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

Lysostaphin (Lst) is a potent bacteriolytic enzyme that kills Staphylococcus aureus, 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 S. aureus 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.IMPORTANCE Lysostaphin (Lst) effectively and selectively kills Staphylococcus aureus, 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 S. aureus 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.

Inhibition of the activity of both domains of lysostaphin through peptidoglycan modification by the lysostaphin immunity protein.

Resistance to lysostaphin, a staphylolytic glycylglycine endopeptidase, is due to a FemABX-like immunity protein that inserts serines in place of some glycines in peptidoglycan cross bridges. These modifications inhibit both binding of the recombinant cell wall targeting domain and catalysis by the recombinant catalytic domain of lysostaphin.

Lysostaphin-based assay of human granulocyte functions: a reevaluation.

Lysostaphin, a staphylococcus-derived staphylocidal substance, has widely been used in assays of granulocyte phagocytic and bactericidal capability. It rapidly kills extracellular bacteria. Thus, a separate determination of intracellular surviving bacteria can be performed. One prerequisite for this approach is the safe inactivation of lysostaphin (usually brought about by trypsin) before the intracellular bacteria are externalized for plating. This inactivation has been found by others to be incomplete. Data are presented demonstrating a safe inactivation of lysostaphin by trypsin, if the pH value is maintained within the alkaline range. A low variation of results is obtained by plotting the total number of bacteria killed per incubate vs the logarithm of initial bacterial inoculum or of the intracellular surviving bacteria, leading to linear regression lines. The variation of the results increases greatly for initial bacteria/granulocyte proportions of greater than 5/1. The results obtained for two different St. aureus strains are significantly different. Dexamethasone pretreatment (12 mg p.o. within 8 h) had no detectable influence, when fresh blood was assayed, while blood storage at room temperature for 12 h (without dexamethasone pretreatment) led to a significant functional impairment, mainly of bactericidal capability when analyzed in a pairwise fashion. A major limitation of this kind of assays is that killed bacteria cannot be determined directly.

The use of lysostaphin in in vitro assays of phagocyte function: adherence to and penetration into granulocytes.

The usefulness of lysostaphin for the removal of cell-adherent and extracellular bacteria in assays performed to measure the intracellular killing of Staphylococcus aureus by granulocytes was investigated. The results showed that the adherence of lysostaphin to the granulocyte surface is effectuated by a temperature-independent process and that bound lysostaphin is still microbicidal. Lysostaphin also penetrates into the granulocytes by a temperature-dependent process and kills ingested S. aureus intracellularly. Therefore, despite reports to the contrary in the literature, lysostaphin is not a reliable agent for the removal of only extracellular S. aureus and should no longer be used in assays to determine the rate of intracellular killing by granulocytes.