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In vitro activity against Staphylococcus aureus of a novel antimicrobial agent, PRF-119, a recombinant chimeric bacteriophage endolysin.

ABSTRACT: Antistaphylococcal activity of the novel chimeric endolysin PRF-119 was evaluated with the microdilution method. The MIC(50) and MIC(90) of 398 methicillin-susceptible Staphylococcus aureus isolates were 0.098 ?g/ml and 0.391 ?g/ml, respectively (range, 0.024 to 0.780 ?g/ml). Both the MIC(50) and MIC(90) values of 776 methicillin-resistant S. aureus isolates were 0.391 ?g/ml (range, 0.024 to 1.563 ?g/ml). All 192 clinical isolates of coagulase-negative staphylococci exhibited MIC values of >50 ?g/ml. In conclusion, PRF-119 exhibited very good activity specifically against S. aureus.

SUBMITTER: Idelevich EA

PROVIDER: S-EPMC3165309 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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In vitro activity against Staphylococcus aureus of a novel antimicrobial agent, PRF-119, a recombinant chimeric bacteriophage endolysin.

Idelevich Evgeny A EA von Eiff Christof C Friedrich Alexander W AW Iannelli Domenico D Xia Guoqing G Peters Georg G Peschel Andreas A Wanninger Ingrid I Becker Karsten K

Antimicrobial agents and chemotherapy 20110711 9

Antistaphylococcal activity of the novel chimeric endolysin PRF-119 was evaluated with the microdilution method. The MIC(50) and MIC(90) of 398 methicillin-susceptible Staphylococcus aureus isolates were 0.098 μg/ml and 0.391 μg/ml, respectively (range, 0.024 to 0.780 μg/ml). Both the MIC(50) and MIC(90) values of 776 methicillin-resistant S. aureus isolates were 0.391 μg/ml (range, 0.024 to 1.563 μg/ml). All 192 clinical isolates of coagulase-negative staphylococci exhibited MIC values of >50 μ ...[more]

PMID: 21746950

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Project description:BACKGROUND: Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAP(K)), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAP(K) cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. METHODS: The CHAP(K) domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants. RESULTS: The structure of CHAP(K) revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAP(K) contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity. CONCLUSIONS: The high-resolution structure of the CHAP(K) domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAP(K) with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.

Project description:The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene (TF) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1L8823-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 (TF gene) to 0.93 (TFnt gene). Incorporation of the TFnt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana, led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC50 25 µg/ml) and 60% (IC50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.

Dataset Information

In vitro activity against Staphylococcus aureus of a novel antimicrobial agent, PRF-119, a recombinant chimeric bacteriophage endolysin.

Publications

In vitro activity against Staphylococcus aureus of a novel antimicrobial agent, PRF-119, a recombinant chimeric bacteriophage endolysin.

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