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 2: Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources. Progress on this project focuses on Problem Statement 2E: Improved Techniques for Genetic Modification and Genetic Engineering of Food Animals. There has been significant advancement on this project to help cure chronic mastitis, an infection of the mammary gland. Mastitis is a costly disease for dairy farming in the United States with over $2 billion in losses to the industry annually. Progress has been made in developing a system for analyzing the eradication of bacterial pathogens residing within cultured bovine mammary gland cells. Antimicrobial enzymes harboring three unique activities that degrade the bacterial cell wall in three unique regions have been created and were fused to 11 different protein fragments (protein transduction domains) that facilitate transport of the antimicrobial enzymes across the cell wall. These triple-acting enzyme fusions were tested and optimal fusions for eradicating pathogens from within mammary cells in culture were identified. Due to solubility problems, the optimal triple-acting enzyme was not able to be produced in sufficiently high amounts and concentrations for injection into the teat canal of cattle infected with the mastitis causing bacteria Staphylococcus aureus. Thus, a suboptimal triple-acting enzyme was introduced into the infected glands; however it was inadequate for eradicating chronic bacterial infections. This work has helped to identify solubility, activity and concentration requirements when creating antimicrobials to cure chronic multi-drug resistant pathogens and other drug resistant Staphylococcus aureus infections, such as osteomyelitis (bone infections) in humans.