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Purification and characterization of Perfrin, a novel bacteriocin from a virulent Clostridium perfringens strain

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Abstract
Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. Single strain dominance is found in the gut of broiler chickens suffering from necrotic enteritis: within an outbreak, all affected animals usually carry the same clonal Clostridium perfringens strain in the affected tissue while clinically healthy chickens can carry different Clostridium perfringens clones in their intestine. It is known that Clostridium perfringens is capable of secreting factors inhibiting growth of other Clostridium perfringens strains and this characteristic is more prevalent in strains isolated from outbreaks of necrotic enteritis compared to strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the purification and the characterization of a bacteriocin from a necrotic enteritis associated Clostridium perfringens strain exhibiting antibacterial activity against other Clostridium perfringens strains. The bacteriocin was purified using cation exchange chromatography followed by hydrophobic interaction and identified with mass spectrometry. The thermal, pH and protease stability and the range of activity was determined. A collection of poultry and non-poultry derived C. perfringens strains was screened for the bacteriocin gene by PCR. Southern Blot was performed to determine the location of the bacteriocin gene. Amino acid sequence analysis indicated that the bacteriocin is a 11.5 kDa fragment of a 22.91 kDa protein and that it constitutes the C-terminal part of this protein. The antibacterial activity of the purified bacteriocin was abolished by proteolytic enzymes trypsin and proteinase K and by heat treatment (10 min at 80 °C). The purified bacteriocin showed inhibitory activity over a wide pH-range (4.0 to 10.0). Since the antibacterial activity of the purified bacteriocin against other Clostridium perfringens strains had a narrower spectrum than the secreting strain, most probably additional bacteriocins are produced by the strain. The bacteriocin gene was identified in 20% (10/50) of the poultry derived strains (22/50 netB positive), and was not found in 45 netB negative C. perfringens type A, B, C, D and E strains isolated from cattle, sheep, pigs, and humans. Southern blotting showed that the bacteriocin gene is not located on a plasmid. A novel bacteriocin from a necrotic enteritis associated Clostridium perfringens strain is purified and characterized. Despite the fact that the 10 strains carrying the bacteriocin gene are all netB positive strains, Perfrin and NetB are not located on the same genetic element since NetB is plasmid encoded.

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Chicago
Timbermont, Leen, Lina De Smet, Anouk Lanckriet, Filip Van Nieuwerburgh, Valeria R Parreira, Gonzalez Van Driessche, Freddy Haesebrouck, et al. 2012. “Purification and Characterization of Perfrin, a Novel Bacteriocin from a Virulent Clostridium Perfringens Strain.” In Bacteriocins and Antimicrobial Peptides, International Scientific Conference, Abstracts.
APA
Timbermont, L., De Smet, L., Lanckriet, A., Van Nieuwerburgh, F., Parreira, V. R., Van Driessche, G., Haesebrouck, F., et al. (2012). Purification and characterization of Perfrin, a novel bacteriocin from a virulent Clostridium perfringens strain. Bacteriocins and Antimicrobial Peptides, International scientific conference, Abstracts. Presented at the International scientific conference on Bacteriocins and Antimicrobial Peptides (BAMP).
Vancouver
1.
Timbermont L, De Smet L, Lanckriet A, Van Nieuwerburgh F, Parreira VR, Van Driessche G, et al. Purification and characterization of Perfrin, a novel bacteriocin from a virulent Clostridium perfringens strain. Bacteriocins and Antimicrobial Peptides, International scientific conference, Abstracts. 2012.
MLA
Timbermont, Leen, Lina De Smet, Anouk Lanckriet, et al. “Purification and Characterization of Perfrin, a Novel Bacteriocin from a Virulent Clostridium Perfringens Strain.” Bacteriocins and Antimicrobial Peptides, International Scientific Conference, Abstracts. 2012. Print.
@inproceedings{3102720,
  abstract     = {Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. Single strain dominance is found in the gut of broiler chickens suffering from necrotic enteritis: within an outbreak, all affected animals usually carry the same clonal Clostridium perfringens strain in the affected tissue while clinically healthy chickens can carry different Clostridium perfringens clones in their intestine. It is known that Clostridium perfringens is capable of secreting factors inhibiting growth of other Clostridium perfringens strains and this characteristic is more prevalent in strains isolated from outbreaks of necrotic enteritis compared to strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the purification and the characterization of a bacteriocin from a necrotic enteritis associated Clostridium perfringens strain exhibiting antibacterial activity against other Clostridium perfringens strains.
The bacteriocin was purified using cation exchange chromatography followed by hydrophobic interaction and identified with mass spectrometry. The thermal, pH and protease stability and the range of activity was determined. A collection of poultry and non-poultry derived C. perfringens strains was screened for the bacteriocin gene by PCR. Southern Blot was performed to determine the location of the bacteriocin gene.
Amino acid sequence analysis indicated that the bacteriocin is a 11.5 kDa fragment of a 22.91 kDa protein and that it constitutes the C-terminal part of this protein. The antibacterial activity of the purified bacteriocin was abolished by proteolytic enzymes trypsin and proteinase K and by heat treatment (10 min at 80 {\textdegree}C). The purified bacteriocin showed inhibitory activity over a wide pH-range (4.0 to 10.0). Since the antibacterial activity of the purified bacteriocin against other Clostridium perfringens strains had a narrower spectrum than the secreting strain, most probably additional bacteriocins are produced by the strain. The bacteriocin gene was identified in 20\% (10/50) of the poultry derived strains (22/50 netB positive), and was not found in 45 netB negative C. perfringens type A, B, C, D and E strains isolated from cattle, sheep, pigs, and humans. Southern blotting showed that the bacteriocin gene is not located on a plasmid.
A novel bacteriocin from a necrotic enteritis associated Clostridium perfringens strain is purified and characterized. Despite the fact that the 10 strains carrying the bacteriocin gene are all netB positive strains, Perfrin and NetB are not located on the same genetic element since NetB is plasmid encoded.},
  author       = {Timbermont, Leen and De Smet, Lina and Lanckriet, Anouk and Van Nieuwerburgh, Filip and Parreira, Valeria R and Van Driessche, Gonzalez and Haesebrouck, Freddy and Ducatelle, Richard and Prescott, John and Deforce, Dieter and Devreese, Bart and Van Immerseel, Filip},
  booktitle    = {Bacteriocins and Antimicrobial Peptides, International scientific conference, Abstracts},
  language     = {eng},
  location     = {Kosice, Slovakije},
  title        = {Purification and characterization of Perfrin, a novel bacteriocin from a virulent Clostridium perfringens strain},
  year         = {2012},
}