Vol. 58, No. 11

INFECTION AND IMMUNITY, Nov. 1990, p. 3779-3787

0019-9567/90/113779-09$02.00/0

Copyright C) 1990, American Society for Microbiology

Streptococcus pyogenes Clinical Isolates and Lipoteichoic Acid OFRA LEON AND CHARLES PANOS* Department of Microbiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received 10 May 1990/Accepted 20 August 1990

Minimally subcultured clinical isolates of virulent nephritogenic and nonnephritogenic Streptococcus pyogenes of the same serotype showed major differences in lipoteichoic acid (LTA) production, secretion, and structure. These were related to changes in coccal adherence to and destruction of growing human skin cell monolayers in vitro. A possible relationship between cellular LTA content and group A streptococcal surface hydrophobicity was also investigated. Nephritogenic S. pyogenes M18 produced twice as much total (i.e., cellular and secretory) LTA as did the virulent, serologically identical, but nonnephritogenic isolate. Also, the LTAs from these organisms differed markedly. The polyglycerol phosphate chain of LTA from the nephritogenic isolate was longer (1.6 times) than was that from the nonnephritogenic isolate. Likewise, both LTAs indicated the presence of alanine and the absence of glucose. Amino sugars were found in LTA from only nephritogenic S. pyogenes. Teichoic acid, as a cellular component or secretory product, was not detected. The adherence of two different nephritogenic group A streptococcal serotypes (M18 and M2) exceeded that of the serologically identical but nonnephritogenic isolates (by about five times), indicating a correlation between virulent strains causing acute glomerulonephritis and adherence to human skin cell monolayers. Likewise, LTA from nephritogenic S. pyogenes M18 was more cytotoxic (1.5 times) than was that from the nonnephritogenic isolate for human skin cells, as determined by protein release. This difference was not perceptible by the more sensitive dye exclusion method (i.e., requiring less LTA), which emphasizes changes in host cell morphology and death. Also, the secretion of LTA by only virulent nephritogenic S. pyogenes M18 was exacerbated by penicillin (a maximum of four times). Finally, while the adherence of nephritogenic S. pyogenes M18 decreased markedly after continued subculturing in vitro, the surface hydrophobicity did not.

Pyrogenic exotoxin A has begun to reappear in certain virulent GAS (35). This toxin has several biological properties in common with streptococcal LTA. These include cytotoxicity, mitogenicity, and immunosuppression. LTA also induces the Shwartzman reaction. Therefore, the maladies indicated above and associated with the resurgent virulence of certain GAS may be due to an additive effect of pyrogenic exotoxin A and other secretions, including LTA. Given the variety of toxins produced by S. pyogenes, this effect is more than just a probability. Studies with fresh isolates of virulent and nonvirulent group B streptococci (GBS) from symptomatic and asymptomatic individuals have already revealed significant differences in the structure and production of their LTAs (22, 23). Also, adherence, like virulence, is a transitory property of only virulent GBS (20). Such information is not available for fresh isolates of S. pyogenes. Changes which enhance the potential role of LTA in disease are of prime importance for our understanding of the current resurgence of GAS virulence, and in this regard toxemia may be an important factor. This study compares the adherence and cytotoxicity of clinical isolates of virulent nephritogenic and nonnephritogenic GAS with differences in their LTA production, structure, and secretion.

A dramatic decline in the prevalence of serious infections caused by group A streptococci (GAS) has occurred during this century. However, streptococcal infections of surprising severity have now begun to reappear. For example, recent outbreaks of acute rheumatic fever among children and military recruits have been recorded (3, 4, 38, 39). In a regional outbreak within the United States, patients with severe GAS infections had soft tissue infections, shock, renal impairment, and acute respiratory distress syndrome. In addition, a mortality rate of 30% was noted. The strains of Streptococcus pyogenes isolated were not of a single serotype. In addition, most produced pyrogenic exotoxin A, a toxin not commonly observed in the recent past (35). Severe GAS outbreaks were also recently reported in Great Britain (9). These were related to changes in other GAS virulence factors. In at least one of these outbreaks, host factors did not appear to explain the increased severity of these streptococcal infections (35). A current consensus is that this resurgence is associated with the emergence of more virulent

organisms. A prerequisite for successful bacterial infection in vivo is adherence to a susceptible host cell. Considerable data have now accumulated indicating that the adherence mechanism within the pathogenic GAS involves lipoteichoic acid (LTA) (2, 6, 26, 41). LTA is a cellular component as well as a secretory product of S. pyogenes. In addition to being amphipathic and amphoteric, LTA is highly cytotoxic for a variety of growing eucaryotic cell monolayers and for glomeruli in tissue cultures (6, 15, 36). Deacylation of LTA abolishes its cytotoxicity. Likewise, treatment of host cells with LTA or intact cells of S. pyogenes with anti-LTA serum

MATERIALS AND METHODS Bacteria and LTA. With one exception, all organisms were virulent isolates. The designations of each as used here, including serotype and pertinent isolation details, were as follows: M18AGN from the throat of a patient with acute glomerulonephritis (AGN), M18NAGN from the peritoneal fluid of a patient with bacteremia and peritonitis (indicating its invasive property) but without AGN (i.e., NAGN), and M2AGN from a patient with AGN. M2NAGN, the NAGN control, was from a classmate of a patient with rheumatic

negates coccal adherence. * Corresponding author. 3779

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LEON AND PANOS

fever. These organisms were supplied by E. Kaplan, World Health Organization Collaborating Center for Reference and Research on Streptococci, University of Minnesota. Additional details and the source of each isolate may be requested from that center. The corresponding center numbers for these strains (in parentheses) were as follows: M18AGN (88-145), M18NAGN (88-126), M2AGN (87-407), and M2NAGN (88-120). From the time of patient isolation until use in these studies, none of these isolates was transferred more than 10 times. Frozen stock inocula of each isolate were prepared, stored, and treated before use exactly as detailed previously (20). When necessary, each experiment was initiated from an original stock inoculum. LTA was isolated from intact M18AGN and M18NAGN cells by the cold phenol method and purified as detailed previously (10). Its high purity was similar to those of previous LTA preparations from S. pyogenes and S. agalactiae (e.g., 0.35% protein, no nucleic acids) (10). Culture media. Viable counts were determined with ToddHewett broth (THB; BBL Microbiology Systems, Cockeysville, Md.) with agar (1.5% [wt/vol] purified Bacto-Agar; Difco, Detroit, Mich.) added. Adherence studies were done with organisms grown in THB (37°C, 5% [vol/vol] inoculum) and harvested during the mid-logarithmic phase of growth (about 3 h). The organisms were harvested by centrifugation (4,340 x g, 10 min) and washed twice with phosphatebuffered saline (PBS) before use. For biochemical studies, 10- to 15-liter batches of THB were inoculated with organisms (5% [vol/vol]) and inoculated overnight at 37°C, corresponding to the late stationary phase of growth. Cells were harvested by centrifugation (6,130 x g, 20 min), washed twice with 0.9% (wt/vol) saline and once with distilled water, and lyophilized. Coccal generation times were calculated from viable counts obtained by inoculating a mid-logarithmic-phase culture of each organism (5% [vol/vol], 2.5 x 108 CFU) into 50 ml of THB in an Erlenmeyer flask (125 ml) equipped with a side arm for determination of the optical density (OD) at 500 ,um and by plating replicates periodically for 24 h. Tissue culture cells. Human (21-year-old male) primary skin (CRL-1474/CCD-25SK) and established mouse fibroblast (CCL-1/L-929) cells from the American Type Culture Collection, Rockville, Md., were used. Skin cells were received at passage number 6 and were used in experiments at passages 9 to 12. These cells were propagated in minimal essential medium (MEM) containing 20% (vol/vol) heatinactivated fetal bovine serum (MEM-20), 2 mM L-glUtamine, 0.1 mM nonessential amino acids, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml. Mouse fibroblasts were grown in the same medium but with 10% (vol/vol) serum and without the nonessential amino acids added. The final pH of all media was 7.2. Media and reagents were from GIBCO Laboratories, Grand Island, N.Y. Both cell lines were grown in flat, 75-cm2, 250-ml plastic bottles (Corning Works, Corning, N.Y.) at 37°C in at atmosphere of CO2 (5%) and air (95%). Viable cell counts were determined with erythrosine B and a hemacytometer. Streptococcal adherence. This assay was conducted as detailed previously except that 104 human skin cells in 1 ml of MEM-20 were seeded into glass Leighton tubes (10, 20). Also, 1 ml of a mid-logarithmic-phase suspension of approximately 2.5 x 108 CFU of each S. pyogenes isolate per ml was prepared and used as described before (20). The distinct advantages of this assay have been detailed previously (10, 20). Results are expressed as the number of attached bacte-

INFECT. IMMUN.

ria and the number of human cells binding bacteria per 100 tissue culture cells. Cytotoxicity studies with LTA. Cytotoxicity was evaluated by measuring radioactivity and viability. For radiolabeling experiments, a confluent monolayer of human skin cells grown in 75-cm2 bottles with supplemented MEM-20 (see above) was trypsinized and 104 cells in 1 ml of medium were seeded into each of 24 wells (Linbro 76-033-05; well area, 2.0 cm 2; Flow Laboratories, Inc., Hamden, Conn.). Incubation for 24 h at 37°C in 5% C02-95% air provided a subconfluent monolayer. Spent medium was removed, and 1 ml of fresh medium containing 2.6 ,uCi L-[355]methionine per ml was added (specific activity, 1,156 Ci/mmol; 10 mCi/ml of H20; ICN Biomedicals Inc., Irvine, Calif.; stored as 1-ml aliquots of 1 mCi/ml of H20 at -80°C and further diluted with MEM-20 to a concentration of 2.6 ,uCi/ml before use). After each monolayer was labeled for 24 h, the medium was removed and each monolayer was washed twice with PBS. Next, 0.5 ml of fresh MEM-0 (i.e., without serum) and 0.5 ml of MEM-0 with various concentrations of LTA adjusted to pH 7.2 with HCO3 were added. Medium without serum was necessary to prevent overgrowth and to maintain subconfluency. Normal skin cell morphology could not be maintained without serum for more than 24 h. All solutions of LTA were prepared prior to use; the concentrations tested ranged from 1 to 200 ,ug per well. After 24 h of incubation with LTA, monolayers in all wells were washed twice with PBS and digested with 0.1 ml of 1 N NaOH for 30 min at 37°C. The digests and the well washings (twice with 0.5 ml of PBS) were combined, transferred to scintillation vials (10 ml of Bray cocktail), and counted in an LKB 1209 Rackbeta liquid scintillation counter. Percent cytotoxicity was calculated as described before (15). Cytotoxicity was also determined by measuring the decline in viability in the multiple-well plates described above. Seeded human skin or mouse fibroblast cells (104 per well) were incubated for 48 and 24 h, respectively, and then refed with MEM-0 containing various concentrations of LTA as described above. After 24 h in the presence of LTA, viability was assessed as follows. Medium was removed, each monolayer was washed twice with PBS, and 0.5 ml of PBS was added to all wells. Following the addition of 0.05 ml of erythrosine B and incubation at room temperature for 20 min, live versus dead cells were counted at a magnification of x 100 with a Nikon inverted microscope equipped with a 21-mm reticle eyepiece, with a control monolayer showing approximately 500 cells. Dead cells were defined as cells retaining the stain. Percent toxicity was calculated as follows: percent toxicity = [(number of cells without LTA number of cells with LTA)/number of cells without LTA] x 100. Secretion of LTA and TA. Direct and indirect passive hemagglutination assays (PHA) were performed essentially as described previously with purified LTA from S. pyogenes M18AGN serving as the standard (21, 26). In brief, the sensitizing activity of LTA in the medium was measured by direct PHA with sheep erythrocytes, whereas indirect PHA assessed the antigenic activity of secreted LTA and teichoic acid (TA). Differences between antigenic and sensitizing activities were taken as the amount of TA present in the medium. For these studies, the anti-LTA serum used previously was used again. This antiserum had been prepared in rabbits with purified LTA from S. agalactiae (10). It crossreacted with LTA from this pathogenic group B type III coccus and a nephritogenic type 12 S. pyogenes to the same extent (titer, 1:256) in PHA with rabbit erythrocytes.

S. PYOGENES CLINICAL ISOLATES AND LTA

VOL. 58, 1990

Secretion of glycerol-labeled LTA with and without penicillin was also determined. First, S. pyogenes M18 isolates were grown overnight in THB (30 ml) containing 10 ,uCi of [3H]glycerol per ml (specific activity, 500 mCi/mmol; Amersham Corp., Arlington Heights, Ill.). Cultures were centrifuged (4,340 x g, 10 min), washed three times with 10 ml of PBS, suspended in fresh THB (30 ml), and divided into 3-ml portions. These portions, with or without penicillin (for concentrations, see Fig. 6) were incubated at 37°C for 3 h. Aliquots of each were centrifuged, the supernatants were inactivated at 56°C for 30 min, and 0.5 ml of each supernatant was counted as described above for assessment of glycerol secretion. Next, aliquots (100 pul) of each supernatant were mixed with 100 ,ul of sheep erythrocytes (108/ml) and incubated at 37°C for 3 h with occasional mixing in Beckman Microfuge tubes. The binding of LTA to sheep erythrocytes was stopped by centrifugation in a Beckman Microfuge B for 2.5 min. Erythrocytes were washed twice with 100 ,ul of PBS, the washes were added to the supernatants, and each was counted as described before. Finally, remaining sheep erythrocytes were lysed with distilled water (100 ,ul), and the lysed cells were transferred to scintillation vials. Also, all Microfuge tubes were washed with 1 N NaOH (100 pu1), and the washes were added to erythrocyte preparations. All erythrocyte-NaOH mixtures were allowed to digest by incubation for 15 min at room temperature before being bleached with 300 p.l of 30% (vol/vol) H202. Radioactivity was measured as described above after the addition of Bray cocktail. Remaining medium aliquots were used to determine LTA and TA contents by direct and indirect PHA (see above). Differences in the hydrophobicity of intact coccal cells. The method of Rosenberg et al. was used to measure hydrophobicity (29). Ten-milliliter cultures (mid-logarithmic and stationary phases) of S. pyogenes M18AGN and M18NAGN were grown in THB. Cells were obtained by centrifugation (4,340 x g, 10 min) and washed twice with 10 ml of PUM buffer (0.1 M potassium phosphate containing 1.8 g of urea and 0.2 g of MgSO4 7H20 per 1,000 ml; final pH, 7.1). After being suspended in this buffer, cells were vortexed to disperse any clumping before being adjusted to an OD at 450 p.m of 0.500 with a Spectronic 20 spectrophotometer (Bausch & Lomb, Rochester, N.Y.). Three-milliliter suspensions of each isolate were placed in a series of 15-ml glass tubes, and increasing volumes of p-xylene (25 to 200 ,ul) were added (see Fig. 7). Each mixture was vortexed for 60 s and allowed to separate at room temperature for 15 min. Comparably aged cells of a laboratory strain of Escherichia coli grown in THB served as a control. The OD of aqueous layers was read at 450 ,um, and changes in hydrophobicity were calculated as follows: percent hydrophobicity = 100 - [(100 x OD after treatment)/OD before treatment]. LTA structure. Chain length determinations (i.e., total phosphorus/organic phosphorus) of each purified LTA were done by alkaline phosphatase digestion as described before (33). The composition of each LTA was established after acid hydrolysis (1 mg of LTA and 0.5 ml of 2 N HCl in a sealed tube at 100°C for 3 h) by descending paper chromatography (n-butanol-pyridine-water, 90:60:45 [vol/vol/vol]). Amino acids and amino sugars were detected by dipping in ninhydrin. Glycerol and sugars were also visualized with ammoniacal AgNO3. One microgram of each standard (alanine, glycerol, glucose, glucosamine, and N-acetyl-D-glucosamine) was easily discernible by this procedure (10, 33, 34). Estimation of the phosphorus/amino sugar ratio of LTA was achieved by visual comparison of chromatograms with -

3781

TABLE 1. Cell-associated and secreted LTA yields of virulent S. pyogenes M18 isolatesa Cell-associated LTA Crude extract Dry cell rafter phenol Pure (mg) wt (g) extraction (mg)'

8 M-Dyygenes

S.

8

AGN NAGN

15.21 17.10

Secreted LTA

Total LTA

(mg)b

(mg)

310.05 (17.86) 77.60 (8.75) 1,440.00 1,517.60 145.96 (13.90) 38.65 (5.68) 720.00 758.65

aPooled batch culture (total, 45 liters); overnight incubation in THB. Determined by PHA. ' Numbers in parentheses represent weight in milligrams of phosphorus. b

incremental increases in the standard (glucosamine) and the hydrolysate (1 to 100 and 5 to 100 ,ug per spot, respectively) and by phosphorus equivalency calculations (see Table 1). Statistics. Statistical analysis was performed with the paired Student t test. RESULTS LTA yields. The generation time of S. pyogenes M18AGN in THB was 20 min; that of S. pyogenes M18NAGN was 40 min. The yields of LTA from these clinical isolates are shown in Table 1. The cellular content of LTA was greater in the former (0.51%) than in the latter (0.23%). Also, the actual amounts of LTA secreted by both isolates were larger than the amounts retained by each as a cellular component, with M18AGN secreting still more (2.13 times) LTA on an equivalent-cell-weight basis. Finally, the total amounts of LTA produced (i.e., cellular plus secreted) by these organisms differed markedly. Overall, M18AGN produced 2.3 times more total LTA than did M18NAGN. The calculated phosphorus equivalency of purified cellular LTA from each of these two clinical isolates was not the same, indicating a difference in molecular weight between their LTAs. One milligram of LTA phosphorus was found to be equivalent to 8.9 and 6.8 mg of LTA from the virulent AGN and NAGN S. pyogenes isolates, respectively (Table 1). Exhaustive studies failed to indicate the secretion of TA (or deacylated LTA) by either of these M18 clinical isolates (see below). LTA structure. Analyses of purified LTA from these M18 clinical isolates substantiated the molecular difference indicated by the phosphorus equivalency calculations. Chain length determinations of LTA from the AGN coccus revealed a chain length of 25 + 1.0 glycerol-phosphate units; the chain length for the NAGN coccus was 16 + 0.7 units (n = 2). In addition, compositional differences were documented. Alanine was barely perceptible and glucose was not detected in acid hydrolysates of LTA from either isolate by paper chromatography. Only LTA from AGN S. pyogenes contained traces of glucosamine and an unidentified but more pronounced amino sugar. The estimated phosphorus/ total amino sugar ratio of this LTA was 1:0.44, with the unidentified but slower-moving (Rf, 0.13) amino sugar being in excess of glucosamine (Rf, 0.25). The method used for the hydrolysis of LTA deacylated N-actylglucosamine (Rf, 0.58) to glucosamine, precluding the establishment of this acetylated derivative as the hexosamine of LTA in this AGN S. pyogenes isolate. Streptococcal adherence to human skin cells. Maximal coccal adherence was achieved with subconfluent monolayers and GAS grown to the mid-logarithmic phase of growth. Two sets of AGN and NAGN S. pyogenes isolates of different serotypes (M18 and M2 GAS) were examined. Two

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LEON AND PANOS 12]

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FIG. 2. Streptococcal distribution per 100 human skin cells binding cocci in tissue cultures. Shown are data for clinical isolates of S. pyogenes M18AGN (M) and M18NAGN (n). Values are averages for one experiment done in duplicate.

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FIG. 1. Streptococcal adherence to human skin cell monolayers in tissue cultures. AGN, Acute glomerulonephritogenic S. pyogenes M18 and M2 isolates; NAGN, nonacute glomerulonephritogenic S. pyogenes M18 and M2 isolates; V(1,2), recent human (neonatal) isolates of virulent S. agalactiae type III (controls; see also reference 20). Values are averages + standard deviations for six experiments done separately.

virulent neonatal clinical isolates of S. agalactiae type III served as controls for adherence specificity, while avirulent M2 was the NAGN control. Since there were no significant differences in adherence within the different coccal groups examined (P c 0.05), data for each group were combined and are presented as averages (Fig. 1). A mean of 48% of the human skin cell population after 24 h of growth was capable of binding S. pyogenes M18AGN and M2AGN (Fig. 1A). Their virulent and avirulent NAGN counterparts and the virulent GBS adhered to not more than 20% of this same cell population. Figure 1B shows the actual CFU (i.e., chains) of each of these coccal groups adhering to 100 human skin cells. Again, the AGN isolates far exceeded the NAGN controls in binding to these host cells. Of note was the almost complete lack of adherence by the virulent GBS controls. Finally, stationary-phase cells of the AGN GAS adhered to the same extent as did mid-logarithmic-phase cells of the NAGN GAS (Fig. 1).

Figure 2 quantitates a typical distribution difference in adherence between the M18AGN and M18NAGN isolates by only that segment of the human skin cell population binding these GAS (approximately 55 and 15%, respectively; see also Fig. 1A). For example, the CFU bound per host cell ranged from 1 to 22. While approximately 10% of the adherent skin cell population was able to bind the maximum number of AGN coccal chains (17 to 22 CFU per cell), none bound the NAGN isolate to this extent. Finally, the AGN isolate was also more uniformly distributed among the adherent host cell population than was the NAGN isolate. Table 2 shows the results of treatments that interfere with coccal adherence. Treatment of S. pyogenes M18AGN with anti-LTA serum or human skin monolayers with LTA inhibited the CFU binding to host cells almost completely. Likewise, the percentage of host cells receptive to this pathogen after each of these treatments was also lowered by approximately 80%. Effect of daily transfers in vitro on coccal adherence to TABLE 2. Cell pretreatments and changes in adherence of S. pyogenes M18AGN' Pretreatment

bound/100 host cells

Mean + SD CFU

SD cells % Mean +host receptive

None Normal rabbit serum (1:128 final dilution) Anti-LTA serum (1:128 final dilution) LTA treatment of skin cell monolayers

379.8 ± 40.8 386.7 ± 35.9 (2)

57.2 ± 6.3 55.2 + 7.6 (2)

5.0 ± 2.8 (2)

11.0 ± 4.2 (2)

7.5 ± 2.1 (2)

10.5 ± 5.0 (2)

a The duration of pretreatments was 30 min in PBS at 37°C. Untreated cells were kept in PBS for the corresponding time period. The numbers of cover slips counted in separate experiments are shown in parentheses. Growing skin monolayers were 24 h old; the coccus was at its mid-logarithmic phase of

growth.

S. PYOGENES CLINICAL ISOLATES AND LTA

VOL. 58, 1990 500 -

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75

100

125

150

175

200

XYLENE CONCENTRATION (i)i) FIG. 7. Changes in the hydrophobicity of S. pyogenes M18 in xylene after 6 (-) and 26 (----) consecutive daily transfers. Symbols: -, AGN cocci; *, NAGN cocci; A, E. coli (control). Values are averages for two separate experiments done in duplicate (standard deviations are not always perceptible).

E 4000-

3 l -2 01 PENi C-I4 PENICILLIN CONCENTRATION

25

1

1

FIG. 6. Assays establishing enhancement of the secretion of radiolabeled LTA from S. pyogenes M18 by penicillin. Intact cells were labeled with [3H]glycerol (see the text). (A) [3H]glycerol secretion. (B) Erythrocyte binding of secreted material. (C) LTA secretion (as determined by direct PHA). Symbols: O, AGN cocci; NAGN cocci. U,

vitro (15, 16). Basement membrane thickening, fusion of epithelial foot processes, etc. also occur in the mouse kidney after repeated injections of native LTA from nephritogenic S. pyogenes (17). Likewise, GAS LTA is believed to regulate diverse functions of mammalian cells by phosphorylating the tyrosine residues of certain proteins (8). Therefore, profuse production and secretion of LTA can only potentiate the pathogenesis of coccal disease. Glucose was found to be a structural component of the hydrophilic and hydrophobic portions of LTA of S. pyogenes type 12 (33, 34). Also, an earlier study reported an amino sugar as the only hexose in glycerol TA from type 3 GAS (18). Glucose was not detected in the LTAs of the virulent GAS M18AGN and M18NAGN isolates in this investigation. Instead, only the former contained small amounts of two amino sugars. The significance of this difference in LTA cytotoxicity is obscure, since LTA from the GAS M18NAGN isolate (without detectable carbohy-

drate) was also cytotoxic, albeit to a lesser degree (see below). Also, in an earlier study, S. pyogenes type 12 LTA with a minimal carbohydrate content was highly cytotoxic for a variety of human and animal cells in vitro (27). Nevertheless, these findings imply that the carbohydrate composition within the hydrophilic portion of the LTA of the GAS is not uniform. It has also been shown that the hydrophilic chain length of LTA can profoundly affect its biological activity (7). Thus, the significantly longer chain length of LTA from the AGN isolate may be one of several factors instrumental in its increased cytotoxicity. Similar differences in length between LTAs from virulent (30 to 35 glycerol phosphate units) and avirulent (10 to 12 glycerol phosphate units) human isolates of S. agalactiae type III have been documented (24). A phosphoglucolipid was established as the hydrophobic component of the LTA of S. pyogenes type 12 (33). However, the compositional differences between the virulent GAS M18 isolates indicate that the complex lipid of the LTA from S. pyogenes need not be a phosphoglucolipid. The differences in the adherence of different serotypes of virulent AGN and NAGN GAS to growing human skin cells were striking. This limited study indicated a correlation between virulent strains causing AGN and adherence to human skin cells. The lack of a similar affinity of the NAGN strains may be due to differences in LTA production and/or exposure (or availability) on the surface of the organism. In turn, it may be related to the structural and compositional differences already mentioned between the GAS M18 isolates. Clearly, although major differences have now been documented in the LTAs from these virulent GAS isolates, a more detailed chemical and structural characterization, especially of the hydrophobic portion, is still needed. Finally, the results of treatment of host cells with coccal LTA or cocci exposed to anti-LTA serum proved that LTA is still involved in the adherence of fresh clinical isolates of AGN GAS to human cells in vitro. Only a certain population within the randomly growing human skin cell monolayer was capable of binding AGN GAS, indicating a limited cyclic expression of receptors on

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LEON AND PANOS

the surfaces of these primary human cells in vitro. While this has indeed been shown to be the case with certain human cells infected with virulent GBS, it is not true of all human cells. Human fetal lung cells, for example, do not exhibit this expression (10, 20). Continuing to transfer an AGN GAS isolate in vitro led to a sudden and almost complete loss of adherence. These data resembled those obtained with the virulent GBS (20). Like virulence, adherence is a transitory property of pathogenic GAS which needs to be preserved if comparative studies of virulent isolates are to be meaningful. Fortunately, adherence of the AGN coccus in vitro remained fairly constant for a period of time before declining rapidly. Thus, a true assessment of this characteristic is still possible after primary isolation and multiple but not indefinite transfer in

vitro. The cytotoxicity of LTA in vitro was established in 1978 (6). Some earlier evidence, however, had suggested that LTA might also be mobile in vivo. An affinity of LTA for kidney tissue was shown by inducing nephrocalcinosis in rabbits after injection of GAS LTA. LTA migration (and affinity) was demonstrated by labeled antibody (40). A recent study with mice injected by different routes with purified coccal LTA tended to confirm this ability to migrate (17). Even earlier, in 1969, streptococcal membrane constituents (and presumably LTA) in the glomeruli of human patients with acute poststreptococcal glomerulonephritis were detected with fluorescent antibody (37). LTA is now known to function as a carrier for streptolysin S. Also, serum albumin binds LTA reversibly and in a specific way and is believed to be a carrier of this amphiphile (32). Therefore, secreted LTA is probably also involved in coccal pathogenesis. Direct contact of membranous and secreted LTA with the host cell surface occurs during and after coccal adherence. Thus, direct transfer of LTA to a host cell during adherence seems likely. Under these conditions, the time required for cytotoxic effects to begin would be less than that following secretion and transportation of LTA to a distant susceptible organ (e.g., the kidneys). Thus, secretion and transportation, plus the direct transfer of LTA to a host cell during adherence, can enhance the destructive effects of this amphiphile in vivo. It has been proposed that coccal LTA in a micellar state may be necessary for disruption of the erythrocyte membrane and for destruction of eucaryotic cells in vitro. Critical micelle concentrations of LTAs from several bacteria were calculated to be 25 to 60 ,ug/ml in PBS (5). The amount of LTA (32 ,ug/ml) normally secreted by the M18AGN GAS isolate (and increased with penicillin; Fig. 6) is within this range. However, arguing against a critical micellar concentration is the fact that appreciably smaller quantities of LTA (8 to 15 ,ug/ml) also caused extensive human cell damage in vitro (see Results). It was established that the GAS secrete LTA and deacylated LTA and that penicillin stimulates this process (1, 11, 14). Also, coccal adherence to host cells is decreased with the loss of LTA. This release of LTA (and lipids) by penicillin is believed to be an "active process rather than a correlate of viability loss, since streptococci tolerant to penicillin also exhibit penicillin-induced release of cell surface components" (11). The present data not only confirm the enhanced secretion of LTA by penicillin but illustrate that the increase is most pronounced in only the M18AGN GAS isolate. Finally, while others observed the secretion of deacylated LTA by GAS, we did not detect this product by serological means in the medium of the virulent M18 clinical

INFECT. IMMUN.

isolates in the presence or absence of penicillin. Apparently not all GAS secrete deacylated LTA. The dramatic differences obtained with the two methods for quantitating LTA cytotoxicity suggest that host cell death (by dye exclusion) occurs before the release of cellular protein with smaller quantities of LTA, which may result from the inhibition of a select enzyme activity (16). Higher concentrations of LTA lead to cell protein leakage after cell death. Thus, a dual cytotoxic effect of LTA which entails a physical perturbation of the host cell membrane is postulated. The differences also suggest that different host cells have different sensitivities to LTA. These findings agree with previous results showing greater changes in cell morphology than in protein release with lower concentrations of LTA. Thus, while bacteria release little or no protein after penicillin treatment, eucaryotic cells lose more protein as the concentration of LTA increases. Hydrophobicity studies with other streptococci showed that slowly growing cells were more hydrophobic than were rapidly growing cells (28) and that bacterial hydrophobicity can be markedly affected by the medium. Since it is generally agreed that a number of mechanisms particularly related to surface characteristics (surface charge, hydrogen bonding, ligands, and hydrophobic bonding) play a role in bacterial adherence, the cell surface hydrophobicities of these coccal clinical isolates were compared. Contrary to previous findings, the more rapidly growing AGN isolate was more hydrophobic than was the slowly growing S. pyogenes M18NAGN in the same medium. Since the AGN coccus contained more membranous LTA and adhered better to human skin cells, suggesting a greater exposure of its surface LTA, these hydrophobicity results were expected. However, unexpected was the lack of change in the hydrophobicity with a loss in the adherence of the AGN isolate after continued daily subtransfers. Apparently, the cell surface hydrophobicity of the AGN isolate is not a determining factor in its adherence to growing human skin cell monolayers in vitro. Nevertheless, the difference in the hydrophobicity of virulent M18AGN and M18NAGN GAS is not due to the presence or absence of a capsule. These collective studies illustrate important biological and biochemical differences between AGN and NAGN clinical isolates of GAS. Also, for the AGN coccus they reveal a change in a parameter directly related to pathogenesis shortly after primary isolation. Such an alteration must be considered for comparative studies in vitro to be meaningfully related to differences in GAS pathogenesis in vivo. ACKNOWLEDGMENTS This investigation was funded by the Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University. We thank E. Kaplan for supplying the clinical isolates used in this investigation. LITERATURE CITED 1. Alkan, M. L., and E. H. Beachey. 1978. Excretion of lipoteichoic acid by group A streptococci. J. Clin. Invest. 61:671-677. 2. Beachey, E. H. 1981. Bacterial adherence: adhesin receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143:325-345. 3. Centers for Disease Control. 1987. Acute rheumatic feverUtah. Morbid. Mortal. Weekly Rep. 36:108-115. 4. Centers for Disease Control. 1988. Acute rheumatic fever at a navy training center-San Diego, California. Morbid. Mortal. Weekly Rep. 37:101-104. 5. Courtney, H. S., W. A. Simpson, and E. H. Beachey. 1986.

VOL. 58, 1990

Relationship of critical micelle concentrations of bacterial lipoteichoic acids to biological activities. Infect. Immun. 51:414418. 6. DeVuono, J., and C. Panos. 1978. Effect of L-form Streptococcus pyogenes and of lipoteichoic acid on human cells in tissue culture. Infect. Immun. 22:255-265. 7. Fiedler, F. 1981. On the participation of lipoteichoic acid in the biosynthesis of wall teichoic acid, p. 195-208. In G. D. Shockman and A. J. Wicken (ed.), Chemistry and biological activities of bacterial surface amphiphiles. Academic Press, Inc., New York. 8. Ganguly, C. L., J. B. Dale, H. S. Courtney, and E. H. Beachey. 1985. Tyrosine phosphorylation of a 94-kDA protein of human fibroblasts stimulated by streptococcal lipoteichoic acid. J. Biol. Chem. 260:13342-13346. 9. Gaworzewska, E., and G. Colman. 1988. Changes in the pattern of infection caused by Streptococcus pyogenes. Epidemiol. Infect. 100:257-269. 10. Goldschmidt, J. C., Jr., and C. Panos. 1984. Teichoic acids of Streptococcus agalactiae: chemistry, cytotoxicity, and effect on bacterial adherence to human cells in tissue culture. Infect. Immun. 43:670-677. 11. Gutmann, L., and A. Tomasz. 1982. Penicillin-resistant and penicillin-tolerant mutants of group A streptococci. Antimicrob. Agents Chemother. 22:128-136. 12. Harrop, P. J., R. L. O'Grady, K. W. Knox, and A. J. Wicken. 1980. Stimulation of lysosomal enzyme release from macrophages by lipoteichoic acid. J. Peridontal Res. 15:492-501. 13. Hauseman, E., 0. Luderitz, K. Knox, A. Wicken, and N. Weinfeld. 1975. Structural requirements for bone resorption by endotoxin and lipoteichoic acid. J. Dent. Res. 54:B94-B99. 14. Horne, D., R. Hakenbeck, and A. Tomasz. 1977. Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J. Bacteriol. 132:704-717. 15. Leon, O., and C. Panos. 1983. Cytotoxicity and inhibition of normal collagen synthesis in mouse fibroblasts by lipoteichoic acid from Streptococcus pyogenes type 12. Infect. Immun. 40:785-794. 16. Leon, O., and C. Panos. 1985. Effect of streptococcal lipoteichoic acid on prolyl hydroxylase activity as related to collagen formation in mouse fibroblast monolayers. Infect. Immun. 50: 745-752. 17. Leon, O., and C. Panos. 1987. An electron microscope study of kidney basement membrane changes in the mouse by lipoteichoic acid from Streptococcus pyogenes. Can. J. Microbiol. 33:709-717. 18. Matsuno, T., and H. D. Slade. 1970. Composition and properties of a group A streptococcal teichoic acid. J. Bacteriol. 102:747752. 19. Miller, G., A. J. Urban, and R. W. Jackson. 1976. Effects of a streptococcal lipoteichoic acid on host responses in mice. Infect. Immun. 13:1408-1417. 20. Miyazaki, S., 0. Leon, and C. Panos. 1988. Adherence of Streptococcus agalactiae to synchronously growing human cell monolayers without lipoteichoic acid involvement. Infect. Immun. 56:505-512. 21. Nealon, T. J., E. H. Beachey, H. S. Courtney, and W. A. Simpson. 1986. Release of fibronectin-lipoteichoic acid complexes from group A streptococci with penicillin. Infect. Immun. 51:529-535. 22. Nealon, T. J., and S. J. Mattingly. 1983. Association of elevated levels of cellular lipoteichoic acids of group B streptococci with human neonatal disease. Infect. Immun. 39:1243-1251. 23. Nealon, T. J., and S. J. Mattingly. 1984. Role of cellular lipoteichoic acids in mediating adherence of serotype III strains of group B streptococci to human embryonic, fetal, and adult

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epithelial cells. Infect. Immun. 43:523-530. 24. Nealon, T. J., and S. J. Mattingly. 1985. Kinetic and chemical analyses of the biologic significance of lipoteichoic acids in mediating adherence of serotype III group B streptococci. Infect. Immun. 50:107-115. 25. Ne'eman, J., and I. Ginsberg. 1972. Red cell sensitizing antigen of group A streptococci. Immunological and immunopathological properties. Isr. J. Med. Sci. 8:1807-1816. 26. Ofek, I., E. H. Beachey, W. Jefferson, and G. L. Campbell. 1975. Cell membrane-binding properties of group A streptococcal lipoteichoic acid. J. Exp. Med. 141:990-1003. 27. Panos, C. 1986. Macromolecular synthesis, survival, and cytotoxicity of an L-form of Streptococcus pyogenes, p. 59-97. In S. Madoff (ed.), The bacterial L-forms. Marcel Dekker, Inc., New York. 28. Rogers, A. H., K. Pilowsky, and P. S. Zilm. 1984. The effect of growth rate on the adhesion of the oral bacteria Streptococcus mutans and Streptococcus milleri. Arch. Oral Biol. 29:147-150. 29. Rosenberg, M., D. Gutnick, and E. Rosenberg. 1980. Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol. Lett. 9:29-33. 30. Silvestri, L. J., K. W. Knox, A. J. Wicken, and E. M. Hoffman. 1979. Inhibition of complement-mediated lysis of sheep erythrocytes by cell-free preparations from Streptococcus mutans BHT. J. Immunol. 122:54-60. 31. Simpson, W. A., J. B. Dale, and E. H. Beachey. 1982. Cytotoxicity of the glycolipid region of streptococcal lipoteichoic acid for cultures of human heart cells. J. Lab. Clin. Med. 99:118-126. 32. Simpson, W. A., I. Ofek, and E. H. Beachey. 1980. Binding of streptococcal lipoteichoic acid to the fatty acid binding sites of serum albumin. J. Biol. Chem. 255:6092-6097. 33. Slabyj, B. M., and C. Panos. 1973. Teichoic acid of a stabilized L-form of Streptococcus pyogenes. J. Bacteriol. 114:934-942. 34. Slabyj, B. M., and C. Panos. 1976. Membrane lipoteichoic acid of Streptococcus pyogenes and its stabilized L-form and the effect of two antibiotics upon its cellular content. J. Bacteriol. 127:855-862. 35. Stevens, D. L., M. H. Tanner, J. Winship, R. Swarts, K. M. Ries, P. M. Schlievert, and E. Kaplan. 1989. Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A. N. Engl. J. Med. 321:1-7. 36. Tomlinson, K., 0. Leon, and C. Panos. 1983. Morphological changes and pathology of mouse glomeruli infected with a streptococcal L-form or exposed to lipoteichoic acid. Infect. Immun. 42:1144-1151. 37. Treser, G., M. Semar, M. McVicar, M. Franklin, A. Ty, I. Sagel, and K. Lange. 1969. Antigenic streptococcal components in acute glomerulonephritis. Science 163:676-677. 38. Veasy, L. G., S. E. Wiedmeier, G. S. Orsmond, H. D. Ruttenberg, M. M. Boucek, S. J. Roth, V. F. Tait, J. A. Thompson, J. A. Daly, E. L. Kaplan, and H. R. Hill. 1987. Resurgence of acute rheumatic fever in the intermountain area of the United States. N. Engl. J. Med. 316:421-427. 39. Wald, E. R., B. Dashefsky, C. Feidt, D. Chiponis, and C. Byers. 1987. Acute rheumatic fever in western Pennsylvania and the tri-state area. Pediatrics 80:371-374. 40. Waltersdorf, R. L., B. A. Fiedel, and R. W. Jackson. 1977. Induction of nephrocalcinosis in rabbit kidneys after long-term exposure to a streptococcal teichoic acid. Infect. Immun. 17: 665-667. 41. Wicken, A. J. 1980. Structure and cell membrane-binding properties of bacterial lipoteichoic acid and their possible role in adhesion of streptococci to eukaryotic cells, p. 137-158. In E. H. Beachey (ed.), Bacterial adherence, receptors, and recognition, series B, vol. 6. Chapman & Hall, Ltd., London.

Streptococcus pyogenes clinical isolates and lipoteichoic acid.

Minimally subcultured clinical isolates of virulent nephritogenic and nonnephritogenic Streptococcus pyogenes of the same serotype showed major differ...
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