The bisis of the bactericidal activity of human lysozyme against Streptococcus sanguis was studied. Experiments were designed to evaluate the role of lysozyme muramidase activity in its bactericidal potency. Inactivation of the muramidase activity of lysozyme was achieved by reduction of essential disulfides with dithiothreitol (DTT) or by incubation with the chitin oligosaccharides chitotriose and chitobiose. Muramidaseinactive lysozyme, prepared by reduction with DTT, was equal in bactericidal potency to native lysozyme. Solutions of native chicken egg white lysozyme and human lysozyme exhibited equal bactericidal potency yet differed ca. fourfold with respect to lytic (muramidase) activity. The above results suggested that the bactericidal activity of lysozyme is not dependent upon muramidase activity. Chitotriose and chitobiose were found to inhibit both lytic and bactericidal activities of lysozyme. The bactericidal activity of mnuramidase-inactive lysozyme (reduction with DTT) was also inhibited by chitotriose and chitobiose. Further investigations demonstrated that chitotriose and chitobiose were also potent inhibitors of the bactericidal activity of the cationic homopolypeptides poly-L-arginine and poly-D-lysine. These latter results suggested that the essential bactericidal property of lysozyme was its extreme cationic nature and that some bacterial endogenous activities, inhibitable by chitotriose and chitobiose, were essential for expression of the bactericidal activity of either native or muramidase-inactive lysozyme or of the cationic homopolypeptides. Experiments with Streptococcus faecalis whole cells, cell walls, and crude autolysin preparations implicated endogenous autolytic muramidases as the bacterial targets of chitotriose and chitobiose. The essentially identical responses of S. sanguis and S. faecalis to chitotriose in bactericidal assays with muramidase-inactive lysozyme and polylysine suggested that muramidase-like enzymes exist in S. sanguis and, furthermore, play an essential role in cationic protein-induced loss of viability of the oral microbe. * Corresponding author. some bacteria to oral surfaces. Lysozyme, with a pI > 10.5, is also a rather extreme example of a cationic protein. Cationic proteins are known bactericidal agents against both gram-positive and gram-negative organisms (15, 17, 38, 41, 49, 50). Two modes of bactericidal action are thus available to lysozyme: a muramidase-dependent mode and a cationicdependent mode. The oral streptococci are typically resistant to direct lysis by either human or chicken lysozyme (3, 25, 40). Lysozyme is, however, bactericidal for the major species of oral streptococci (3, 22, 25, 40; N.

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Several strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii plus fresh isolates of Streptococcus salivarius were surveyed for their abilities to deplete lysozyme from human-whole-saliva supematant. Bacteria were incubated in saliva for 60 min at 37°C and then removed by centrifugation, and the recovered supernatant solutions were assayed for lysozyme activity by using whole cells of Micrococcus lysodeikticus as the substrate. Mean lysozyme depletions by bacterial strains varied over a wide (eightfold) range. The greatest mean depletion of lysozyme (60 to 70%) was observed with S. sanguis (biotype I), serotype b of S. mutans, and the fresh S. salivarius isolates. The lowest mean depletion was noted with S. mitis (15%) and biotype II S. sanguis (ca. 30%). The remaining species and strains exhibited an intermediate degree of depletion. In studies with S. sanguis 903, lysozyme was depleted by normal or heated (90°C, 30 min) bacteria and could be recovered from the organism. Furthermore, under appropriate conditions, lysozyme depletion by cells at 0 and 37°C was very similar. On the basis of these observations, we concluded that depletion was due to the adsorption of lysozyme by the organism. With S. sanguis 903, lysozyme adsorption depended on the concentration of bacteria, time of incubation, and the ionic strength of the medium. The extent of adsorption, however, was independent of pH's of 3.9 to 8.3. When a low concentration of S. sanguis 903 was used, lysozyme adsorption reached saturation (4 pLg of adsorbed lysozyme per 107 cells) at 20 jig of lysozyme

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The first step in the life cycle of a bacteriophage is its attachment to an outer surface of the bacterial cell. In gram-positive bacteria the cell wall peptidoglycan, or this molecule complexed with other macromolecules, often serves as the phage receptor (1). Previous reports have suggested that the group-specific carbohydrate may play a role as receptor site for the virulent group A streptococcal phage, A25. The inhibition of adsorption by group A antisera and the inability of group A variant cells to adsorb phage A25, provided indirect evidence supporting this view (2). Direct evidence, the isolation and characterization of the receptor site, was not obtained because heat-killed or mechanically disrupted cells failed to inactivate phage particles irreversibly, even though the potential to adsorb this phage reversibly was retained (2, 3).Because it is now clear that phage A25 can infect and transduce antibiotic resistance between different groups of streptococci, such as groups C and G, it seemed essential to identify the phage A25 receptor in group A organisms (4-6). In contrast to earlier reports, the findings reported here demonstrate the irreversible attachment of phage A25 to heat-killed group A, G, and A-variant cells. The complexity of the adsorption process is exemplified by the fact that mechanical disruption of heat-killed cells completely destroyed the irreversible nature of this process. Receptors for the reversible attachment of phage A25 survive mechanical disruption, and experiments including electron microscopy are described which demonstrate the specificity of reversible adsorption and identify the responsible receptor as peptidoglycan. The evidence suggests that adsorption of phage A25 to group A streptococci occurs in a two-step reaction, the first step is the reversible attachment of the phage tail to peptidoglycan exposed on the cell surface, followed by the irreversible inactivation of the phage particle. The latter is still not understood. Materials and MethodsBacterml Strains and Bactermphages. The virulent phage designated A25 was originally described and kindly supphed by Dr. W. R. Maxted (7,8). This virulent phage was used for all adsorption experiments and has been further described by Malke (5) and Read and Reed (9).