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Expression and delivery of an endolysin to combat Clostridium perfringens.

Gervasi T, Horn N, Wegmann U, Dugo G, Narbad A, Mayer MJ - Appl. Microbiol. Biotechnol. (2013)

Bottom Line: The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract.The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media.The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.

View Article: PubMed Central - PubMed

Affiliation: Gut Health and Food Safety Programme, Institute of Food Research, Colney, Norwich, NR4 7UA, UK.

ABSTRACT
Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.

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Related in: MedlinePlus

CP25L stability. Endolysin activity was measured in turbidity reduction assays using 33 μg/ml Ni-NTA-purified protein. Results represent the activity of samples stored at room temperature as a percentage of the activity of endolysin stored at 4 °C assayed at the same time; activity was calculated as the percent decrease in OD600 over 4 min of linear lysis using the mean of duplicate assays ± standard deviation
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Fig3: CP25L stability. Endolysin activity was measured in turbidity reduction assays using 33 μg/ml Ni-NTA-purified protein. Results represent the activity of samples stored at room temperature as a percentage of the activity of endolysin stored at 4 °C assayed at the same time; activity was calculated as the percent decrease in OD600 over 4 min of linear lysis using the mean of duplicate assays ± standard deviation

Mentions: The Ni-NTA-purified CP25L was stable at 4 °C for several months, and its activity was not affected by a 30-min incubation at 37 °C or 45 °C; however, treatment at 65 °C for 30 min, or 42 °C overnight, or a pasteurization treatment at 72 °C for 20 s all abolished lytic activity (data not shown). Stability at room temperature was monitored over 3 weeks, the activity remaining largely unchanged after 8 days compared with a sample stored at 4 °C, but, after 15 and 22 days storage at room temperature, the endolysin activity had reduced to c. 83 % and c. 62 % respectively, compared with the protein samples stored at 4 °C (Fig. 3).Fig. 3


Expression and delivery of an endolysin to combat Clostridium perfringens.

Gervasi T, Horn N, Wegmann U, Dugo G, Narbad A, Mayer MJ - Appl. Microbiol. Biotechnol. (2013)

CP25L stability. Endolysin activity was measured in turbidity reduction assays using 33 μg/ml Ni-NTA-purified protein. Results represent the activity of samples stored at room temperature as a percentage of the activity of endolysin stored at 4 °C assayed at the same time; activity was calculated as the percent decrease in OD600 over 4 min of linear lysis using the mean of duplicate assays ± standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3936119&req=5

Fig3: CP25L stability. Endolysin activity was measured in turbidity reduction assays using 33 μg/ml Ni-NTA-purified protein. Results represent the activity of samples stored at room temperature as a percentage of the activity of endolysin stored at 4 °C assayed at the same time; activity was calculated as the percent decrease in OD600 over 4 min of linear lysis using the mean of duplicate assays ± standard deviation
Mentions: The Ni-NTA-purified CP25L was stable at 4 °C for several months, and its activity was not affected by a 30-min incubation at 37 °C or 45 °C; however, treatment at 65 °C for 30 min, or 42 °C overnight, or a pasteurization treatment at 72 °C for 20 s all abolished lytic activity (data not shown). Stability at room temperature was monitored over 3 weeks, the activity remaining largely unchanged after 8 days compared with a sample stored at 4 °C, but, after 15 and 22 days storage at room temperature, the endolysin activity had reduced to c. 83 % and c. 62 % respectively, compared with the protein samples stored at 4 °C (Fig. 3).Fig. 3

Bottom Line: The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract.The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media.The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.

View Article: PubMed Central - PubMed

Affiliation: Gut Health and Food Safety Programme, Institute of Food Research, Colney, Norwich, NR4 7UA, UK.

ABSTRACT
Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.

Show MeSH
Related in: MedlinePlus