Title: Antimicrobials for staphylococcal pathogens that are refractory to resistance development Authors
|Foster Frey, Juli|
|Almeida, Raul -|
|Marriott, Ian -|
|Dong, Shengli -|
|Pritchard, David -|
Submitted to: International Symposium on Antimicrobial Peptides
Publication Type: Abstract Only
Publication Acceptance Date: April 20, 2009
Publication Date: June 17, 2009
Citation: Becker, S.C., Foster Frey, J.A., Willard, R.R., Lease, R.A., Almeida, R., Marriott, I., Dong, S., Pritchard, D.G., Donovan, D.M. 2009. Antimicrobials for staphylococcal pathogens that are refractory to resistance development. International Symposium on Antimicrobial Peptides. Interpretive Summary: C. Problem— Bovine mastitis results in annual costs between $1.7 billion and $2 billion in the United States only. One of the most relevant causative agents of mastitis is Staphylococcus aureus. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Therefore, alternative antimicrobials for the treatment of mastitis are needed. C. Accomplishment-- The principle of domain shuffling for creation of optimized antimicrobials based on modular peptidoglycan hydrolases has been demonstrated with bacteriophage endolysins directed against bacteria of the genus Listeria. The cell wall binding domains of selected endolysins were characterized regarding their binding properties, and functional modules were deliberately combined, yielding fusion proteins with broader binding ranges, higher affinities to the bacterial cell wall, and increased lytic activities against the target bacteria. C. Contribution of Accomplishment to Solving the Problem-- These findings provide new evidence that optimized antimicrobials can be created by molecular engineering of modular peptidoglycan hydrolases, and suggest successful applicability of domain shuffling also against other Gram-positive bacteria such as Staphylococcus aureus. Characterization of staphylococcal and phage derived SH3b domains will contribute to identifying functional modules that can enhance lytice activity of heterologous fusion constructs against this mastitis causing pathogen.
Technical Abstract: Bacteriophages are viruses exclusively infecting bacteria and therefore offer suitable tools for their detection and control. At the end of their multiplication cycle, most phages lyse their hosts from within by means of an endolysin (peptidoglycan hydrolase), thereby enabling release of the phage progeny. In case of Gram-positive bacteria which lack an outer membrane, these enzymes also work when applied from without. Endolysins from a Gram-positive background show a modular architecture, consisting of one or more enzymatically active domains (EADs), which degrade the bacterial peptidoglycan, and often a cell wall binding domain (CBD), which directs the enzyme to its substrate and confers specificity for the target cells. This modular organization allows creation of proteins with new properties for detection and control of pathogens by artificial recombination of functional domains. Fusing CBDs from endolysins specific for Listeria, a bacterial genus comprising human and animal pathogens, to fluorescent proteins such as the GFP (green fluorescent protein) made it possible to determine the binding specificities and affinities of these domains to the cell wall, and yielded tools for detection and differentiation of Listeria strains in mixed bacterial cultures. By combination of selected CBDs within heterologous fusion constructs proteins with extended binding ranges and up to fifty times higher affinities were created. Furthermore, the deliberate combination of EADs and CBDs resulted in enzymes with up to threefold higher lytic activity against certain Listeria strains. These results with Listeria phage endolysins suggest that the principle of domain shuffling could also be successfully applied to enzymes active against other Gram-positive organisms. In our current research, we focus on phage endolysins and bacterial peptidoglycan hydrolases directed against Staphylococcus aureus, one of the most relevant causative organisms of bovine mastitis, a disease which results in annual losses of up to $2 billion in the United States only. Based on sequence homology, over fifty enzymes of staphylococcal and phage origin were classified into five highly repetitive groups and one group of stand alone proteins for which no homologies were found.