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

Abstract

Lysostaphin (Lst) is a potent bacteriolytic enzyme that kills , a common bacterial pathogen of humans and animals. With high activity against both planktonic cells and biofilms, Lst has the potential to be used in industrial products, such as commercial cleansers, for decontamination. However, Lst is inhibited in the presence of monoethanolamine (MEA), a chemical widely used in cleaning solutions and pharmaceuticals, and the underlying mechanism of inhibition remains unknown. In this study, we examined the cell binding and killing capabilities of Lst against ATCC 6538 in buffered salt solution with MEA at different pH values (7.5 to 10.5) and discovered that only the unprotonated form of MEA inhibited Lst binding to the cell surface, leading to low Lst activity, despite retention of its secondary structure. This reduced enzyme activity could be largely recovered via a reduction in wall teichoic acid (WTA) biosynthesis through tunicamycin treatment, indicating that the suppression of Lst activity was dependent on the presence and amount of WTA. We propose that the decreased cell binding and killing capabilities of Lst are associated with the influence of uncharged MEA on the conformation of WTA. A similar effect was confirmed with other short-chain alkylamines. This study offers new insight into the impact of short-chain alkylamines on both Lst and WTA structure and function and provides guidance for the application of Lst in harsh environments. Lysostaphin (Lst) effectively and selectively kills , the bacterial culprit of many hospital- and community-acquired skin and respiratory infections and food poisoning. Lst has been investigated in animal models and clinical trials, industrial formulations, and environmental settings. Here, we studied the mechanistic basis of the inhibitory effect of alkylamines, such as monoethanolamine (MEA), a widely used chemical in commercial detergents, on Lst activity, for the potential incorporation of Lst in disinfectant solutions. We have found that protonated MEA has little influence on Lst activity, while unprotonated MEA prevents Lst from binding to cells and hence dramatically decreases the enzyme's bacteriolytic efficacy. Following partial removal of the wall teichoic acid, an important component of the bacterial cell envelope, the inhibitory effect of unprotonated MEA on Lst is reduced. This phenomenon can be extended to other short-chain alkylamines. This mechanistic report of the impact of alkylamines on Lst functionality will help guide future applications of Lst in disinfection and decontamination of health-related commercial products.

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).…”

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

“…The resulting plasmid pGS21a−Lst was PCR-amplified using the Quick-Change Mutagenesis kit (Agilent Technologies) with primers Lst−NL-F/R (1 and 2 for two PCR reactions) and Lst−GSA-F/R to obtain plasmids pGS21a−Lst−NL and pGS21a−Lst−GSA. The Lst binding domain (LBD) was fused to EGFP on the pCDFDuet vector as previously reported, 34 forming the pCDFDuet−EGFP−LBD plasmid.…”

“…Protein purification was performed as previously reported. 34 Briefly, cells were thawed and sonicated and the His-tagged proteins of interest were purified with NiNTA agarose beads. The purified enzymes were then dialyzed against phosphate-buffered saline (PBS, pH 7.4) (or PBS supplemented with additional 150 mM NaCl for SiBP-containing constructs) with a dilution factor of >10 000 000, sterilized with 0.22 μm filters (EMD Millipore), and stored at 4 °C.…”

“…Lst was essentially inactive in the presence of C12 (Figure 7a). Circular dichroism analysis revealed that the typical random coil spectrum of native Lst in aqueous buffer 34 was shifted to a predominantly β-sheet spectrum (Figure 7b) in the presence of 0.2% C12, indicating significant structural change of the enzyme but not complete denaturation. Thus, it is consistent with the low, yet nonzero, enzyme activity in low concentrations of C12.…”