Partially Unfolded Lysozyme at Neutral pH Agglutinates and Kills Gram-Negative and Gram-Positive Bacteria through Membrane Damage Mechanism

Abstract

The antimicrobial mechanism and structural changes of hen egg white lysozyme irreversibly inactivated at 80 °C and at different pHs were investigated. We found that heat denaturation of lysozyme at increasing temperatures for 20 min at pH 6.0 results in progressive loss of enzyme activity while greatly promotes its antimicrobial action to Gram-negative bacteria. Interestingly, lysozyme devoid of enzyme activity (heated at 80 °C and pH 7.0 or at pH 6.0 over 90 °C) exhibited strong bactericidal activity against Gram-negative and -positive bacteria, suggesting action independent of catalytic function. The most potent antimicrobial lysozyme to either Gram-negative or -positive bacteria was that heated at 80 °C and pH 6.0 (HLz80/6), retaining 50% of the native enzymatic activity, which exhibited a 14-fold increase in surface hydrophobicity, with two exposed thiol groups. HLz80/6-induced agglutination coincided with severe reduction in colony-forming ability of the susceptible bacteria in a dose-dependent manner. Denatured lysozyme HLz80/6 showed promoted binding capacity to peptidoglycan of Staphylococcus aureus and lipopolysaccharide of Escherichia coli as assessed by ELISA. Addition of HLz80/6 to E. coli phospholipid vesicles resulted in a blue shift in the intrinsic tryptophan fluorescence accompanied by an increase in the size of the vesicles, indicating enhanced protein−membrane binding and subsequent fusion of liposomes. Direct membrane damage of E. coli membrane by HLz80/6 was revealed by electron microscopy observation. Thus, the results introduce an interesting finding that partial unfolding of lysozyme with the proper acquisition of the hydrophobic pocket to the surface can switch its antimicrobial activity to include Gram-negative bacteria without a detrimental effect on the inherent bactericidal effect against Gram-positive ones. The data suggest that the unique antimicrobial action of unfolded lysozyme attributes to membrane binding and subsequent perturbation of its functions. Keywords: Lysozyme; conformational changes; antimicrobial action; agglutination; membrane interaction and fusion

Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

“…Reduction of enzymatic activity by heat or ␤-mercapto-ethanol denaturation clearly leads to a reduction in the observed bactericidal effect, and complete elimination of enzymatic activity by extended heat treatment (60 min, 100°C) completely eliminates the bactericidal effect. At this point, therefore, our results do not allow us to confirm the hypothesis raised by other authors that the antimicrobial activity of lysozyme and/or heat-denatured lysozyme would consist partly of a mechanism that is independent of enzymatic activity (10,13,21). The second observation from these experiments is that partial heat denaturation can extend the spectrum of lysozyme bactericidal activity under pressure to a wider range of bacteria.…”

“…Therefore, the values shown for the control treatment are taken from Table 1. E. coli et al (10,13), but we failed to confirm any of the bactericidal or bacteriostatic effects under atmospheric pressure that were reported by these authors. Denaturation with ␤-mercapto-ethanol also did not endow lysozyme with antimicrobial activity at atmospheric pressure.…”

“…This seems not to be the case, since even after a harsh treatment at 600 MPa and 60°C during 15 min, 100% of the enzymatic activity was retained (Table 2). Lysozyme was denatured with heat in a way similar to that described by Ibrahim et al (10,13) and, in addition, with ␤-mercaptoethanol. The different treatments of lysozyme, the designations used for each denatured product, and the corresponding remaining enzymatic activities are shown in Table 2.…”

“…Second, the bacteriostatic and bactericidal properties of lysozyme have been the subject of many studies, and over the last 10 years, several authors have proposed a novel antibacterial mechanism of action for lysozyme that is independent of its 1,4-␤-Nacetylmuramidase activity. These reports are based on unique bactericidal properties observed with partially or completely denatured lysozymes having reduced or no enzymatic activity against both gram-positive and gram-negative bacteria (2,10,13,16). For example, Ibrahim et al (10,13) demonstrated inactivation by heat-denatured lysozyme of an Escherichia coli K-12 strain, which was relatively insensitive to native lysozyme.…”

“…These reports are based on unique bactericidal properties observed with partially or completely denatured lysozymes having reduced or no enzymatic activity against both gram-positive and gram-negative bacteria (2,10,13,16). For example, Ibrahim et al (10,13) demonstrated inactivation by heat-denatured lysozyme of an Escherichia coli K-12 strain, which was relatively insensitive to native lysozyme. Düring et al (2) showed that native and heat-treated enzymatically inactive lysozyme from coliphage T4 caused similar inactivation levels on a particular E. coli strain.…”