Location: Molecular Plant Pathology Laboratory
Project Number: 8042-22000-318-001-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Aug 1, 2019
End Date: Jul 31, 2023
Objective:
Clostridium perfringens (Cp) is a gut commensal that is also a causative agent of necrotic enteritis (NE), a significant problem to the poultry, pig and beef industry. The disease can manifest as an acute or chronic enterotoxaemia, resulting in significant levels of mortality, and the chronic disease leads to poor health and lower body weight. Alternative antimicrobials to fight this disease organism are sorely needed. Bacteriophage lytic enzymes (Ply) (peptidoglycan hydrolases; PGHs) degrade the major structural component of the bacterial cell wall (peptidoglycan; PG) and can kill Gram positive bacteria (Cp) via osmolysis, either when synthesized by the phage inside the bacteria (during the phage lytic cycle) or when recombinant enzymes are synthesized /purified and exposed to the bacteria externally. The central hypothesis of this proposal is Cp-specific lytic enzymes (bacteriophage endolysins), fed to chickens will reduce intestinal carriage of Cp. A major concern with protein antimicrobials in animal feed, is the harsh conditions of animal feed production (90' x 15 min) and the gut environs (acid pH and proteases). To overcome these hurdles, we have expressed lytic enzymes in both yeast (Saccharomyces cerevisiae) and plants (Nicotiana benthamiana) to serve as carrier organisms for delivery of the enzyme to the lower gut. ARS will perform preliminary feeding trials in chickens, separately with transiently expressing PlyCP41 plants, and transiently expressing PlyCP41 yeast, and assay for the ability of these feed additives to reduce Cp gut carriage via monitoring of feces and gut contents for a reduction of a Cp inoculum strain. We predict that the intracellular milieu within these carrier organisms (e.g. naturally occurring chaperone proteins in yeast or N. bethamiana) will help protect the enzyme from denaturation or degradation allowing them to reduce the lower gut carriage of Cp. ARS Beltsville, MD has the expertise to produce the PlyCP41 protein in plants using a plant virus-based transient vector system; ARS Peoria, IL is producing PlyCP41 in transgenic yeast; University of Maryland Eastern Shore is expert in chicken feeding, and Morgan State University has background in microbiology.
Approach:
ARS will develop optimal experimental methods to produce phage endolysins in Nicotiana benthamiana plants using plant virus-based transient expression vectors, develop methods to preserve functionally active endolysins in N. benthamiana plant tissue or in plant extracts, and provide sufficient quantities of plant-produced endolysin materials for poultry feeding trials in cooperation with collaborators at the University of Maryland Eastern Shore, Morgan State University, and USDA ARS in Peoria, IL.
