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A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method.

Yang H, Linden SB, Wang J, Yu J, Nelson DC, Wei H - Sci Rep (2015)

Bottom Line: The increasing emergence of multi-drug resistant streptococci poses a serious threat to public health worldwide.ClyR is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen as well as the first lysin that kills all four of the bovine mastitis-causing pathogens.This study demonstrates the success of the screening method resulting in a powerful lysin with potential for treating most streptococcal associated infections.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

ABSTRACT
The increasing emergence of multi-drug resistant streptococci poses a serious threat to public health worldwide. Bacteriophage lysins are promising alternatives to antibiotics; however, their narrow lytic spectrum restricted to closely related species is a central shortcoming to their translational development. Here, we describe an efficient method for rapid screening of engineered chimeric lysins and report a unique "chimeolysin", ClyR, with robust activity and an extended-spectrum streptococcal host range against most streptococcal species, including S. pyogenes, S. agalactiae, S. dysgalactiae, S. equi, S. mutans, S. pneumoniae, S. suis and S. uberis, as well as representative enterococcal and staphylococcal species (including MRSA and VISA). ClyR is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen as well as the first lysin that kills all four of the bovine mastitis-causing pathogens. This study demonstrates the success of the screening method resulting in a powerful lysin with potential for treating most streptococcal associated infections.

No MeSH data available.


Related in: MedlinePlus

High lytic activity of ClyR.(A) Time-kill curves of ClyR against S. dysgalactiae ATCC 35666. Bacterial cells were washed once with PBS, treated with 25 μg/ml ClyR, and the change of OD600 (right Y-axis) were monitored by a microplate reader at 37 °C for 30 min. Triangles: ClyR; Circles: PBS controls. In parallel, the viable cell numbers (squares, left Y-axis) were calculated by plating onto BHI agar plates at different time points. (B) Dose-dependent lytic efficacy of ClyR against S. agalactiae S12, S. dysgalactiae ATCC 35666, or an equal mixture of both strains in market pasteurized milk at 30 °C for 1 h. (C) TEM images of S. dysgalactiae ATCC 35666 cells exposed to ClyR. Bar sizes: 500 nm. (D) Time-killing efficacy of ClyR (40 μg/ml) against S. agalactiae S12, S. dysgalactiae ATCC 35666, or an equal mixture of both strains in market pasteurized milk at 30 °C. (E) Lytic efficacy of ClyR (40 μg/ml) in pasteurized cow’s milk against S. agalactiae S12, or S. dysgalactiae ATCC 35666 at 30 °C for 1 h. Fresh cow milk samples A, B, and C were taken from healthy cows, and samples D and E were from mastitic cows. (F) Comparison of the activity of 0.89 μM ClyR or PlyCAC against various strains. (G) Comparison of the activity of 0.89 μM ClyR or PlyGBS-180 against S. dysgalactiae ATCC 35666. (H) Efficacy of PlyGBS-180 against S. dysgalactiae ATCC 35666 in market pasteurized milk at 30 °C for 60 min.
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f3: High lytic activity of ClyR.(A) Time-kill curves of ClyR against S. dysgalactiae ATCC 35666. Bacterial cells were washed once with PBS, treated with 25 μg/ml ClyR, and the change of OD600 (right Y-axis) were monitored by a microplate reader at 37 °C for 30 min. Triangles: ClyR; Circles: PBS controls. In parallel, the viable cell numbers (squares, left Y-axis) were calculated by plating onto BHI agar plates at different time points. (B) Dose-dependent lytic efficacy of ClyR against S. agalactiae S12, S. dysgalactiae ATCC 35666, or an equal mixture of both strains in market pasteurized milk at 30 °C for 1 h. (C) TEM images of S. dysgalactiae ATCC 35666 cells exposed to ClyR. Bar sizes: 500 nm. (D) Time-killing efficacy of ClyR (40 μg/ml) against S. agalactiae S12, S. dysgalactiae ATCC 35666, or an equal mixture of both strains in market pasteurized milk at 30 °C. (E) Lytic efficacy of ClyR (40 μg/ml) in pasteurized cow’s milk against S. agalactiae S12, or S. dysgalactiae ATCC 35666 at 30 °C for 1 h. Fresh cow milk samples A, B, and C were taken from healthy cows, and samples D and E were from mastitic cows. (F) Comparison of the activity of 0.89 μM ClyR or PlyCAC against various strains. (G) Comparison of the activity of 0.89 μM ClyR or PlyGBS-180 against S. dysgalactiae ATCC 35666. (H) Efficacy of PlyGBS-180 against S. dysgalactiae ATCC 35666 in market pasteurized milk at 30 °C for 60 min.

Mentions: Clone AK104, containing a chimeolysin we named ClyR, showed the highest lytic activity against S. dysgalactiae on screening plates (Fig. 2B), and was therefore selected for further characterization. ClyR is well expressed as a soluble protein in E. coli and purification of >95% could be achieved as observed by 12% SDS-PAGE gel (Supplementary Fig. S4A). As shown in Fig. 3A, ClyR rapidly kills S. dysgalactiae; the turbidity decreased from 1.0 to near 0.2 within 30 min when treated with 25 μg/ml ClyR, corresponding to a 4 log reduction in CFU. Notably, a reduction of over 2 logCFU was observed in the first minute.


A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method.

Yang H, Linden SB, Wang J, Yu J, Nelson DC, Wei H - Sci Rep (2015)

High lytic activity of ClyR.(A) Time-kill curves of ClyR against S. dysgalactiae ATCC 35666. Bacterial cells were washed once with PBS, treated with 25 μg/ml ClyR, and the change of OD600 (right Y-axis) were monitored by a microplate reader at 37 °C for 30 min. Triangles: ClyR; Circles: PBS controls. In parallel, the viable cell numbers (squares, left Y-axis) were calculated by plating onto BHI agar plates at different time points. (B) Dose-dependent lytic efficacy of ClyR against S. agalactiae S12, S. dysgalactiae ATCC 35666, or an equal mixture of both strains in market pasteurized milk at 30 °C for 1 h. (C) TEM images of S. dysgalactiae ATCC 35666 cells exposed to ClyR. Bar sizes: 500 nm. (D) Time-killing efficacy of ClyR (40 μg/ml) against S. agalactiae S12, S. dysgalactiae ATCC 35666, or an equal mixture of both strains in market pasteurized milk at 30 °C. (E) Lytic efficacy of ClyR (40 μg/ml) in pasteurized cow’s milk against S. agalactiae S12, or S. dysgalactiae ATCC 35666 at 30 °C for 1 h. Fresh cow milk samples A, B, and C were taken from healthy cows, and samples D and E were from mastitic cows. (F) Comparison of the activity of 0.89 μM ClyR or PlyCAC against various strains. (G) Comparison of the activity of 0.89 μM ClyR or PlyGBS-180 against S. dysgalactiae ATCC 35666. (H) Efficacy of PlyGBS-180 against S. dysgalactiae ATCC 35666 in market pasteurized milk at 30 °C for 60 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660466&req=5

f3: High lytic activity of ClyR.(A) Time-kill curves of ClyR against S. dysgalactiae ATCC 35666. Bacterial cells were washed once with PBS, treated with 25 μg/ml ClyR, and the change of OD600 (right Y-axis) were monitored by a microplate reader at 37 °C for 30 min. Triangles: ClyR; Circles: PBS controls. In parallel, the viable cell numbers (squares, left Y-axis) were calculated by plating onto BHI agar plates at different time points. (B) Dose-dependent lytic efficacy of ClyR against S. agalactiae S12, S. dysgalactiae ATCC 35666, or an equal mixture of both strains in market pasteurized milk at 30 °C for 1 h. (C) TEM images of S. dysgalactiae ATCC 35666 cells exposed to ClyR. Bar sizes: 500 nm. (D) Time-killing efficacy of ClyR (40 μg/ml) against S. agalactiae S12, S. dysgalactiae ATCC 35666, or an equal mixture of both strains in market pasteurized milk at 30 °C. (E) Lytic efficacy of ClyR (40 μg/ml) in pasteurized cow’s milk against S. agalactiae S12, or S. dysgalactiae ATCC 35666 at 30 °C for 1 h. Fresh cow milk samples A, B, and C were taken from healthy cows, and samples D and E were from mastitic cows. (F) Comparison of the activity of 0.89 μM ClyR or PlyCAC against various strains. (G) Comparison of the activity of 0.89 μM ClyR or PlyGBS-180 against S. dysgalactiae ATCC 35666. (H) Efficacy of PlyGBS-180 against S. dysgalactiae ATCC 35666 in market pasteurized milk at 30 °C for 60 min.
Mentions: Clone AK104, containing a chimeolysin we named ClyR, showed the highest lytic activity against S. dysgalactiae on screening plates (Fig. 2B), and was therefore selected for further characterization. ClyR is well expressed as a soluble protein in E. coli and purification of >95% could be achieved as observed by 12% SDS-PAGE gel (Supplementary Fig. S4A). As shown in Fig. 3A, ClyR rapidly kills S. dysgalactiae; the turbidity decreased from 1.0 to near 0.2 within 30 min when treated with 25 μg/ml ClyR, corresponding to a 4 log reduction in CFU. Notably, a reduction of over 2 logCFU was observed in the first minute.

Bottom Line: The increasing emergence of multi-drug resistant streptococci poses a serious threat to public health worldwide.ClyR is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen as well as the first lysin that kills all four of the bovine mastitis-causing pathogens.This study demonstrates the success of the screening method resulting in a powerful lysin with potential for treating most streptococcal associated infections.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

ABSTRACT
The increasing emergence of multi-drug resistant streptococci poses a serious threat to public health worldwide. Bacteriophage lysins are promising alternatives to antibiotics; however, their narrow lytic spectrum restricted to closely related species is a central shortcoming to their translational development. Here, we describe an efficient method for rapid screening of engineered chimeric lysins and report a unique "chimeolysin", ClyR, with robust activity and an extended-spectrum streptococcal host range against most streptococcal species, including S. pyogenes, S. agalactiae, S. dysgalactiae, S. equi, S. mutans, S. pneumoniae, S. suis and S. uberis, as well as representative enterococcal and staphylococcal species (including MRSA and VISA). ClyR is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen as well as the first lysin that kills all four of the bovine mastitis-causing pathogens. This study demonstrates the success of the screening method resulting in a powerful lysin with potential for treating most streptococcal associated infections.

No MeSH data available.


Related in: MedlinePlus