Role of divalent cations in the novel bactericidal activity of the partially unfolded lysozyme

It was recently found that partial denaturation of hen egg white lysozyme at 80 degrees C for 20 min at pH 6.0 produces a strong bactericidal lysozyme (HLz) not only against Gram-positive bacteria but also against the Gram-negative ones. The novel antimicrobial action of the mildly denatured lysozyme was found to operate through a membrane-disrupting mechanism independent of its muramidase activity. To evaluate the role of Ca2+ and Mg2+, which are known to stabilize the structure of bacterial membranes, in the antimicrobial activity of HLz, the antimicrobial activity in the presence of divalent cations and chelator EDTA was tested. The antimicrobial activity of HLz against test microorganisms Staphylococcus aureus and Escherichia coli K12 progressively decreased with an increase in Ca2+ or Mg2+ concentration, whereas 1 mM cation produced nonbactericidal lysozyme, either the native (NLz) or the heated enzyme (HLz). However, the inhibitory effect of divalent cations on the antimicrobial activity was more pronounced on HLz than on NLz. The antimicrobial activity of HLz against Gram-negative E. coli, which has been abolished in the presence of free Ca2+ (1 mM), was recovered by the addition of 1 mM EDTA, indicating the competition between free and membrane-bound Ca2+ on the part of HLz molecule. Addition of increasing concentration of NaCl up to 10 mM had no inhibitory effect on the strong antimicrobial activity of HLz. Thus, the inhibitory effect of divalent cations on the activity of HLz is unlikely to be simply due to charge suppression. Intrinsic fluorescence analysis revealed that Ca2+ induces conformational change of the HLz molecules, thus providing evidence that HLz exerts its antimicrobial action against Gram-negative bacteria by disrupting the normal electrostatic interactions between divalent cations and components of the outer membrane. Structural changes relevant to divalent cations sensitization of HLz are discussed.