Human parotid saliva histidine-rich polypeptides exerted antifungal activity against Candida albicans at concentrations similar to the known antifungal activity of the imidazole antibiotics. Inhibition of both growth and viability could be demonstrated by optical density monitoring and plating assays. Inhibition of growth was observed to be greatest when the histidine-rich polypeptides were added to the inoculum before addition to the growth media. However, complete inhibition by these polypeptides was still noted during active growth at turbidities of C. albicans corresponding to 106 CFU/ml. At higher cell densities, growth was delayed but not halted under the experimental conditions investigated. Candidacidal activity was observed with both growing and nongrowing cells. With respect to the latter, reaction of cells in buffer with the histidine-rich polypeptides for a period of 30 min resulted in killing of >90% of two different strains of C. albicans, whereas a third strain was found to be less susceptible. Moreover, the kinetics of loss of cell viability correlated with the loss of potassium from the cells. In addition to the histidine-rich polypeptides, hen egg white lysozyme, poly-L-lysine, and poly-L-histidine affected C. albicans. Both of the polyamino acids completely inhibited the growth of the yeast whereas lysozyme was not as potent. Where delays in growth were observed for all of these agents, including the histidine-rich polypeptides, turbidities reached those of untreated controls after a 24-h period. Enhanced effects were noted if C. albicans was preincubated with these agents in 0.025 2-(N-morpholino)-ethanesulfonic acid buffer, pH 5.2, before growth in the yeast synthetic medium.

Growth inhibition and cell viability assays demonstrate that the histidine-rich polypeptides isolated from human parotid saliva are bacteriostatic and bactericidal for strains of Streptococcus mutans belonging to the serotype b and c classifications. Both inhibition of growth and cell division are enhanced by preincubation of bacteria with these polypeptides in low-ionic-strength buffers of acidic and neutral pH before dilution into enriched growth media. With prior exposure at pH 6.8, inhibition by these polypeptides of the serotype c strains, S. mutans GS5 and SB, as well as the serotype b strain, S. mutans BHT, is reversible over time under the experimental conditions selected. With similar exposure at pH 5.2, however, irreversible damage is manifested by complete inhibition of both growth and cell viability. At concentrations of 250 jxg of the mixture of histidine-rich polypeptides per 5 x 105 bacterial cells per ml in the acidic preincubation buffer, bacterial lethality is maintained for a period of 48 h in the enriched growth media. At a 50-,ug/ml concentration of these salivary agents, approximately 80% killing of S. mutans SB is noted after a 24-h incubation; however, surviving bacteria multiply and reach turbidities of untreated control cells when examined at the 48-h growth point. Similarly, hen egg white lysozyme is also found to be bactericidal for these microorganisms when preincubation is carried out under acidic conditions. However, in contrast to the histidine-rich polypeptides, lysozyme under these experimental conditions does not inhibit growth of S. mutans SB at neutral pH, although it does inhibit growth of both S. mutans BHT and S. mutans GS5 at this pH. Preexposure of S. mutans SB to the peptides in buffer at ionic strengths of 0.025 to 0.125, followed by either viability assays under nongrowing conditions or growth inhibition studies, suggests that there is very little effect of ionic strength on the antibacterial function of these peptides. In contrast to the inhibition of viability noted under growing conditions, lower concentrations of the histidine-rich polypeptides were required to elicit immediate cell death under nongrowing conditions.

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After extraction of the membranes of Escherichia coli K12 strain 44 by Brij-36T, each of the four enzyme activities (natural transpeptidase, unnatural transpeptidase, carboxypeptidase and endopeptidase) of the wall peptide crosslinking system, OCCUTS in two forms characterized by large differences in their sensitivity to ampicillin (but much smaller differences in their sensitivity to cephalothin). The fractionation of the enzyme activities into two groups of low and high sensitivity to ampicillin is achieved essentially by chromatography of the membrane extract on DEAEcellulose.I n Escherichia coli K12, strain 44, the DD-carboxypeptidase, transpeptidase and endopeptidase activities are membrane bound [l, 21. Fractionation of the enzyme complex revealed the existence of two systems : a Dwcarboxypeptidase-transpeptidase system for which the glutamate-amidated tetrapeptide L-D-alanine was a substrate and/or an inhibitor, and a DD-carboxypeptidase-endopeptidase system on which the glutamate-amidated tetrapeptide had no effect [ 2 ] . The use of /?-lactam antibiotics that are known to interfere with the wall peptide crosslinking reaction in E. coli [3] was likely to provide further information on the properties and the functioning of the enzymes involved. The results of these investigations are presented in this paper. E. coli K12, strain 44, a /?-lactamase-negative mutant and the fractions derived from its membrane [ 2 ] , were of particular interest in this type of research. Indeed, the study of the action of /?-lactam antibiotics on the peptide crosslinking system of wild strains of E. coli is complicated by the presence of cell bound /?-lactamases [4-61 which hydrolyze the penicillin molecule into penicilloic acid [7]. The isolation of the p-lactamase-negative mutant, strain 44, is also described in the present paper.Abbreviation. Brij-36T, polyoxyethylated (6 = 10) laurglether. MATERIALS AND METHODS StrainE. coli K12 F-, strain PA601 (originally from Dr E. Wollmann, Institut Pasteur, Paris) was selected because it exhibited a wide number of markers (thr-, leu-, his-, arg-, pro-