Kenneth W. Knox

Characteristics of a High Molecular Weight Extracellular Protein of Streptococcus mutans

A high molecular weight protein antigen, designated P1, has been isolated from the culture fluid of chemostat-grown Streptococcus mutans strain Ingbritt and shown to be free of other antigens including glucosyltransferase. Antiserum against the protein was used in rocket immunoelectrophoresis to confirm and extend the previous observation that there were major differences in the amount of the protein produced under different growth conditions. Physico-chemical and serological studies indicated that protein P1 was indistinguishable from antigens B, I/II and IF isolated in other laboratories. Mammalian tissue cross-reactivity of protein P1 was demonstrated by binding of antiserum to P1 to sections of normal rabbit tissues, particularly heart. There was also a statistically significant increase in the number of mononuclear leucocytes in heart tissue of rabbits which had been injected with protein P1, when compared with the levels in control uninjected rabbits; injection with whole cells of S. mutans Ingbritt did not produce this effect.

Comparative Studies on the Protein Profiles and Hydrophobicity of Strains of Streptococcus mutans Serotype c

Twelve strains of Streptococcus mutans serotype c were grown in batch culture with glucose at constant pH (6.0) and a number of properties compared. On the basis of their cellular and extracellular protein profiles, the strains were divided into three groups, I, II and III, containing five, four and three strains, respectively. The extracellular protein profiles for a particular strain differed if the organisms were grown either at pH 6.0 with fructose instead of glucose or with glucose but without pH control. The total amount of extracellular protein produced by group III strains grown in glucose-containing medium at pH 6.0 was several times that produced by strains of groups I and II, which were also more hydrophobic. One of the potentially important proteins is P1, also called antigen B or I/II, and it was shown to be entirely in the culture fluid of group III strains but mostly cell-associated from strains of groups I and II. Approximately half of the cell-associated fraction of P1 could be removed with hot sodium dodecyl sulphate.

Effect of Tween 80 on the Morphology and Physiology of Lactobacillus salivarius Strain IV CL-37 Grown in a Chemostat under Glucose Limitation

The effect of Tween 80 on Lactobacillus salivarius strain IV CL-37 growing in a chemostat under various conditions was investigated. The organisms could grow under glucose limitation in the absence of Tween 80 at pH 6.0 or lower anaerobically but not aerobically. Aerobic growth under glucose limitation and in the presence of Tween 80 occurred in complete MRS medium but not in the dialysable fraction of MRS medium. The morphology of cells differed from coccal to filamentous and branched structures according to the growth condition. The possible effect of Tween 80 on membrane components was examined by estimating the cellular and extracellular lipoteichoic acid contents. In both batch and continuous culture the amounts of cellular lipoteichoic acid were inversely related to the amount of Tween 80 whereas the amounts of extracellular lipoteichoic acid were influenced by other factors in addition to Tween 80.

Amphipathic antigens of oral microorganisms - immunogenicity and other biological properties.

Amphipathic antigens are characterized by possessing both hydrophilic and hydrophobic regions in their molecular structure. These dual features dictate their physicochemical properties and, in turn, play a determinative role in their biological properties. The most well known examples of such antigens are the lipopoly-saccharides of Gram-negative bacteria and the lipoteichoic acids found in many genera of Gram-positive bacteria. Both types of amphophile are membrane associated through hydrophobic interaction of their lipid moieties with phospholipid bilayer; in the case of lipopolysaccharides with the outer cell membrane of the cell envelope and with lipoteichoic acids the plasma cell membrane of the Gram-positive bacterial cell (1,2,3). More recently examples of another class of amphipathic molecule have been reported (4,5), the lipomannans of Micrococcus spp. Like lipoteichoic acids, lipomannans are membrane associated. All three classes of amphophile are known to be immunogenic and can act as surface antigens (3,4). In the case of lipopolysaccharides a surface location is perhaps obvious from the association with the outer cell membrane.

Comparison of Extracellular Protein Profiles of Seven Serotypes of Mutans Streptococci Grown under Controlled Conditions

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.

Effect of Growth Conditions on the Antigenic Components of Streptococcus Mutans and Lactobacilli

1) The release of LTA from chemostat-grown cultures of S. mutans and L. fermentum is dependent on the generation time and pH. 2) The growth rate affects the immunogenicity of LTA and the wall polysaccharide of L. fermentum. 3) The growth of L. plantarum under glucose limitation influences the glucose content of the wall teichoic acid. The composition of the wall polysaccharide of S. mutans Ingbritt may also be changed under these conditions. 4) The conditions of autoclaving used to release cell wall antigens can hydrolyse the covalent linkage between polysaccharide and peptidoglycan.

Identification of the group G antigen of Lactobacilli.

The group G antigen of lactobacilli was identified as a negatively-charged cell wall polysaccharide. The components of the preparation isolated from cell walls of L. salivarius subsp. salivarius by mild acid hydrolysis were glucose, galactose and lesser amounts of rhamnose, N-acetylglucosamine and phosphate. Quantitative serological studies on acid-released polysaccharide and enzymic lysates of cell walls showed that rhammose was the immunodominant component. The antigen was also detected in L. salivarius subsp. salicinus but not in a recent isolate of L. salivarius, strain IV CL-37.