A VersaTile-driven platform for rapid hit-to-lead development of engineered lysins.

Health care authorities are calling for new antibacterial therapies to cope with the global emergence of antibiotic-resistant bacteria. Bacteriophage-encoded lysins are a unique class of antibacterials with promising (pre)clinical progress. Custom engineering of lysins allows for the creation of variants against potentially any bacterial pathogen. We here present a high-throughput hit-to-lead development platform for engineered lysins. The platform is driven by VersaTile, a new DNA assembly method for the rapid construction of combinatorial libraries of engineered lysins. We constructed approximately 10,000 lysin variants. Using an iterative screening procedure, we identified a lead variant with high antibacterial activity against Acinetobacter baumannii in human serum and an ex vivo pig burn wound model. This generic platform could offer new opportunities to populate the preclinical pipeline with engineered lysins for diverse (therapeutic) applications.

Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa.

AIMS: To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi-resistant) Pseudomonas aeruginosa. METHODS AND RESULTS: We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N-phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 10(6) Ps. aeruginosa cells ml(-1) in presence of 10 mmol l(-1) ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 µg ml(-1) endolysin EL188 led to a strain-dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 µg ml(-1) further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units). CONCLUSIONS: Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min. SIGNIFICANCE AND IMPACT OF THE STUDY: This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as 'enzybiotics' must not be limited to gram-positive pathogens.

Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment.

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35-45 degrees C), pressure (10.5-21.0 MPa) and treatment time (0-60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.

Stability analysis of the bacteriophage phiKMV lysin gp36C and its putative role during infection.

The kinetic, thermodynamic and structural stability of gp36C, the virion-associated peptidoglycan hydrolase domain of bacteriophage phiKMV, is analyzed. Recombinant gp36C is highly thermoresistant (k = 0.595 h(-1) at 95 degrees C), but not thermostable (T(m) = 50.2 degrees C, DeltaH(cal) = 6.86 x 10(4) cal mol(-1)). However, aggregation influences kinetic stability in an unusual manner since aggregation is more pronounced at 55 degrees C than at higher temperatures. Furthermore, gp36C reversibly unfolds in a two-state endothermic transition, and circular dichroism analysis shows that gp36C almost completely refolds after a 3-h heat treatment at 85 degrees C. These properties are in agreement with gp36C being part of the extensible tail which is ejected in an unfolded state during phage infection.

Identification and characterization of a highly thermostable bacteriophage lysozyme.

Pseudomonas aeruginosa bacteriophage phiKMV is a T7-like lytic phage. Liquid chromatography-mass spectrometry of the structural proteins revealed gene product 36 (gp36) as part of the phiKMV phage particle. The presence of a lysozyme domain in the C terminal of this protein (gp36C) was verified by turbidimetric assays on chloroform-treated P. aeruginosa PAO1 and Escherichia coli WK6 cells. The molecular mass (20,884 Da) and pI (6.4) of recombinant gp36C were determined, as were the optimal enzymatic conditions (pH 6.0 in 16.7 mM phosphate buffer) and activity (4800 U/mg). Recombinant gp36C is a highly thermostable lysozyme, retaining 26% of its activity after 2 h at 100 degrees C and 21% after autoclaving. This thermostability could prove an interesting characteristic for food conservation technology.