Streptococci Grown in Different Environments

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1 INFECTION AND IMMUNITY, June 1982, p /82/6864-6$2./ Vol. 36, No. 3 Production of Lipoteichoic Acid by Lactobacilli and Streptococci Grown in Different Environments ANTHONY J. WICKEN,l* KEVIN W. BROADY,1t ANITA AYRES,' AND KENNETH W. KNOX2 School of Microbiology, University of New South Wales, Kensington, New South Wales 233,1 and Institute of Dental Research, United Dental Hospital, Sydney, New South Wales 21,2 Australia Received 18 January 1982/Accepted 24 February 1982 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. 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. case NIRD R94 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. case 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. case. However, in fructose-limited cultures L. fermentum formed less total LTA and L. case 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. case strains is also indicated by differences in the yield of organisms at different dilution rates in continuous culture. The identification of lipoteichoic acid (LTA) as a membrane-derived surface antigen of a number of gram-positive bacteria (22) led to its subsequent recognition as a component present in significant amounts in the culture fluid of cultures of streptococci and lactobacilli grown in batch culture with glucose (1, 8, 9, 15). Extracellular LTA can exist in two forms with respect to fatty acid substitution, namely, acylated micellar LTA, which displays a wide range of biological properties, and deacylated LTA, which is generally inactive (22, 23). Acylated LTA will sensitize erythrocytes and by means of a semiquantitative procedure it was shown that strains of Streptococcus mutans, particularly BHT, generally produced considerably more extracellular acylated LTA than other streptococci, including S. sanguis, or lactobacilli, including L. fermentum, L. case, and L. plantarum (15). Subsequent studies confirmed and extended the results with S. mutans strains by testing batch cultures grown at ph 6. to mimimize the effect of ph on LTA production (4), employing quantitative rocket immunoelectrophoresis to estimate both acylated and deacylated LTA (6), and examining cultures grown in glucose and fructose, which showed that some strains produced even more LTA when grown in fructose (4). t Present address: Clinpath Pty. Ltd., Paddington, New South Wales 221, Australia. 864 One aspect of the present study was to reexamine cultures of batch-grown strains of S. sanguis and Lactobacillus spp. by rocket immunoelectrophoresis to allow a better comparison with the results for S. mutans strains. The other aspect concerns an examination of cultures of L. fermentum and L. case grown in continuous culture in a chemostat at defined generation times and ph values with growth-limiting glucose or fructose. This overcomes a number of the uncontrollable variables generally associated with growth in batch culture. Chemostat studies on S. mutans strain BHT (6) and Ingbritt (4, 5) have shown that the production of both cellular and extracellular LTA can be influenced by the generation time, ph of growth, and also, in the case of strain Ingbritt (4, 5), by the carbohydrate source, particularly with a change from glucose to fructose. L. fermentum and L. case were chosen for detailed examination as these two species have already been the subject of a number of studies in our laboratories which showed, among other things, that the LTA component of L. fermentum NCTC 6991 is the group F antigen of lactobacilli (13) and is detectable as a major surface component (18), whereas the LTA component of L. casei (strains NCTC 6375 and NIRD R94) is a cross-reacting antigen (21) which is usually not present in significant Downloaded from on January 31, 219 by guest

2 VOL. 36, 1982 amounts on the surface (18). Further, the immunogenicity of the LTA component of L. fermentum is influenced by the generation time as shown by examination of organisms grown in continuous culture (11). MATERIALS AND METHODS Organisms. The strains of lactobacilli were representatives of four serological groups that had been used in earlier studies: group C, L. case subsp. case NIRD R94 and L. case subsp. rhamnosus NCTC 6375 (1, 21); group D, L. plantarum NCIB 722 (11, 14); group F, L. fermentum NCTC 6991 (13); group G, L. salivarius subsp. salivarius ATCC (12). Strains of S. sanguis serotype 2 (17) were supplied by B. Rosan, School of Dental Medicine, University of Pennsylvania, Philadelphia, and were designated Blackburn (NCTC 1231), Challis (NCTC 7868), 1558 (NCTC 7865), and M5. Growth conditions. Lactobacilli were grown in continuous culture with limiting (.5%) glucose or fructose in a medium consisting of the diffusible components of the complex MRS medium (2). The dilution rate (D) in the chemostat is related to the generation time by the following formula: generation time = In 2/D; thus, D =.1 h-1 corresponds to a generation time of approximately 7 h. Batch culture experiments were carried out in the same chemostat apparatus but without medium flow. Lactobacilli were grown at 37C and constant ph (6.) in the same medium as used in the continuous culture studies, except that the carbohydrate concentration was increased to 1%. L. salivarius was grown under anaerobic conditions (95% N2 plus 5% C2). S. sanguis strains were also grown under anaerobic conditions in a medium containing 2% carbohydrate and the diffusible components of the complex medium (19) previously employed in studies on strains of S. mutans (5). By following previous procedures (4), we monitored the rate of growth in batch culture experiments by recording alkali addition to maintain constant ph (6.), and cultures were harvested within 1 h of growth entering the stationary phase as judged by the cessation of acid production. By this means, changes that would otherwise occur in the stationary phase (8, 15) were avoided. Rocket immunoelectrophoresis. For comparing the relative amounts of LTA in organisms grown under different conditions, cell suspensions (approximately 1 mg/ml) were extracted with hot aqueous phenol (6, 2). After dialysis of the aqueous phase against.85% NaCl, appropriate volumes were examined by rocket immunoelectrophoresis against antiserum to L. casei LTA (6). Corrections for variations in serological activity of different batches of antisera were made by inclusion of a standard homologous L. casei LTA preparation in each run. From the known dry weight of extracted cells, results for rocket heights were calculated as centimeters per 5 plg of cells. Culture fluids were concentrated by membrane filtration (6), and appropriate volumes were examined by rocket immunoelectrophoresis. To allow a direct comparison with the cellular values, we standardized results by making a calculation based on the volume of culture fluid that yielded 5 pig of cells (4-6). Rocket immunoelectrophoresis will detect acylated micellar LTA and deacy- LTA OF LACTOBACILLI AND STREPTOCOCCI 865 TABLE 1. Production of LTA by strains of S. sanguis grown in batch culture at ph 6. with either 2% glucose or 2% fructose Rocket height (cm/5,ug of cells) Strain Carbohydrate Cellular Extracellular LTA LTA Blackburn Glucose Fructose Challis Glucose Fructose Glucose Fructose M5 Glucose Fructose lated LTA, both of which are present in cell extracts and culture fluids (6). RESULTS Production of LTA by batch-grown cultures of S. sanguis. Four strains of S. sanguis serotype 2 were grown in batch culture at ph 6. with 2% glucose or fructose and both cellular and extracellular LTA estimated by rocket immunoelectrophoresis. The results (Table 1) showed that extracellular LTA generally represented less than 25% of the cellular value, the exception being strain M5 grown in fructose. For two of the strains, namely, Challis and 1558, growth in fructose resulted in a net increase in the total amount of cellular and extracellular LTA, with the major effect being on the cellular component. Production of LTA by batch-grown cultures of lactobacilli. A similar comparison was made of the effect of growth in 1% glucose and fructose on the production of LTA by representative strains of L. fermentum, L. case, L. plantarum, and L. salivarius. These results (Table 2) also provided evidence of variation in response by the different organisms. The amount of extracellular LTA was generally low for glucose-grown cultures and enhanced by growth in fructose. For three strains, namely, L. fermentum NCTC 6991, L. casei NIRD R94, and L. salivarius ATCC 11741, growth in fructose resulted in at least a threefold increase in the total amount of cellular and extracellular LTA. Production of LTA by L. fermentum and L. casei grown in continuous culture. L. fermentum NCTC 6991 and L. casei NIRD R94 were grown in continuous culture at 37C and ph 6. at dilution rates of.5 to.5 h-1 and with either glucose (.5%) or fructose (.5%) as the growth-limiting nutrient. Cellular and extracellular LTA fractions were compared by rocket Downloaded from on January 31, 219 by guest

3 866 WICKEN ET AL. TABLE 2. Production of LTA by Lactobacillus strains grown in batch culture at ph 6. with either 1% glucose or 1% fructose Rocket height (cm/ Organism Carbohydrate 5 Ftg of cells) Cellular Extra- LTA LA cellular LTA L. fermentum NCTC 6991 Glucose Fructose L. casei NIRD R94 Glucose Fructose L. case NCTC 6375 Glucose Fructose L. plantarum Glucose NCIB 722 Fructose L. salivarius Glucose.9.4 ATCC Fructose immunoelectrophoresis (Fig. 1). The results for glucose-grown cells showed that L. fermentum (Fig. la) contained more detectable cellular LTA than L. case (Fig. lb), with the greatest amount in each case being obtained at intermediate dilution rates. The major difference between the two strains, however, was the relatively high level of extracellular LTA produced by L. fermentum, particularly at low dilution rates (.5 and.1 h-1), compared with the very low levels for L. case. The results with L. case NIRD R94 have been reinforced by a similar examination of L. case NCTC 6375, when the amounts of cellular LTA were similar to those for strain NIRD R94, but extracellular LTA ranged from 1% of the cellular level at low dilution rates down to undetectable levels at higher dilution rates. A comparison of the results obtained for L. fermentum grown with limiting fructose (Fig. ic) and limiting glucose (Fig. la) showed that the major effect was a decrease in the amount of extracellular LTA produced on growth in fructose. For L. case NIRD R94, however, a comparison with the glucose values (Fig. lb) shows that growth in fructose resulted in a general increase in the amount of cellular LTA (Fig. id). The net effect was that the overall difference in LTA produced by the two fructosegrown cultures was much less than for the glucose-grown cultures. Other evidence for a difference between the response of the two organisms to glucose and fructose was provided by their yields (Table 3). For example, with organisms growing relatively fast at D =.5 h-1, when the generation time was 1.4 h, there was very little difference in the co w -i E 1 15s (a) Cc) _ ~~~~~~(b) DILUTION RATE (h-1) FIG. 1. Effect of growth in continuous culture at ph 6. with different dilution rates and with either limiting glucose or fructose on the amounts of LTA detectable by rocket immunoelectrophoresis. (a) L. fermentum NCTC 6991 grown in glucose; (b) L. casei NIRD R94 grown in glucose; (c) L. fermentum NCTC 6991 grown in fructose; (d) L. case NIRD R94 grown in fructose. Solid bars, cellular LTA; hatched bars, extracellular LTA. yields. At the low D =.5 h-1, equivalent to a 14-h generation time, L. fermentum would not grow in fructose whereas the yield of L. case was more than 5% greater than in glucose. The effect of variation in the ph on the production of LTA at constant dilution rate (.1 h-1) in growth-limiting glucose was also examined. As shown in Fig. 2, L. fermentum produced more extracellular than cellular LTA over the ph range of 5. to 7.5, with a large increase in both fractions at ph 7.5, which was the maximum ph for growth. In contrast, the amount of extracellular LTA formed by L. case NIRD R94 was minimal over the ph range of 5. to 7.5 but did rise to nearly the cellular level at ph 8.. DISCUSSION In most of our previous studies on the production of LTA by cultures of streptococci and lactobacilli (4-7), two serological methods were used, namely, a semiquantitative hemagglutination procedure, which detects only acylated micellar LTA (the culture fluid titer), and rocket immunoelectrophoresis, which is a more accurate procedure and detects both acylated and deacylated LTA. The latter was the preferred method for the current study, but, although data are not shown, the presence of acylated micellar LTA was confirmed by examination of a number of cultures by the hemagglutination procedure. I INFECT. IMMUN. Cd) Downloaded from on January 31, 219 by guest

4 VOL. 36, 1982 LTA OF LACTOBACILLI AND STREPTOCOCCI 867 TABLE 3. Effect of dilution rate (D) and growth-limiting carbohydrate on the yield of L. fermentum NCTC 6991 and L. case NIRD R94 in continuous culture at ph 6. Yield (mg [dry wtj/ml) D (h-1) L. fermentum NCTC 6991 L. case NIRD R94 Glucose Fructose Glucose Fructose.5.27 a a Culture did not stabilize. The presence of both acylated and deacylated LTA was shown by analysis (2) of the products separated by gel filtration on AcA22 (from LKB Instruments Co.). In the initial studies that compared the amounts of LTA detectable in the culture fluid of glucose-grown batch cultures by the hemagglutination procedure, the values for strains of S. sanguis and Lactobacillus spp. were uniformly low, and only 1/3 to 1/1 of those for many S. mutans strains (15). The current results, coupled with those of an earlier examination of similarly grown S. mutans cultures by rocket immunoelectrophoresis (4), confirm these differences. Wa E (a) FIG. 2. Effect of growth in continuous culture at a dilution rate of.1 h-1 with limiting glucose and different ph values on the amounts of LTA detectable by rocket immunoelectrophoresis. (a) L. fermentum NCTC 6991; (b) L. case NIRD R94. Solid bars, cellular LTA; hatched bars, extracellular LTA. ph For example, rocket heights of 5 cm/5 1Lg of cells were obtained for S. mutans Ingbritt culture fluid (4) compared with the current results of.5 to 1.7 and.2 to 1.7 cm/5 jig of cells for S. sanguis and Lactobacillus spp., respectively. Such comparisons are considered justified as cultures were grown at constant ph until they had just entered the stationary phase. The amount of extracellular LTA can increase markedly during the stationary phase (8, 15), and an example is provided by studies (7) on another strain of L. salivarius where the value after 18 h in the stationary phase was four times that for exponential-phase organisms as shown by both rocket immunoelectrophoresis and the hemagglutination procedure; for comparison, there was also a twofold increase in cellular LTA (7). The importance of the carbohydrate source on the production of LTA, both cellular and extracellular, has been shown by growing S. mutans strains in batch culture (4) and confirmed in continuous culture (4, 5). The enhancement by fructose in batch culture was particularly pronounced for certain strains such as Ingbritt (serotype c) where there was a threefold increase in total LTA, although there was no increase for GS5 (serotype d). The current studies on S. sanguis employed four strains from the same serotype to remove one area of genotypic variability, but there was still a marked variation between the response of the different strains. Thus, there was a twofold increase in the cellular LTA content of strains Challis and 1558 when grown in fructose instead of glucose, but for the other two, such an effect was not obtained. In view of the above variations within a species, the variations in the response to fructose of strains representing different species of lactobacilli are not surprising; in this context, it should be noted that the strains of L. case represent two subspecies which differ in physiological and serological properties (1). The greatest effect of fructose is achieved with L. fermentum NCTC 6991 and L. case NIRD R94, where the differ- Downloaded from on January 31, 219 by guest

5 868 WICKEN ET AL. ences are much greater than could be accounted for by any minor variations in growth conditions. These two organisms are also the ones that have been studied in greater detail in the chemostat. For cultures grown in glucose at ph 6. and different dilution rates (generation times), the values for cellular LTA span the corresponding values for batch-grown cultures. A major difference, however, is obvious with the results for extracellular LTA, where the difference between L. fermentum and L. case was much greater than that for batch cultures. The amounts of L. fermentum extracellular LTA were also greatest at the lower dilution rates of.5 and.1 h-1 (corresponding to generation times of 14 and 7 h, respectively), a result which is similar to the result previously obtained with S. mutans BHT (6). In an earlier study on L. fermentum (11), it was shown that injection of fast-growing organisms (1.4-h generation time) into rabbits led to a significantly higher antibody production to the LTA component (group F antigen) compared with the corresponding results for slow-growing organisms (14 and 21 h generation times). Based on the current studies, these variations cannot be explained in terms of the amounts of detectable LTA but may relate to differences in surface-associated proteins, as the immunogenicity of LTA is influenced by the amount of associated protein (2), and studies with S. mutans Ingbritt have shown an effect of growth conditions on protein production (4). The ph of growth can also affect LTA production as shown by studies on S. mutans BHT (6) and Ingbritt (4, 5). With strain Ingbritt, the greatest increase in cellular and extracellular LTA was obtained at ph values greater than 7.. A similar effect has been obtained with L. fermentum, but for L. casei the effect on the total amount of LTA is minimal apart from an increase in the proportion of extracellular LTA at ph 8.. The effect of generation time and ph on the production of cellular and extracellular LTA means that the results obtained in batch culture represent a composite picture, and unless some controls are instituted, such as constant ph and collection of cultures at the end of the exponential phase, the interpretation of such results is rendered difficult. In addition to ph and generation time, previous studies on S. that LTA production in continuous culture is influenced by the carbohydrate source, particularly with a change from glucose to fructose (5). The current studies have shown that growth in limiting fructose in a chemostat influences LTA production, although the effect is not as marked mutans Ingbritt have shown INFECT. IMMUN. as for batch cultures with excess fructose. The major point to note is that the differences between fructose-grown L. fermentum and L. casei are much less than for the corresponding glucose-grown chemostat cultures. The reasons for the differences in the response of both batch and continuous cultures of L. fermentum and L. case may relate to differences in the mechanisms of carbohydrate uptake and metabolism. The yields of organisms (Table 3) certainly point to differences. At a dilution rate of.5 hi1, there were only minor variations, whereas at.5 hid, the yield of glucosegrown L. case was twice as high as for L. fermentum and increased by a further 5% in fructose. L. fermentum would not stabilize at this low dilution rate in fructose. When grown in batch culture, L. casei is a homofermenter, whereas L. fermentum is a heterofermenter, differences which contribute to their classification. Batch culture conditions more closely correspond to continuous culture at a high dilution rate, but whether the metabolic pathways are the same at low dilution rates is not known. With S. mutans it is known that slow-growing cultures do not display the typical properties of a homofermenter as acetate, formate, and ethanol are major fermentation products (3). A further important point is the mechanism of carbohydrate uptake. The phosphoenol pyruvate (PEP) sugar phosphotransferase plays an important role in sugar uptake in S. mutans, where its contribution is influenced by the growth conditions (3) and has also been detected in batch cultures of an L. case strain (16). A survey of a number of fermentative bacteria led to the conclusion that this uptake mechanism only occurs in homofermenters with the Embden-Meyerhof-Parnas mode of glucose fermentation and is lacking in heterofermenters with the Entner-Doudoroff pathway (16). Thus, it would be expected that L. fermentum would lack the PEP phosphotransferase system. A consequent difference in the cell's energy balance could account for differences in yield of cells and either directly or indirectly influence the synthesis of LTA, where the ultimate source of the glycerol phosphate component is dihydroxyacetone phosphate, an intermediate in the glycolytic pathway. ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia and U.S. Public Health Service grants RO1 DE 4174 and RO1 DE 4175 from the National Institute of Dental Research. LITERATURE CITED 1. Alkan, M., and E. Beachey Excretion of lipoteichoic acid by group A streptococci. J. Clin. Invest. 61: Downloaded from on January 31, 219 by guest

6 VOL. 36, 1982 LTA OF LACTOBACILLI AND STREPTOCOCCI de Man, J. C., M. Rogosa, and M. E. Sharpe A medium for the cultivation of lactobacilli. J. Apple. Bacteriol. 23: Hamilton, I. R., P. J. Phipps, and D. C. Ellwood Effect of growth rate and glucose concentration on the biochemical properties of Streptococcus mutans Ingbritt in continuous culture. Infect. Immun. 26: Hardy, L., N. A. Jacques, H. Forester, L. K. Campbell, K. W. Knox, and A. J. Wicken The effect of fructose and other carbohydrates on the surface properties, lipoteichoic acid production and extracellular proteins of Streptococcus mutans Ingbritt grown in continuous culture. Infect. Immun. 31: Jacques, N. A., L. Hardy, L. K. Campbell, K. W. Knox, J. D. Evans, and A. J. Wicken Effect of carbohydrate source and growth conditions on the production of lipoteichoic acid by Streptococcus mutans Ingbritt. Infect. Immun. 26: Jacques, N. A., L. Hardy, K. W. Knox, and A. J. Wicken Effect of growth conditions on the formation of extracellular lipoteichoic acid by Streptococcus mutans BHT. Infect. Immun. 25: Jacques, N. A., L. Hardy, K. W. Knox, and A. J. Wicken Effect of Tween 8 on the morphology and physiology of Lactobacillus salivarius strain IV CL-37 grown in a chemostat under glucose limitation. J. Gen. Microbiol. 119: Joseph, R., and G. D. Shockman The synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species. Infect. Immun. 12: Kessler, R. E., and G. D. Shockman Precursorproduct relationship of intracellular and extracellular lipoteichoic acids of Streptococcus faecium. J. Bacteriol. 137: Knox, K. W Isolation of group specific products from Lactobacillus case and L. case var. rhamnosus. J. Gen. Microbiol. 63: Knox, K. W., L. K. Campbell, K. W. Broady, and A. J. Wicken Serological studies on chemostat-grown cultures of Lactobacillus fermentum and Lactobacillus plantarum. Infect. Immun. 24: Knox, K. W., L. K. Campbell, J. D. Evans, and A. J. Wicken Identification of the group G antigen of lactobacilli. J. Gen. Microbiol. 119: Knox, K. W., M. J. Hewett, and A. J. Wicken Studies on the group F antigen of lactobacilli: antigenicity and serological specificity of teichoic acid preparations. J. Gen. Microbiol. 6: Knox, K. W., and A. J. Wicken Serological studies on the teichoic acids of Lactobacillus plantarum. Infect. Immun. 6: Markham, J. L., K. W. Knox, A. J. Wicken, and M. J. Hewett Formation of extracellular lipoteichoic acid by oral streptococci and lactobacilli. Infect. Immun. 12: Romano, A. H., J. D. Trifone, and M. Brustolon Distribution of the phosphoenolpyruvate: glucose phosphotransferase system in fermentative bacteria. J. Bact. 139: Rosan, B Relationship of the cell wall composition of group H streptococci and Streptococcus sanguis to their serological properties. Infect. Immun. 13: van Driel, D., A. J. Wicken, M. R. Dickson, and K. W. Knox Cellular location of the lipoteichoic acids of Lactobacillus fermentum NCTC 6991 and Lactobacillus case NCTC J. Ultrastruct. Res. 43: van Houte, J., and C. A. Saxton Cell wall thickening and intracellular polysaccharide in microorganisms of the dental plaque. Caries Res. 5: Wicken, A. J., J. W. Gibbens, and K. W. Knox Comparative studies on the isolation of membrane lipoteichoic acid from Lactobacillus fermentum. J. Bacteriol. 113: Wicken, A. J., and K. W. Knox A serological comparison of the membrane teichoic acids from lactobacilli of different serological groups. J. Gen. Microbiol. 67: Wicken, A. J., and K. W. Knox Lipoteichoic acids-a new class of bacterial antigens. Science 187: Wicken, A. J., and K. W. Knox Bacterial cell surface amphiphiles. Biochim. Biophys. Acta 64:1-26. Downloaded from on January 31, 219 by guest