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Humoral immune responses to lipoteichoic acid (LTA) as a surface antigen of Lactobacillus fermentum were assessed in rabbits and mice. Intravenous injection of rabbits with whole bacteria was effective, over a wide range of doses (10(4) to 10(10) cells), in eliciting antibodies to LTA. In mice, significant levels of anti-LTA antibodies were induced only following intraperitoneal injection of 10(8) to 10(9) L. fermentum cells. Rabbit antibodies to LTA were predominantly of the IgM class and were specific for the polyglycerol phosphate and carbohydrate moieties of the LTA. In contrast, both IgM and IgG anti-LTA antibodies were produced in the mouse, and antibody specificity was restricted to the polyglycerol phosphate sequence.

Regimens of intravenous injections of saline-washed Lactobacillus fermentum elicited hypersensitivity reactions in rabbits. Pathological investigation revealed evidence consistent with induction of aggregate anaphylaxis, characterised by acute cor pulmonale. Additional evidence of similar tissue injury was observed in livers of rabbits which had received several intravenous injections of L. fermentum. Deposition of immune complexes in kidney glomeruli was demonstrated in only 1 out of 11 animals. Skin testing experiments revealed that lipoteichoic acid was involved in type I and type II antibody-mediated hypersensitive states. The involvement of bacterial cell surface components and extracellular products in such reactions implies a potential role in host tissue injury.

We examined the effect of growth conditions in chemostat culture on the quantity and composition of the cell wall teichoic acids of Streptococcus mutans BHT and Lactobacillus plantarum NCIB 7220 and the membrane lipoteichoic acid from S. mutans Ingbritt. With the cell wall teichoic acids, which are covalently linked to peptidoglycan, the amount of teichoic acid is independent of the growth conditions employed. However, the extent of glucosyl substitution of the polymer from L. plantarum was dependent on growth conditions. S. mutans Ingbritt lipoteichoic acid, on the other hand, was little affected by growth conditions in terms of composition or serological activity, but the amount produced was markedly affected by changes in growth conditions.

Lipoteichoic acid acids with a range of chemical compositions have been studied using 1H; 13C- and 31P-nuclear magnetic resonance. Proton spectroscopy provided a rapid method for demonstrating whether alanine in a sample is covalently bound to the polyglycerophosphate chains and for monitoring hydrolysis of alanine. The nature of sugar substituents can be determined, with some limitations, from the 13C spectra, and the proportions of glycerol residues substituted by alanine and sugar can be measured. The 31P spectra of lipoteichoic acid provided information about both the degree of substitution and the distribution of the substituent along the polyglycerophosphate chain, except when the substituent was galactose. The polyglycerophosphate chains were shown to undergo rapid internal rotation and no evidence for tertiary structure was found either in the presence or absence of magnesium ions. Magnesium ions exchange rapidly between the bound and free state and the binding constant to lipoteichoic acid of 64 M-1 is typical for monophosphates in aqueous solution. There was no evidence that alanine substitution affects the binding constant for magnesium ions.

Intravenous immunisation of rabbits with 10(9) lactobacillus fermentum cells elicited a response, specific for lipoteichoic acid (LTA), detectable as IgM plaque-forming cells (PFC) in the spleen by day 2 and as thiol-sensitive 19s antibodies in sera by day 3. Direct PFC responses peaked at day 6, with no indirect PFC demonstrable at the time. Specific IgG PFC appeared after 14 days. A second intravenous injection 5 weeks later induced a 10-fold higher IgM PFC response to LTA which reached a maximum on day 4. An enhanced specific IgG PFC response was also observed. Serum analysis showed further evidence of the anamnestic IgM response to LTA. The results are discussed in terms of the T-dependence of the LTA immunogen.

Representative strains of Streptococcus sanguis serotype 2 and of four Lactobacillus species were examined for the production of cellular and extracellular lipoteichoic acid (LTA) when grown at pH 6.0 in batch culture to the stationary phase with either glucose or fructose. Extracellular LTA was a minor component in all cases except for L. fermentum and L. casei NIRD R094 grown in fructose. The total amount of LTA (cellular and extracellular) produced by fructose-grown cultures was also considerably greater for these two strains, for L. salivarius, and also two of the S. sanguis strains. Growth of L. fermentum and L. casei in continuous culture in a chemostat showed that generation time and pH of growth can influence the total amount of LTA and the proportion of extracellular material. The results for glucose-limited cultures were quite disparate, with L. fermentum forming considerably more extracellular LTA than L. casei. However, in fructose-limited cultures L. fermentum formed less total LTA and L. casei more so that the differences were only minor. A difference in the utilization of glucose and fructose by the heterofermentative L. fermentum and the homofermentative L. casei strains is also indicated by differences in the yield of organisms at different dilution rates in continuous culture.

Decreases in electrophoretic mobilities of intracellular lipoteichoic acid, intracellular deacylated lipoteichoic acid, and extracellular deacylated lipoteichoic acid were observed during inhibition of protein synthesis in Streptococcus faecium after exposure to chloramphenicol or valine deprivation. Increased carbohydrate content, and thus an increased mass-to-charge ratio, rather than changes in ester alanine content or novel fatty acid substitutions, appeared to account for the decreased electrophoretic mobilities. The increase in carbohydrate content, as judged from mobility measurements, was progressive over time and appeared to occur on biosynthetically new lipoteichoic acid as well as on lipoteichoic acid made before inhibition of protein synthesis.

Streptococcus mutans BHT was grown in a chemostat with glucose limitation and at defined dilution rates and pH values. Lipoteichoic acid was estimated by determining the ability of dilutions of culture fluid to sensitize erythrocytes. The greatest amounts of extracellular lipoteichoic acid were produced by organisms growing at a low dilution rate and at pH 6.0 or 6.5. To enable a more accurate estimation of the total amount of extracellular material, rocket immunoelectrophoresis was employed. These results confirmed that the greatest amounts of reactive material were produced by slow-growing organisms, although there were discrepancies between these results and those obtained by hemagglutination. The extracellular material was fractionated by column chromatography and membrane ultrafiltration to yield a lipoteichoic acid-containing fraction and a presumptive deacylated lipoteichoic acid fraction. The relative proportions detected by rocket immunoelectrophoresis differed with the growth conditions, particularly the dilution rate. Analysis of the phenol-extracted cellular material also indicated the presence of deacylated lipoteichoic acid, although less than in the culture fluid.

Streptococcus mutans Ingbritt was grown in a chemostat at destined dilution rates in either 0.5% fructose or 0.5% sorbitol and at destined pH values in 0.5% fructose. The yield of cells was affected by the carbohydrate source, as well as by the pH, with the lowest yield being at pH 5.5 in 0.5% fructose. Fructose-grown cells showed greater susceptibility to lysis by a muramidase than the corresponding glucose-grown cells, but there were no marked differences in the lytic susceptibilities of the corresponding cell wall preparations or in the serological reactivities of wall lysates with antiserum to S. mutans Ingbritt. The greatest amounts of cellular lipoteichoic acid were obtained at high dilution rates in both fructose and sorbitol, as well as at high pH values in fructose. The greatest amounts of extracellular lipoteichoic acid were found at low dilution rates, as estimated by rocket immunoelectrophoresis and also by hemagglutination. Three major extracellular protein components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the effects of growth conditions on these components were determined. Results for batch-grown cultures showed that there was genotypic variation in the susceptibility of cells to lysis by a muramidase. The enhancement of lipoteichoic acid production by fructose and sorbitol in batch cultures was not identical in representative strains of S. mutans serotype c, nor was the effect of fructose found uniformly in representative strains of the different S. mutans serotypes.

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.

Streptococcus mutans Ingbritt was grown in a chemostat at defined dilution rates and pH values and under carbohydrate limitation. At a constant dilution rate of D = 0.1 h-1 and with either 0.5% glucose or 0.5% sucrose, the amounts of both cellular and extracellular lipoteichoic acid increased as the culture pH increased from 5.0 to 7.5. At a constant pH of 6.0, the amount of cellular lipoteichoic acid formed by cultures growing in 0.2% or 0.5% glucose was relatively constant over a range of dilution rates, although the amount of extracellular lipoteichoic acid formed in 0.2% glucose at intermediate dilution rates was less than that formed in 0.5% glucose. Organisms grown in 0.5% sucrose at pH 6.0 contained increasing amounts of cellular lipoteichoic acid as the dilution rate was increased. A comparison of the amounts of cellular lipoteichoic acid formed by organisms growing at D = 0.5 h-1 and pH 6.0 in glucose, sucrose, fructose, or mixtures of glucose and fructose in limiting amounts suggested that the enhanced production of lipoteichoic acic by sucrose-grown organisms was due to the fructose component. The culture fluids from both glucose- and sucrose-grown organisms contained detectable amounts of serotype c antigen, whereas glucose-grown cultures also contained significant amounts of an extracellular hexose-containing polymer.

Purified lipoteichoic acids (LTAs) from several gram-positive organisms have been shown, by methods involving spectral changes of an added merocyanine dye probe, to have critical micelle concentrations in the range of 1 to 10 micrograms/ml, suggesting that acylated LTAs in their monomer forms may represent the major configuration of extracellular LTAs in bacterial culture fluids. The critical micelle concentrations obtained did not differ markedly with degree of carbohydrate substitution of the polymers. The significance of these findings in relation to the biological properties of LTA is discussed.