1a. Objectives (from AD-416)
Create triple-lytic-domain PG hydrolase-PTD fusions and test in cultured cells. Create triple-lytic-domain-PTD fusions via molecular techniques. Verify that the triple-lytic-domain-PTD fusions maintain three enzymatic activities. Test efficacy of the triple-lytic-domain-PTD fusions against extracellular staphylococci (S. aureus and CoNS) in broth cultures and against internalized S. aureus in cultured bovine mammary epithelial cells. Test efficacy of the Lyso-PTD fusion protein to cure chronic bovine mastitis. Induce S. aureus mastitis in cows and evaluate the triple-lytic-domain-PTD fusion for the ability to cure chronic mastitis via intramammary injections at dry off. Determine if the triple-lytic-domain-PTD kills staphylococci that reside in milk somatic cells. Characterize S. aureus isolates from this study that are resistant to the triple-lytic-domain-PTD. Determine if triple-lytic-domain-PTD fusion can kill staphylococci within mammary abscesses. Examine proteome differences between S. aureus grown in ex vivo milk vs. within chronically infected quarters.
1b. Approach (from AD-416)
Identify pregnant lactating cattle for purchase, house and monitor lactating cattle, infect them with S. aureus, monitor for chronic status of infection, dry-off the cattle, inject the cattle with the antimicrobial fusion protein during dry-off period, birth the calves, and monitor for eradication of pathogen in colostrum and milk following birth.
3. Progress Report
This work supports the National Program 101 mission statement in the area of developing information, tools, and technologies that can be used to improve animal production systems. Significant progress was made on Component 1: Understanding, improving, and effectively using animal genetic and genomic resources. Progress on this project focuses on Problem 1A, the need for developing genome-enabling tools and reagents for livestock (pig and cattle). These tools will not only be useful for traditional animal production research applications (reproduction, growth and development, nutrient intake and utilization, product quality), but will also be used to decrease the environmental footprint of animal production, improve animal health, well-being and resistance to disease, and enhance food safety. There has been significant advancement on this project to help cure chronic mastitis. Mastitis is a costly disease to the U.S. dairy farming with over $2 billion in losses to the industry annually. Progress has been made in developing a system for analyzing intracellular uptake and eradication of staphylococcus bacteria residing within mammary epithelial cells. Two unique staphylolytic enzyme antimicrobials harboring three enzymatic activities that destroy the bacterial cell wall have been created and are each being fused to 11 different protein domains that allow the enzymes to be transduced across the cell membrane. This work will help to create antimicrobials to cure chronic methicillin resistant staphylococcus aureus and other drug resistant staphylococcal infections. Activity on this grant is monitored through e-mails, conference calls, and in-house meetings with staff.