|FILATOVA, LYUBOV - LOMONOSOV UNIVERSITY|
|ISHNAZAROVA, NADIYA - LOMONOSOV UNIVERSITY|
|Foster Frey, Juli|
|PUGACHEV, VLADIMIRE - FEDERAL STATE BUDGETARY SCIENTIFIC INSTITUTION "RESEARCH INSTITUTE OF EXPERIMENTAL AND CLINICAL MEDICINE"|
|DMITRIEVA, NATALIA - MOSCOW STATE UNIVERSITY|
|KLYACHKO, NATALIA - LOMONOSOV UNIVERSITY|
Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2015
Publication Date: 5/11/2016
Citation: Filatova, L.Y., Donovan, D.M., Ishnazarova, N., Foster Frey, J.A., Becker, S.C., Pugachev, V.G., Dmitrieva, N.F., Klyachko, N.L. 2016. A chimeric LysK-lysostaphin fusion enzyme lysing Staphylococcus aureus cells: A study of both kinetics of inactivation and specifics of interaction with anionic polymers. Enzyme and Microbial Technology. doi: 10.1007/s12010-016-2115-7.
Interpretive Summary: Due to the high incidence of drug-resistant bacteria, there is a need for to identify or create antimicrobials (agents that kill microorganisms) towards different strains of the Staphyloccocus bacterium that are highly refractory to resistance development. In our research a putative antimicrobial was engineered by isolating distinct biological activities from multiple enzymes involved in the killing of bacteria and fusing them together; the fusion enzyme was shown to be highly refractory to resistance development. For therapeutic applications, it is important to characterize the conditions under which the fusion enzyme has optimal activity and devise a way to conceal the protein from the human immune system, such that it does not result in an adverse immune response. Optimal conditions for the fusion enzyme’s activity and optimal storage were determined. Approaches were also determined that are predicted to help the fusion enzyme avoid initiating an immune response while maintaining high antimicrobial activity. Further studies will be carried out to investigate the application of this fusion enzyme as an effective antimicrobial agent.
Technical Abstract: A staphylolytic fusion protein (K-L) was created, harboring three unique lytic activities comprised of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of K-L was investigated. As a protein antimicrobial with potential antigenic properties, the biophysical effect of including K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. K-L has a high lytic activity under the following optimal (opt) conditions: pHopt 6.0-10.0, Topt 20-30°C, and NaClopt 400-800 mM. At the functioning temperature of 37°C K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7±3.0 hrs. At storage temperatures of 22°C and 4°C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the K-L inactivation. The optimal storage conditions under which the enzyme retains 100% activity after 140 days of incubation (4°C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. K-L included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol maintains enzyme activity and stability, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.