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A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability.

Swift SM, Seal BS, Garrish JK, Oakley BB, Hiett K, Yeh HY, Woolsey R, Schegg KM, Line JE, Donovan DM - Viruses (2015)

Bottom Line: The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures.PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl.The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens.

View Article: PubMed Central - PubMed

Affiliation: Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 10300 Baltimore Avenue, Bldg. 230, BARC-East, Beltsville, MD 20705, USA. steven.swift@ars.usda.gov.

ABSTRACT
Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

No MeSH data available.


Related in: MedlinePlus

Effect of pH and NaCl on the ability of PlyGVE2CpCWB to lyse Clostridium perfringens. Top panel shows the activity of the endolysin over a range of pH values. Bottom panel shows the activity of the endolysin over a range of NaCl concentrations. Lytic activity of the endolysin was determined by the turbidity reduction assay and all activities were normalized to the maximal activity achieved (=100%).
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viruses-07-02758-f003: Effect of pH and NaCl on the ability of PlyGVE2CpCWB to lyse Clostridium perfringens. Top panel shows the activity of the endolysin over a range of pH values. Bottom panel shows the activity of the endolysin over a range of NaCl concentrations. Lytic activity of the endolysin was determined by the turbidity reduction assay and all activities were normalized to the maximal activity achieved (=100%).

Mentions: PlyGVE2CpCWB lytic activity was characterized for pH and NaCl concentration dependence by utilizing turbidity reduction assays. The pH range for PlyGVE2CpCWB was assayed from pH 4.0 to 11.0 and substantial activity (10%–80% of maximum) was observed from pH 4.0 to pH 10.0 with the greatest activity occurring at pH 8.0 (Figure 3, top panel). Interestingly, the PlyGVE2 endolysin, from which the catalytic domain of PlyGVE2CpCWB is derived, had a broad pH range for activity, but had greatest activity at pH 6.0 [20]. Some differences were expected between PlyGVE2 and PlyGVE2CpCWB because of the substitution of the CWB domain and because the PlyGVE2 endolysin was characterized at 60 °C. Since PlyGVE2CpCWB maintains activity at low pH, it may well survive passage through the gizzard of a chicken, pH ~3, into the intestines, which are between pH 6.0 and 6.8 [49] and once there, it could then be able to lyse C. perfringens that might be present in the gastrointestinal tract. Based on the pH data, further characterization, for NaCl concentration dependence and thermostability, was completed at pH 8.0. Since NaCl in solution can have an effect on enzyme solubility and activity, we next examined the influence of NaCl on the activity of PlyGVE2CpCWB over a range of 10 mM to 600 mM NaCl in 50 mM NaH2PO4 pH 8.0 (Figure 3, bottom panel). Interestingly, the highest lytic activity was observed at the lowest salt concentration, 10 mM NaCl. Lytic activity decreased with the increase of salt in solution, with activity decreasing to 37% maximal at 150 mM NaCl, and still retained 16% activity at 600 mM NaCl. Clostridium perfringens has been reported in the caeca of broiler chickens [50]. The caeca, a pair of sacs or extensions off of the chicken intestine, have been reported to have 27–64 mM sodium ions, 22–36 mM potassium ions, and 17–25 mM chloride ions, depending on the diet of the chickens [51]. This low concentration of sodium chloride would be compatible with PlyGVE2CpCWB activity in the caeca.


A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability.

Swift SM, Seal BS, Garrish JK, Oakley BB, Hiett K, Yeh HY, Woolsey R, Schegg KM, Line JE, Donovan DM - Viruses (2015)

Effect of pH and NaCl on the ability of PlyGVE2CpCWB to lyse Clostridium perfringens. Top panel shows the activity of the endolysin over a range of pH values. Bottom panel shows the activity of the endolysin over a range of NaCl concentrations. Lytic activity of the endolysin was determined by the turbidity reduction assay and all activities were normalized to the maximal activity achieved (=100%).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488725&req=5

viruses-07-02758-f003: Effect of pH and NaCl on the ability of PlyGVE2CpCWB to lyse Clostridium perfringens. Top panel shows the activity of the endolysin over a range of pH values. Bottom panel shows the activity of the endolysin over a range of NaCl concentrations. Lytic activity of the endolysin was determined by the turbidity reduction assay and all activities were normalized to the maximal activity achieved (=100%).
Mentions: PlyGVE2CpCWB lytic activity was characterized for pH and NaCl concentration dependence by utilizing turbidity reduction assays. The pH range for PlyGVE2CpCWB was assayed from pH 4.0 to 11.0 and substantial activity (10%–80% of maximum) was observed from pH 4.0 to pH 10.0 with the greatest activity occurring at pH 8.0 (Figure 3, top panel). Interestingly, the PlyGVE2 endolysin, from which the catalytic domain of PlyGVE2CpCWB is derived, had a broad pH range for activity, but had greatest activity at pH 6.0 [20]. Some differences were expected between PlyGVE2 and PlyGVE2CpCWB because of the substitution of the CWB domain and because the PlyGVE2 endolysin was characterized at 60 °C. Since PlyGVE2CpCWB maintains activity at low pH, it may well survive passage through the gizzard of a chicken, pH ~3, into the intestines, which are between pH 6.0 and 6.8 [49] and once there, it could then be able to lyse C. perfringens that might be present in the gastrointestinal tract. Based on the pH data, further characterization, for NaCl concentration dependence and thermostability, was completed at pH 8.0. Since NaCl in solution can have an effect on enzyme solubility and activity, we next examined the influence of NaCl on the activity of PlyGVE2CpCWB over a range of 10 mM to 600 mM NaCl in 50 mM NaH2PO4 pH 8.0 (Figure 3, bottom panel). Interestingly, the highest lytic activity was observed at the lowest salt concentration, 10 mM NaCl. Lytic activity decreased with the increase of salt in solution, with activity decreasing to 37% maximal at 150 mM NaCl, and still retained 16% activity at 600 mM NaCl. Clostridium perfringens has been reported in the caeca of broiler chickens [50]. The caeca, a pair of sacs or extensions off of the chicken intestine, have been reported to have 27–64 mM sodium ions, 22–36 mM potassium ions, and 17–25 mM chloride ions, depending on the diet of the chickens [51]. This low concentration of sodium chloride would be compatible with PlyGVE2CpCWB activity in the caeca.

Bottom Line: The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures.PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl.The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens.

View Article: PubMed Central - PubMed

Affiliation: Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 10300 Baltimore Avenue, Bldg. 230, BARC-East, Beltsville, MD 20705, USA. steven.swift@ars.usda.gov.

ABSTRACT
Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

No MeSH data available.


Related in: MedlinePlus