Partially Unfolded Lysozyme at Neutral pH Agglutinates and Kills Gram-Negative and Gram-Positive Bacteria through Membrane Damage Mechanism Hisham R. Ibrahim,* ,† Shinji Higashiguchi, ‡ Mamoru Koketsu, ‡ Lekh R. Juneja, ‡ Mujo Kim, ‡ Takehiko Yamamoto, ‡ Yasushi Sugimoto, † and Takayoshi Aoki † Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890, Japan, and Central Research Laboratories, Taiyo Kagaku Company, Ltd., Yokkaichi, Mie 510, Japan 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 INTRODUCTION Among the antimicrobial proteins, the mechanism by which lysozyme kills the sensitive bacteria is known to be the degradation of the glycosidic -linkage between N-acetylhexosamines of the peptidoglycan layer in the bacterial cell wall (Fleming, 1922; Jolles and Jolles, 1984). Its antimicrobial function is limited to certain Gram-positive bacteria owing to the differences found in composition as well as the accessibility of the pepti- doglycan to the enzyme action (Salton and Pavlik, 1960; Salton et al., 1968). Aside from its bacteriolytic action, it has been demonstrated that lysozymes have many other functions, including inactivation of certain viruses by forming insoluble complexes (Hasselberger, 1978), important roles in surveillance of membranes of mam- malian cells (Osserman et al., 1974), enhancement of phagocytic activity of polymorphonuclear leukocytes (Kokoshis et al., 1978) and macrophages (Thacore and Willet, 1966), and stimulation of proliferation and antitumor functions of monocytes (Lemarbre et al., 1981). Recently, studies have shown that lysozyme interacts with and induces fusion of phospholipid vesicles (Posse et al., 1994). In parallel, immunochemical in vivo studies using confocal microscopy have demonstrated that lysozyme is synthesized and secreted with surfac- tant apoprotein A by rat alveolar type II epithelial cells and alveolar macrophages, suggesting its role in the extracellular remodeling of surfactant phospholipids in the air spaces of lung (Gibson and Phadke, 1994). The latter studies have also shown that lysozyme was distributed peripherally within the lamellar body in its native molecular weight, 14 kDa, and a considerable amount of a dimer of 2 times the molecular weight. Lysozyme is known to be specifically bactericidal to certain Gram-positive bacteria but less effective against Gram-negative ones, despite its strong interaction with and disruptive effect on lipopolysaccharides (LPS) of the later species (Ohno and Morrison, 1989) and the positive correlation between the increased level of lysozyme secretion in many tissues and bacterial infections (Brouwer et al., 1984). Though in small amount, an irreversible dimeric form of lysozyme was detected in avian egg whites and within the lumen of the cystlike aggregates of alveolar macrophages (Back, 1984; Gibson and Phadke, 1994), presumably through intermolecular disulfide exchange. The tendency of lysozyme to as- sociate into dimers and higher polymers as a function of pH, concentration, and temperature has also been * Author to whom correspondence should be ad- dressed (telephone, +81 (99) 285-8658; fax, +81 (99) 285-8525; e-mail, hishamri@chem.agri.kagoshima-u.ac.jp). † Kagoshima University. ‡ Taiyo Kagaku Co., Ltd. 3799 J. Agric. Food Chem. 1996, 44, 3799-3806 S0021-8561(96)00133-1 CCC: $12.00 © 1996 American Chemical Society