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Title: Expression of Two Bacteriophage Enzymes that Lyse Clostridium perfringens which Share Sequences in the Cell Binding Domain of the Molecules but are Dissimilar in their Catalytic Enzymatic Domain

Author
item Simmons, Ibn Abdul
item SIRAGUSA, GREGORY - Agtech Products, Inc
item Donovan, David
item Seal, Bruce

Submitted to: International Symposium on Antimicrobial Peptides
Publication Type: Abstract Only
Publication Acceptance Date: 3/12/2009
Publication Date: 6/17/2009
Citation: Simmons, I.M., Siragusa, G., Donovan, D.M., Seal, B.S. 2009. Expression of Two Bacteriophage Enzymes that Lyse Clostridium perfringens which Share Sequences in the Cell Binding Domain of the Molecules but are Dissimilar in their Catalytic Enzymatic Domain. International Symposium on Antimicrobial Peptides.

Interpretive Summary:

Technical Abstract: Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins which are responsible for disease symptoms and pathogenesis in a variety of animals, humans and poultry. The organism is the third leading cause of food-borne bacterial disease among humans. In chickens C. perfringens is the presumptive etiologic agent of necrotic enteritis which can cause increased mortality among broiler flocks in its acute form. The sub-clinical form can lead to decreased digestion and reduced weight gain due to damage of the intestinal mucosa. Although C. perfringens can be controlled by antibiotics, there is increasing pressure to ban growth promoting antimicrobials in poultry. This ban has yielded an increased incidence of C. perfringens-associated necrotic enteritis in poultry from countries that have curtailed the use of antibiotic growth promoters in feed. To address this need, we are identifying new antimicrobial agents, putative lytic enzymes from the genomes of bacteriophages. Two putative phage lysin (ply) genes from phiCP39O and phiCP26F were cloned, expressed in E. coli and the resultant proteins were purified to homogeneity. Gene and protein sequencing revealed that the actual and predicted amino acid sequences of the two recombinant proteins were identical at the C-terminus (cell-wall binding domain), but only 55 per cent similar to each other at 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 turbidity reduction assays. This reduction in turbidity was accompanied by up to a 3 log cfu/ml reduction in viable C. perfringens on BHI (brain heart infusion) agar plates. Both enzymes had activity that was restricted to C. perfringens but did not lyse other members of the clostridia.