A Chimeric LysK-Lysostaphin Fusion Enzyme Lysing Staphylococcus aureus Cells: a Study of Both Kinetics of Inactivation and Specifics of Interaction with Anionic Polymers

Abstract

A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed 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 chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal () conditions: pH 6.0-10.0, t 20-30 °C, NaCl 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme 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. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.

Are Phage Lytic Proteins the Secret Weapon To Kill Staphylococcus aureus ?

“…The interactions between the protein and the polymers can break down enzyme aggregates, increasing the lytic activity and keeping full activity for at least 4 months ( 74 ). Similar results were obtained for the chimeric protein K-L (containing the LysK CHAP endopeptidase and amidase domains, as well as the lysostaphin glycyl-glycine endopeptidase domain), which showed increased stability in the presence of block copolymers of poly- l -glutamic acid and polyethylene glycol ( 75 ). On the other hand, incorporation of the lytic proteins into these polymers has the advantage of reducing their immunogenicity by hindering recognition by the immune system.…”

“…On the other hand, incorporation of the lytic proteins into these polymers has the advantage of reducing their immunogenicity by hindering recognition by the immune system. This also prevents the inactivation of the protein caused in some cases by the polyethylene glycol used to reduce protein immunogenicity ( 75 ).…”

Unprotonated Short-Chain Alkylamines Inhibit Staphylolytic Activity of Lysostaphin in a Wall Teichoic Acid-Dependent Manner

“…Lst (246 amino acids) consists of an N-terminal catalytic domain (amino acids 1 to 137) and a C-terminal substrate-binding domain (amino acids 154 to 246) joined by a flexible linker (amino acids 138 to 153) (13,14). Each domain is functional on its own and can be fused with other protein domains to generate novel chimeric enzymes (15)(16)(17)(18). Attributed to its superior activity and specificity, and in addition to its formulation into an antibacterial paint (11), Lst has been exploited for integration into end products in health-related industries, such as in personal/pet care products, either as a direct treatment of S. aureus infections or for preservation of the products from bacterial contamination over long-term storage and use.…”

Engineering of the CHAPk Staphylococcal Phage Endolysin to Enhance Antibacterial Activity against Stationary-Phase Cells

“…Nevertheless, resistance to lysostaphin has been reported [ 29 , 30 ]. Previous studies demonstrated how the SH3b cell-binding domain of lysostaphin could improve the lytic activity of staphylococcal lysins [ 12 , 27 , 31 , 32 , 33 , 34 ]. The SH3b cell-binding domain of lysostaphin possesses an unusual binding mechanism that allows a synergistic and structurally dynamic recognition of S. aureus peptidoglycan, as the pentaglycine cross-bridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain [ 35 ].…”