Flexible Peptide Linkers Enhance the Antimicrobial Activity of Surface-Immobilized Bacteriolytic Enzymes

Abstract

Chemical linkers are frequently used in enzyme immobilization to improve enzyme flexibility and activity, whereas peptide linkers, although ubiquitous in protein engineering, are much less explored in enzyme immobilization. Here, we report peptide-linker-assisted noncovalent immobilization of the bacteriolytic enzyme lysostaphin (Lst) to generate anti-Staphylococcus aureus surfaces. Lst was immobilized through affinity tags onto a silica surface (glass slides) and nickel nitrilotriacetic acid (NiNTA) agarose beads via silica-binding peptides (SiBPs) or a hexahistidine tag (Histag) fused at the C-terminus of Lst, respectively. By inserting specific peptide linkers upstream of the SiBP or His-tag, the immobilized enzymes killed >99.5% of S. aureus ATCC 6538 cells (10 8 CFU/mL) within 3 h in buffer and could be reused multiple times without significant loss of activity. In contrast, immobilized Lst without a peptide linker was less active/stable. Molecular modeling of Lst−linker−affinity tag constructs illustrated that the presence of the peptide linkers enhanced the molecular flexibility of the proximal Lst binding domain, which interacts with the bacterial substrate, and such increased flexibility correlated with increased antimicrobial activity. We further show that Lst immobilized onto NiNTA beads retained the ability to kill ∼99% of a 10 8 CFU/mL microbial challenge even in the presence of 1% of a commercial anionic surfactant, C12-14 alcohol EO 3:1 sodium sulfate, when the Lst construct contained a decapeptide linker containing glycine, serine, and alanine residues. This linker-assisted immobilization strategy could be extended to an unrelated lytic enzyme, the endolysin PlyPH, to target Bacillus anthracis Sterne cells either in buffer or in the presence of anionic surfactants. Our approach, therefore, provides a facile route to the use of antimicrobial enzymes on surfaces.

Reducing Staphylococcus aureus resistance to lysostaphin using CRISPR‐dCas9

“…The expression and purification of Lst was similar to previously reported protocols (X. Wu, Fraser, et al, ; X. Wu, Kwon, et al, ). Briefly, the lst gene purchased from Genscript was cloned into pGS21a between Nde I and Xho I, and transformed into E. coli BL21 Star (DE3) cells for expression.…”

“…After dialysis against phosphate‐buffered saline (PBS; pH 7.4), Lst was sterilized through a 0.22 μm filter and stored at 4°C. The peptidoglycan‐binding domain of Lst (LBD) with an N‐terminal enhanced green fluorescence protein (EGFP) fusion (EGFP‐LBD; X. Wu, Fraser, et al, ; X. Wu, Kwon, et al, ) was expressed and purified in the same way as Lst, except that protein expression was induced for 16 hr in E. coli BL21 (DE3).…”

“…EGFP‐LBD binding onto the S. aureus cell surface was measured using a modified method of a previously reported protocol (X. Wu, Kwon, et al, ). S. aureus ATCC 6538 overnight culture was sub‐cultured at a 1:50 dilution factor into 3 ml TSB, 3 ml TSB + 3 µl dimethyl sulfoxide (DMSO), or 3 ml TSB + 3 µl of tunicamycin (0.25 mg/ml stock dissolved in DMSO), and grown at 37°C with 220 rpm shaking.…”

“…The activity of Lst on isolated cell wall fragments was measured based on a previously reported turbidity assay (X. Wu et al, ; X. Wu, Kwon, et al, ). Briefly, 20 μl of isolated cell wall fragments and 10 μl of Lst (30 μg/ml) were mixed with 170 μl of PBS or TSB, and the turbidity decrease was monitored on a Molecular Devices plate reader at 600 nm for 10 min with 3 s intervals.…”

“…However, some bacteria are resistant to lytic enzymes under certain circumstances, such as in the presence of human serum or industrial emulsifiers, although the enzymes retain full catalytic activity under these conditions (Osipovitch, Therrien, & Griswold, ; Proenca et al, ; Proença, Leandro, Garcia, Pimentel, & São‐José, ; J. A. Wu, Kusuma, Mond, & Kokai‐Kun, ; X. Wu, Fraser, Zha, & Dordick, ; X. Wu, Paskaleva, Mehta, Dordick, & Kane, ).…”