Lysozyme is a cationic enzyme that is a prominent member of the antibacterial complex of human saliva. It has the potential to kill bacteria either by direct lysis, or by mechanisms that are independent of its lytic activity. The objectives of this application are (1) to uncover the non-lytic bactericidal mechanisms of lysozyme, and (2) to evaluate the effects of lysozyme in acquired salivary pellicle on adherence of oral bacteria to hydroxyapatite and their subsequent survival. We reported earlier that chitotriose inhibited the non-lytic bactericidal action of human lysozyme and of polylysine and polyarginine against Streptococcus sanguis and S. faecalis. These and other results suggested that some cell-associated activity, inhibitable by chitotriose, was necessary for expression of the bactericidal activity of active and inactive lysozyme. We proposed to survey strains of S. mutans, S. sanguis, S. mitis, S. salivarius and A. viscosus for chitotriose protection from the bactericidal action of muramidase-inactive human lysozyme and polylysine. Cell surface-associated proteins that interact (e.g., bind) with chitin and chitotriose will be identified and isolated from selected bacteria and tested for N-acetylmuramidase and N-acetylglucosaminidase activity using radiolabeled whole cells and peptidoglycan. Bacteria treated with inactive lysozyme or polylysine, in the presence and absence of chitotriose, will be evaluated for membrane damage as judged by leakage of low molecular weight charged substances, permeability to external phosphoenolpyruvate, and ability to maintain transmembrane pH and cation gradients. In concert with viability determinations, the above experiments will allow us to identify those phenomena that are directly associated with lysozyme-dependent loss of microbe viability. Effects of lysozyme in salivary pellicles on in vitro adherence of oral bacteria and their subsequent likelihood of survival will be estimated by a high salt elution and lysis procedure developed by us. We anticipate the results of this study will have wide-spread application to understanding the bactericidal mechanisms of host cationic proteins of phagocytic cells and of the secretions that bathe mucosal surfaces.
| Wang, Y B; Germaine, G R (1993) Effects of pH, potassium, magnesium, and bacterial growth phase on lysozyme inhibition of glucose fermentation by Streptococcus mutans 10449. J Dent Res 72:907-11 |
| Wang, Y B; Germaine, G R (1991) Effect of lysozyme on glucose fermentation, cytoplasmic pH, and intracellular potassium concentrations in Streptococcus mutans 10449. Infect Immun 59:638-44 |
| Tompkins, G R; O'Neill, M M; Cafarella, T G et al. (1991) Inhibition of bactericidal and bacteriolytic activities of poly-D-lysine and lysozyme by chitotriose and ferric iron. Infect Immun 59:655-64 |
