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

Abstract

Bacteriophage endolysins and their derivatives have strong potential as antibacterial agents considering the increasing prevalence of antibiotic resistance in common bacterial pathogens. The peptidoglycan degrading peptidase CHAPk, a truncated derivate of staphylococcal phage K endolysin (LysK), has proven efficacy in preventing and disrupting staphylococcal biofilms. Nevertheless, the concentration of CHAPk required to eliminate populations of stationary-phase cells was previously found to be four-fold higher than that for log-phase cells. Moreover, CHAPk-mediated lysis of stationary-phase cells was observed to be slower than for log-phase cultures. In the present study, we report the fusion of a 165 amino acid fragment containing CHAPk with a 136 amino acid fragment containing the cell-binding domain of the bacteriocin lysostaphin to create a chimeric enzyme designated CHAPk-SH3blys in the vector pET28a. The chimeric protein was employed in concentrations as low as 5 μg/mL, producing a reduction in turbidity in 7-day-old cultures, whereas the original CHAPk required at least 20 μg/mL to achieve this. Where 7-day old liquid cultures were used, the chimeric enzyme exhibited a 16-fold lower MIC than CHAPk. In terms of biofilm prevention, a concentration of 1 μg/mL of the chimeric enzyme was sufficient, whereas for CHAPk, 125 μg/mL was needed. Moreover, the chimeric enzyme exhibited total biofilm disruption when 5 μg/mL was employed in 4-h assays, whereas CHAPk could only partially disrupt the biofilms at this concentration. This study demonstrates that the cell-binding domain from lysostaphin can make the phage endolysin CHAPk more effective against sessile staphylococcal cells.

Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity

“…According to previous reports, some chimeric proteins exhibit increased activity compared to their parental endolysins ( Daniel et al, 2010 ; Mao et al, 2013 ; Rodríguez-Rubio et al, 2013 ; Arroyo-Moreno et al, 2021 ; Lee et al, 2021 ) or also show the ability to penetrate eukaryotic cells and kill intracellular bacteria, when fused to a cell penetrating peptide ( Röhrig et al, 2020 ). Following a stepwise funnel approach combining turbidity reduction, MIC, time-kill, and biofilm assays, we eventually identified a leading candidate, LysRODIΔAmi, which contains the CHAP and CBD domains from LysRODI but lacks the amidase domain.…”

“…Protein engineering based on the shuffling of the enzymatic modules of phage lytic proteins has been proven to be an efficient strategy to improve their lytic activity or stability compared to the natural proteins from where the domains are derived (Mao et al, 2013;Yang et al, 2014;Arroyo-Moreno et al, 2021;Lee et al, 2021), when used alone or in combination with antibiotics like oxacillin (Daniel et al, 2010) or bacteriocins like lysostaphin (Schmelcher et al, 2012b). Recently, a third generation of antimicrobials against S. aureus has been developed by immobilizing the engineered protein AuresinePlus (enzymatic activity domain from the staphylococcal autolysin LytM fused to the SH3b of lysostaphin) into a poly(lactideco-glycolide)/chitosan matrix to be used as wound dressing (Urbanek et al, 2021).…”

Lysostaphin: Engineering and Potentiation toward Better Applications

“…The SH3b domain of lysostaphin has been fused to various lytic enzymes or their catalytic domains (Figure A) to increase the overall catalytic efficiency, − to alter specificity, , or to change the compatibility of the enzyme with different pH and salt concentrations . These chimeric enzymes are less active than lysostaphin, albeit more active than the lytic enzymes that donate their catalytic domains for fusion engineering.…”

LYZ2-SH3b as a novel and efficient enzybiotic against methicillin-resistant Staphylococcus aureus

“…Due to the challenges mentioned above, in the last decade, researchers have paid special attention to the use of peptidoglycan hydrolases (PGHs) generated by bacteriophages rather than the direct use of phages. Numerous investigations have revealed that these enzymes are antibacterial against gram-positive pathogens [ 8 – 14 ]. Through the specific destruction of the PG layer in the bacterial cell wall, these enzymes cause bacteria lysis and death.…”

“…Numerous studies have been conducted on the role and production of chimeric endolysins in fighting S. aureus bacteria [ 3 , 8 , 9 , 18 ]. Meanwhile, few studies have focused on using exolysins as enzybiotic agents comparable to endolysins.…”