Triple-acting lytic enzyme treatment of drug-resistant and intracellular Staphylococcus aureus

Authors: BECKER, STEPHEN - US Department Of Agriculture (USDA); Roach, Dwayne; CHAUHAN, VINITA - University Of North Carolina; SHEN, YANG - University Of Maryland; Foster Frey, Juli; Powell, Anne; BUCHANNAN, GARY - US Department Of Agriculture (USDA); LEASE, RICHARD - US Department Of Agriculture (USDA); Mohammadi, Homan; Harty, William; Simmons, Chad; Schmelcher, Mathias; Camp, Mary; SHIELDS, KELLY - Brigham & Women'S Hospital; DOWD, MEGAN - Harvard Medical School; DONG, SHENGLI - University Of Alabama; BAKER, JOHN - University Of Alabama; SHEEN, TAMSIN - San Diego State University; DORAN, KELLY - San Diego State University; PRITCHARD, DAVID - University Of Alabama; ALMEIDA, RAUL - University Of Tennessee; NELSON, DANIEL - University Of Maryland; MARRIOTT, IAN - University Of North Carolina; LEE, JEAN - Brigham & Women'S Hospital; Donovan, David

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/11/2016
Publication Date: 4/28/2016
Citation: Becker, S.C., Roach, D.R., Chauhan, V., Shen, Y., Foster Frey, J.A., Powell, A.M., Buchannan, G., Lease, R., Mohammadi, H., Harty, W.J., Simmons, C.B., Schmelcher, M., Camp, M.J., Shields, K., Dowd, M., Dong, S., Baker, J.R., Sheen, T.R., Doran, K.S., Pritchard, D.G., Almeida, R., Nelson, D.C., Marriott, I., Lee, J.C., Donovan, D.M. 2016. Triple-acting lytic enzyme treatment of drug-resistant and intracellular Staphylococcus aureus. Scientific Reports. 6:25063.

Interpretive Summary: Staphylococcus aureus (S. aureus) is a notorious pathogen for both animal and human health with multi-drug resistant strains highly prevalent. This pathogen has also evolved another way to evade antibiotic treatment by invading and residing intracellularly within animal cells. New treatments that reduce resistance development and tackle intracellular pathogens are sorely needed. We have identified three enzyme activities that cut the cell wall structural element (peptidoglycan) of S. aureus in three unique regions. Any one of the three enzymatic activities can kill the cells when exposed externally. When fused these three enzyme domains maintain each of their activities in the final triple-acting fusion. We have shown that these triple-acting enzyme constructs greatly reduce resistance development compared to the parental enzymes alone and are effective at reducing S. aureus nasal colonization 97%. We also show that when these enzymes (single, double or triple domain harboring) are fused to protein transduction domains, they can enter animal cells and reduce the intracellular load of the pathogen up to 97% in three different cell types from three different species. The effect is maintained in two different animal models (osteomyelitis and mastitis of mice) as well as staphylococcal biofilms. The findings of this work identify the necessary components and methodology for generating multiple domain fusions with elements to address intracellular and multi-drug resistant pathogens. These findings will aid scientists in the development of these and similar molecules as commercializable antimicrobials for use in animal production, animal and human health and food safety.

Technical Abstract: Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over-used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein, effectively reducing the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of the triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured cells and in mouse models of staphylococcal mastitis and osteomyelitis. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics.