Recombinant expression of two bacteriophage proteins that lyse clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal domain

Authors: Simmons, Ibn Abdul; DONOVAN, DAVID - Animal Health Institute; SIRAGUSA, GREGORY - Agtech Products, Inc; Seal, Bruce

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2010
Publication Date: 9/8/2010
Citation: Simmons, I.M., Donovan, D.M., Siragusa, G.R., Seal, B.S. 2010. Recombinant expression of two bacteriophage proteins that lyse clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal domain. Journal of Agricultural and Food Chemistry. 58(19):10330-10337.

Interpretive Summary: The bacterium Clostridium perfringens is the third leading cause of bacterial food-borne disease among humans and is a leading cause of gastrointestinal diseases among poultry during production. Countries that have complied with the ban on using antimicrobial growth promoters (AGP) in animal feeds have had increased incidences of C. perfringens-associated gastrointestinal disease in poultry during production. Also the increase in antibiotic resistant bacteria has lead to concerns on how to control these organisms. To address these issues we are identifying new antimicrobial agents, putative lytic enzymes from the genomes of bacteriophages. Consequently bacteriophages that lyse and kill Clostridium perfringens were screened utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Analyses of the bacteriophage DNA genomes demonstrated that the bacterial viruses had genes encoding proteins potentially capable of lysing the host bacterium Clostridium perfringens. Two putative phage lysin (ply) genes from the clostridial phages phiCP39O and phiCP26F were cloned, expressed in E. coli and the resultant proteins were purified to homogeneity. Both recombinant lytic enzymes were capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays. The complete lysis of the pathogenic bacterium was also visualized with the light microscope. Also of importance is that these enzymes are highly species specific and did not kill other members of the clostridial bacteria family or other beneficial bacteria. Hence the bacteriophage enzymes can be directed to kill specific pathogenic bacteria and do not have the more generalized effect of most antibiotics by potentially having a negative effect on other beneficial microbes.

Technical Abstract: Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins that are responsible for disease symptoms and pathogenesis in a variety of animals, humans and poultry. The organism is the third leading cause of human food-borne bacterial disease and C. perfringens is the presumptive etiologic agent of necrotic enteritis among chickens which in the acute form can cause increased mortality among broiler flocks. Countries that have complied with the ban on antimicrobial growth promoters (AGP) in feeds have had increased incidences of C. perfringens-associated necrotic enteritis in poultry. To address this issue, we are identifying new antimicrobial agents, putative lytic enzymes from the genomes of bacteriophages. Two putative phage lysin (ply) genes from the clostridial phages phiCP39O and phiCP26F were cloned, expressed in E. coli and the resultant proteins were purified to homogeneity. Gene and protein sequencing revealed that the predicted and chemically determined amino acid sequences of the two recombinant proteins were identical in the C-terminus cell-wall binding domain, but only 55 per cent identical to each other in the N-terminal catalytic domain. Both recombinant lytic enzymes were capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays. The observed reduction in turbidity was correlated with up to a 3 log cfu/ml reduction in viable C. perfringens on brain heart infusion agar plates. However other members of the clostridia were resistant to the enzymes by both assay methods.