F1-3961. Multiple Domain Fusion Proteins as Targeted Antimicrobials for Gram-positive Pathogens
Session: Poster Session: New Classes of Membrane-Active or Cell Wall Targeting Agents
Tuesday, October 28, 2008: 12:00 AM
Room: Hall C
Background: MRSA is an important MDRP. One approach to combating MRSA uses phage endolysins. These peptidoglycan (PG) hydrolases lyse bacteria by degrading cell wall PG with near species-specificity. No bacteria have been identified that can develop resistance to their phage lysins. Lysins are modular, often with two lytic domains and a cell wall binding (CWB) domain. PG hydrolase fusions maintain both parental lytic activities. Methods: LysK, a phage endolysin with two lytic domains lyses many staphylococcal strains including MRSA. Lysostaphin, a bacteriocin secreted by S. simulans, is also a potent MRSA antimicrobial. Fusing LysK to lysostaphin creates a chimeric enzyme with three active lytic domains as determined by analysis of PG peptide digestion products. Fusion enzymes are stable enough for topical agent tests. Similarly, replacing the λSa2 streptococcal phage lysin Cpl-7 CWB domains with the lysostaphin SH3b CWB domain, allows the weakly staphylo-lytic λSa2 endopeptidase domain to increase its lysis of S. aureus 10-fold. Evaluation of these two classes of lytic fusion proteins includes the following stability assessments: across a relevant pH range, photostability, and in carrier media relevant for topical and/or systemic use. Results: Multi-domain fusion proteins were produced that kill S. aureus, and streptococci. A range of MIC values was obtained for S. aureus [lab strains and MRSA (USA300)], LysK (94 -33 ug/ml), lysostaphin (0.1- 0.3 ug/ml) and a LysK-lysostaphin fusion (23.4 µg/ml). Conclusions: These results confirm a proof of concept and indicate the potential utility of lytic fusion proteins as a highly targeted topical and/or systemic intervention against HA- and CA-MRSA as well as streptococcal pathogens. The domains utilized determine the antibacterial spectrum, and in theory could target any Gram positive species, increasing the versatility of such fusion proteins.
Anton Leighton, MD1, David Pritchard, PhD2, David Donovan, PhD3, Hans Hager, MD4, John Baker, PhD2, Shengli Dong2, Steve Becker, PhD3 and  A. Leighton,
AdRem Role(s): Shareholder (excluding diversified mutual funds)., (1)Exponential Biotherapies, (2)Biochem. & Mol. Genetics, UAB, (3)ABBL, ARS, USDA, (4)CC-ID, Anchorage, AK


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