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. This effectively reduces 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 a 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 mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics.
Highlights
Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of diseases including mastitis, osteomyelitis, and endocarditis[1]
There is a need for antimicrobials that are refractory to resistance development and can kill intracellular multi-drug resistant (MDR) bacteria
LysK and lysostaphin are known to be inactive against Gram negative bacteria, but show strong lytic activity against antibiotic-sensitive and antibiotic-resistant S. aureus and Coagulase negative strains (Supplementary Table 1), and in combination demonstrate synergy in killing S. aureus cells[13]
Summary
After 10 rounds of sub-lethal exposure in liquid culture, parental LysK (two lytic domains) and lysostaphin (one lytic domain) yielded cultures with 42-fold and 585-fold increases in MICs, respectively. Parental enzymes (lysostaphin, LysK) and triple-acting fusions K-L and L-K were each modified by addition of 11 different C-terminal PTD sequences (Table 1; schematic Fig. 1A) and were tested for their ability to reduce intracellular S. aureus in multiple cultured cells known to support S. aureus intracellular invasion (Fig. 2A,B and C). The ability of LysK or L-K fusion to eradicate intracellular S. aureus in MAC-T cells was inhibited by the addition of a PTD (Fig. 2A). Triple-acting fusion K-L reduced the intracellular bacteria recovered from either MAC-T cells or murine osteoblasts (mOB), and this effect was not significantly enhanced with the addition of a PTD (Fig. 2A,C)
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