high level of antibiotic resistance. The possible reasons for the marked difference between the lytic performance of EC300 and that of the amidase are discussed.

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“…Variables such as the density of the cell suspension, bacterial strain, growth phase, buffer formulation, assay temperature, and enzyme concentration can profoundly influence results. Nonetheless, the kinetics of GAGS and LytLys are seemingly on par with or exceed that of many top-performing lysins, including λSa2 (Becker et al , 2009), 2638A (Abaev et al , 2013), and LysK (Becker et al , 2009). …”

Section: Resultsmentioning

confidence: 99%

Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent

Osipovitch

Griswold

2014

FEMS Microbiology Letters

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Despite intense efforts by the medical and pharmaceutical communities, Staphylococcus aureus continues to be a pervasive pathogen that causes a myriad of diseases and a high level of morbidity and mortality among infected patients. Thus, discovering or designing novel therapeutics able to kill both drug-resistant and drug-sensitive S. aureus remains a top priority. Bacteriolytic enzymes, mostly from phage, have shown great promise in preclinical studies, but little consideration has been given to cis-acting autolytic enzymes derived from the pathogen itself. Here, we use the S. aureus autolysin LytM as a proof-of-principal to demonstrate the antibacterial potential of endogenous peptidoglycan degrading enzymes. While native LytM is only marginally bactericidal, fusion of LytM to the lysostaphin cell wall binding domain enhances its anti-staphylococcal activity approximately 540-fold, placing it on par with many phage lysins currently in preclinical development. The potential to therapeutically co-opt a pathogen’s endogenous peptidoglycan recycling machinery opens the door to a previously untapped reservoir of antibacterial drug candidates.

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Staphylococcal Phage 2638A endolysin is lytic for Staphylococcus aureus and harbors an inter-lytic-domain secondary translational start site

Cited by 53 publications

References 22 publications

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

EC300: a phage-based, bacteriolysin-like protein with enhanced antibacterial activity against Enterococcus faecalis

Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent

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