Vol. 22, No. 1
INFECTION AND IMMUNITY, Oct. 1978, p. 255-265 0019-9567/78/0022-0255$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Effect of L-Form Streptococcus pyogenes and of Lipoteichoic Acid on Human Cells in Tissue Culture JOANNA DEVUONO AND CHARLES PANOS* Department ofMicrobiology, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania 19107 Received for publication 2 August 1978
These studies showed the destruction of growing primary and established human cell lines with a predilection for the group A streptococci by an L-form of Streptococcus pyogenes adapted to grow in isotonic media. Also, this L-form was detected by fluorescent antibody for longer periods of time than by viable count in infected but recovered tissue culture monolayers. Additional studies with human heart cells showed changes in their protein profile and fatty acid content (but not composition) after L-form infection. This report is the first to show that the morphological changes and death of human kidney cells by this viable L-form were mimicked by the structurally different lipoteichoic acids from this organism and its parental streptococcus. These lipoteichoic acids were also equally effective in preventing attachment of S. pyogenes to human cell monolayers, but their deacylation obviated these two activities. Finally, the attachment of the isotonic L-form, as well as the parental streptococcus, to growing human kidney cells suggested that a rigid cell wall is not a prerequisite for host attachment in vitro. This laboratory was the first to document the destructive effect of an L-form of Streptococcus pyogenes, which was able to grow in ordinary, physiologically osmolar media (i.e., isotonic Lform), on human heart cells in tissue culture (13). Also, we quantitated and characterized the teichoic acid and the lipoteichoic acid (Lp-TA) from the membrane of this streptococcus and its L-form (25, 26). Pertinent findings were that: (i) the teichoic acid moiety of Lp-TA from the Lform is only one-half as long and lacks D-alanine as compared with that from the parental coccus; (ii) teichoic acid from this coccus and L-form contains D-glucose, but the L-form has only onesixth the teichoic acid or Lp-TA content of the parental coccus; and (iii) the lipid of Lp-TA from both organisms is unusual, it being a phosphoglycolipid, glycerophosphoryl diglucosyl diglyceride. Much work has been done involving Lp-TA in the binding or adherence of group A streptococci to human platelets and cells scraped from body tissues (8-11). Also, data have been obtained indicating that group A streptococci bind to epithelial cells because of their content of LpTA rather than M protein (9, 12) and that this polymer induces nephrocalcinosis in rabbit kidneys (27). Until recently (20), nothing was known on the destructive nature of this anionic polymer for growing human cells in tissue culture. Likewise, no information was available on the continued ability of an L-form of a bacterial
pathogen to bind to human cells. This report examines the destructive effect of an isotonic Lform of S. pyogenes on growing tissues with a predilection for the group A streptococci. It also compares this destruction with that caused by the Lp-TA from this organism and its parental coccus. In addition, the inhibition of streptococcal binding by Lp--TA from S. pyogenes and its L-form and the ability of these two organisms to bind to human kidney cells are compared. (Parts of this investigation were presented in preliminary form at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, La., 8-13 May 1977, and elsewhere
[20].) MATERLALS AND METHODS Bacteria, media, and viable counts. S. pyogenes type 12, its stabilized but osmotically fragile L-form, and this L-form adapted to grow in physiological isotonic medium (i.e., the isotonic L-form) were used (13). Usually, from 20 to 90 liters of culture (in 10-liter
batches) was incubated at 370C for 18 h and harvested by centrifugation (10,000 x g, 40C). The streptococcus was cultured in regular brucella broth (Pfizer Diagnostics, Brooklyn, N.Y.) with 8 g of bovine serum albumin per liter (fraction V; Armour Pharmaceutical Co., Chicago, Ill.). The isotonic and osmotically fragile L-forms were grown in the same medium but with the addition of 0.35 or 3.0% (wt/vol) NaCl, respectively. Viable counts were performed in duplicate by addition of 1.5% agar to the media described above. Extraction and purification of Lp-TA. S. py255
256
DEVUONO AND PANOS
ogenes and its osmotically fragile L-form were washed with saline and 3.0% (wt/vol) NaCl, respectively, and extracted with phenol at 40C, and the crude Lp-TA was purified as done previously (25, 28). Deacylation of Lp-TA was performed as detailed by Ganfield and Pieringer (8). The Lp-TA from either source contained from 2 to 5% protein, and each was devoid of nucleic acids. Tissue culture cells, medium, and infectivity studies. Because of the relationship between streptococcal infection and acute glomerulonephritis, human embryonic kidney cells (Grand Island Biological Co., Grand Island, N.Y.) were used. The same Girardi human heart cells which we used earlier (13) and Chang human liver cells (CCL 27 and CCL 13, respectively; American Type Culture Collection Repository, Rockville, Md.) were also employed. Cells were grown in flat 75-cm2, 250-ml plastic bottles (Corning Glass Works, Corning, N.Y.) with 25 ml of Eagle base medium (10x) diluted 1:10 plus 10% (vol/vol) inactivated fetal calf serum (Grand Island Biological Co.), 0.29 mg of glutamine per ml, and the final pH adjusted to 7.5 with sodium bicarbonate. Cells were also grown in multi-well plates (16-mm diameter per well; Costar; Rochester Scientific Co., Rochester, N.Y.) with 1 to 2 ml of medium per well. Heart, liver, and kidney control cells were subcultured at 5- to 7-day intervals with a split ratio of 1:3 after trypsinization (0.25% solution). Viable cell counts were determined by the dye exclusion method with Erythrocin B and a hemocytometer (23). Cells were prepared for photography by fixing with methanol and staining with the May-Gruenwald and Giemsa blood stains. To assess cytological damage, 1-day-old tissue cultures were refed and infected with an 18-h culture of the isotonic L-form and incubated as detailed by us earlier (13). The infectivity ratios of L-form to tissue culture cells used were 100:1 and 40:1 (13). Finally, identical infectivity ratios with heat-killed (1 h, 70'C) L-form cells were also employed. Morphological changes of viable and heatkilled L-form-infected tissue cultures were followed by light microscopy in situ (inverted microscope, x100) and by fixation with methanol and staining with the May-Gruenwald and Giemsa blood stains. Destruction of kidney cells by Lp-TA. One-dayold human kidney cells were subjected to various concentrations (see below) of Lp-TA and chemically deacylated Lp-TA. Lp-TA from the coccus and L-form was used. Medium (1 ml) was decanted from tissue culture cells growing in wells and replaced with fresh medium containing dissolved Lp-TA or deacylated LpTA after the pH was adjusted to 7.2 with sodium bicarbonate. Resulting morphological changes of kidney cells after these additions were noted as described above after 3 days of incubation. Microbial binding assay. The ability of S. pyogenes and its isotonic L-form to bind to human embryonic kidney cells was determined as previously described (9, 17), with modifications. Streptococcal and L-form cells from overnight cultures were suspended in phosphate-buffered saline (PBS), pH 7.2 (17), to a concentration of approximately 2 X 108 colony-forming units per ml. Human embryonic kidney cells were counted with a hemocytometer as described above and seeded into Leighton tubes. Each
INFECT. IMMUN. Leighton tube contained approximately 2 x 106 tissue culture cells per ml in a total volume of 1 ml. After overnight incubation, kidney cells were then washed twice with PBS, 1 ml of streptococcal or L-form suspension was added, and each tube was incubated at 370C for 0.5 h with constant end-over-end agitation on a rotary apparatus (1 rpm). After washing kidney cells twice with PBS to remove unattached streptococcal or L-form cells, microslides from each Leighton tube were fixed, stained with the Giemsa stain, and examined by bright-field microscopy (x400, x1,000). Control cells were incubated with PBS but without organisms. The ratio of tissue cells to organisms employed was critical if nonspecific binding of organisms to the microslide surface was to be avoided. This modified binding assay permitted, for the first time, use of monolayers of growing host cells instead of tissue scrapings for assessing the binding capabilities of this coccus and its isotonic L-form. Quantitation of organisms attached to kidney cells was made by counting individual host cells (at least 10) from enlarged photographs of growing monolayers (similar to Fig. 5) and expressing the results as number of organisms per kidney cell. Inhibition of streptococcal binding to human kidney cells by b-form Lp-TA. To test for inhibitory effects on binding by Lp-TA before and after deacylation, human embryonic kidney cells were seeded into Leighton tubes as described above and incubated at 37°C overnight. Tissue culture cells were then washed twice with PBS, and 1 ml of a PBS solution containing 1 mg of Lp-TA or deacylated LpTA per ml (adjusted to pH 7.2 with sodium bicarbonate) was added. After incubation and washing of kidney cells as described above to remove excess Lp-TA, 1 ml of streptococcal suspension (approximately 2 x 108 colony-forming units per ml) was added, and the mixture was incubated (0.5 h at 37°C) with agitation on the rotating apparatus as described above. Controls included experiments without Lp-TA or deacylated Lp-TA. After washing kidney cells twice to remove unattached streptococci, microslides were fixed and stained with the Giemsa stain and examined by brightfield microscopy (x400). L-form detection of infected cells by fluorescent antibody. Antibody to the L-form was prepared, and the indirect fluorescent antibody technique described by us elsewhere was utilized (7). Polyacrylamide disc gel electrophoresis. Gels with 7.5% (wt/vol) acrylamide, 35% (vol/vol) acetic acid, and 5 M urea in glass tubes (0.6 by 7.0 cm; Canalco Inc., Rockville, Md.) were prepared as described previously (21, 24), including solubilization of L-form and heart cells before and after infection with phenol-acetic acid-water. For these experiments, heart cell controls and cells 4 days after infection (infectivity ratio, 100:1) were removed by scraping with a rubber policeman in lieu of trypsin, washed with PBS, and lyophilized. Each gel was loaded with from 0.3 to 0.4 mg of protein and run for 165 min. A current of 5 mA/gel was used, and all gels were stained with amido black. Destaining was done nonelectrically with acetic acid (7%, vol/vol). Fatty acid studies. The same infected heart cells utilized for polyacrylamide disc gel electrophoresis
COCCAL L-FORM AND HUMAN CELLS
VOL. 22, 1978 were used for fatty acid studies. Methods for their extraction and quantitation and identification of their fatty acids by highly resolving capillary column gas chromatography have been detailed previously (22). Protein determinations. The method of Lowry et al. (14) was used for protein determinations, with bovine serum albumin as the standard.
257
RESULTS L-form infectivity and detection. Figure 1 illustrates a primary kidney cell line before and on day 4 after infection with the intact L-form. In addition to the confluent layer becoming scattered after infection, other effects noted were
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258
INFECT. IMMUN.
DEVUONO AND PANOS
In addition to the morphological differences observed.with the primary kidney cell line, these infected cells displayed a cytoplasm which appeared to be more scattered or diffuse than defined, as compared with infected kidney cells (Fig. 1). Approximately one-half of all kidney and liver
the appearance of enlarged, heavily staining nuclei, the cytoplasm becoming spindle shaped, the presence of giant cells (at least two times normal size), and the dinucleation of approximately 6% of the infected cells. Figure 2 shows the effect of L-form infectivity on an established cell line of human liver cells.
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VOL. 22, 1978
cells inoculated with heat-killed L-form cells equivalent to an infectivity ratio of 100:1 showed cell damage characterized by vacuolated cytoplasm, enlarged nuclei, and granulation within the cytoplasm after 7 days of incubation. Dinucleated and giant cells were also observed. Refeeding alone of such cells did not result in recovery, but recovery was achieved after trypsinization plus refeeding (see below). To detect or isolate the L-form in infected liver and kidney cells for an extended time period, an infectivity ratio of 40:1 was used. This was necessary to decrease or minimize the rapid destruction of the host cells observed with the higher (100:1) infectivity ratio. Such infected cultures showed the same type of morphological changes indicated above, but after 10 to 14 days (instead of 4 days), and the effects were not as prevalent. In contrast to experiments with the higher infectivity ratio, host cells returned to a normal morphology after at least two trypsinizations and two refeedings (i.e., about 1 month). In these long-term experiments, viable L-form cells could be recovered at 10 to 12 days after infection in quantitatively similar fashion to those from infected human heart cells detailed by us earlier (13). In contrast to the parental coccus and as in previous studies (13), this Lform did not grow (i.e., divide) in tissue culture medium with these host cells, and the final viable count obtained never reached the inoculum size used. When fluorescent antibody was used, the L-form was detected up to 1 month after the time possible by viable count in infected cells that had regained their typical morphology after trypsin treatment and multiple refeedings. Detection was not possible after 3 months by fluorescent antibody. Positive preparations appeared similar to those from the organs of immunosuppressed mice infected with the L-form as photographed by us earlier (7). Biochemical changes of heart cells after infection. Our earlier morphological study had established the destructive nature of this isotonic L-form for human heart cells in vitro (13), but no information was at hand concerning biochemical changes. The polyacrylamide disc gel electrophoresis results with human heart cells after L-form infection are illustrated in Fig. 3. Gels A and B are L-form and heart cell controls, respectively. In addition to the L-form-infected heart cell (gel C) being a composite of the controls, a major difference in its protein profile is an overtly intensified, rapidly migrating band not predominant in either control. Other more subtle quantitative differences are also apparent in this gel relative to the controls. On a dry weight basis, the fatty acid content
COCCAL L-FORM AND HUMAN CELLS
259
of these infected heart cells increased by 62% (Table 1), whereas their total protein content decreased by only 16% (i.e., 62% and 52% for control and infected cells, respectively). The
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Myristic 3.77 4.32 Palmitic 30.57 32.76 Palmitoleic 6.33 8.58 Stearic 15.66 13.91 Oleic 29.79 27.19 cis-Vaccenic 10.11 10.93 Linoleic 3.78 2.31 Palmitic + oleic 60.36 59.95 % Total fatty acids 5.3 8.6 aAverage of two determinations. Components below 2% are omitted. h Infectivity ratio, 100:1.
260
DEVUONO AND PANOS
sizes of a heart cell and this L-form are markedly different (and similar to that for this L-form and kidney cell [Fig. 4]) but their fatty acid content is the same (each about 5% of its dry weight). Also, the viability of this L-form slowly decreases with time in heart cell tissue cultures (13). These facts and the fact that the fatty acid composition (but not content) of these heart cells remained unchanged after infection (Table 1) suggest that the fatty acids of the L-form inoculum are not solely responsible for the increased fatty acid content of these infected heart cells. Palmitic and oleic acids continued to account for over one-half of the long-chain fatty acids of this human cell line after microbial infection (Table 1). Destruction of human cell monolayers by Lp-TA. The destructive effect upon human heart (13), kidney, and liver cells by the heatkilled L-form suggested a toxic cellular component. Therefore, purified Lp-TA from the parental coccus and its osmotically fragile L-form was tested for its destructive effect on human kidney cell monolayers. Results with the Lp-TA's from the coccus and L-form were identical. Maximum destruction was achieved with 250 tg of Lp-TA per ml of tissue culture medium, with proportionally less destruction occurring with lesser amounts (e.g., 142 jg of Lp-TA resulted in 50% destruction of cells after 3 days of incubation).
INFECT. IMMUN.
The lowest amount of Lp-TA causing no discernible effect was 75 tig per ml of tissue culture medium. Complete destruction was characterized by less than 10% of the initial cell population still remaining attached and their atypical appearance being identical to that described earlier for kidney cells infected with viable L-form cells after 4 days of incubation, including the presence of dinucleated and giant cells (e.g., identical to Fig. 1B). Figures 5A and B illustrate at a higher magnification the effect of 250 tg of Lp-TA from either organism per ml on these human kidney cells after 3 days of incubation; characteristic spindle-shaped (Fig. 5A) and rounded-up cells with enlarged nuclei and a granular, vacuolated cytoplasm (Fig. 5B) are obvious. These remaining infected cells could not be rescued by trypsin treatment and refeedings as was done with kidney cells infected with heat-killed L-form preparations (see above). Conversely, with 50% destruction the changes were identical to those seen with the heat-killed inoculum (see above). There was no difference in the rate or amount of destruction observed when these experiments were repeated in an enriched (5%) CO2 atmosphere. Lp-TA from the streptococcus, after fatty acid and alanine removal, and deacylated Lp-TA from the L-form with its fatty acids removed failed to alter the morphology or growth rate of
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FIG. 4. An individual human kidney cell with the much smaller isotonic L-form attached. Arrows indicate areas of maximum attachment. Bar, 25 [im.
COCCAL L-FORM AND HUMAN CELLS
VOL. 22, 1978
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these three cell lines. A maximum concentration of 285 pg of deacylated Lp-TA per ml, from intact Lp-TA of the coccus or L-form, was inactive. Binding of the coccus and L-form to human kidney cells. Experiments by Ofek et al. (18) had shown Lp-TA to be involved in the
binding of group A streptococci to scrapings of buccal mucosal cells. Therefore, and in addition to its destructive capability, this anionic polymer from this coccus and L-form was also tested for its ability to inhibit streptococcal binding to a host cell with a predilection for S. pyogenes. Kidney cells first treated with coccal or L-form
262
INFECT. IMMUN.
DEVUONO AND PANOS
rapidly and in significant numbers (Fig. 6). Figure 4 also illustrates the binding of the much smaller L-form to an untreated kidney cell. This was confirmed by use of an oil immersion lens. Also, this procedure illustrates the new tensile
Lp-TA failed to bind S. pyogenes; an average of not more than 1 organism per tissue culture cell was observed. However, in the absence of pretreatment with Lp-TA from either organism, these cells did bind suspensions of S. pyogenes ax
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263
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COCCAL L-FORM AND HUMAN CELLS
strength of a membrane once responsible for the osmotic fragility of this intact organism (13). Finally, no marked quantitative difference was apparent between this coccus and its L-form in their affinities for human kidney cell monolayers (42 ± 6 streptococci bound per human kidney cell, compared with 50 ± 19 for the isotonic Lform). Pretreatment of kidney cells with deacylated Lp-TA (1 mg/ml), prepared from L-form Lp-TA, failed to prevent or lessen the binding of the coccus to kidney cells. DISCUSSION The death of human kidney and liver cells in vitro caused by the L-form shows this organism to be equally effective against established and primary cell lines with a predilection for the group A streptococci. A major difference between this and our earlier study with human heart cells (13) was that only these kidney and liver cells became enlarged and/or dinucleated after infection with the L-form. The significance of this at present is obscure. Earlier, we showed this L-form to be detectable in various organs from immunosuppressed mice by fluorescent antibody for a maximum of 17 days (7). Use of L-form-infected kidney and liver monolayers with fluorescent antibody now extends this time of detection beyond 1 month. Before this, viable L-forms had only been recovered after from 10 to 14 days from infected human heart cells and organs of immunosuppressed mice (7, 13). The altered protein profile of human heart cells infected with the L-form is in accord with the most recent findings of others showing protein alterations in liver and L-cell plasma membranes during infection with Coxiella burnetii (15). Such changes might also be related to the increased fatty acid content of these infected heart cells (altered regulatory mechanism[s]?). Nevertheless, these changes and our earlier electron microscope studies showing marked deterioration of heart cells after L-form infection (13) prove that supposedly innocuous wall-less organisms of pathogenic bacteria can induce morphological and biochemical changes within a susceptible host. This report is the first to show the marked destructive capability of Lp-TA for human cells in tissue culture. Also, it is clear that loss of Dalanine and a significantly shorter glycerol-phosphate chain length do not affect the toxicity of Lp-TA, whereas chemically deacylated Lp-TA from both organisms does. Therefore, although the teichoic acid moiety of Lp-TA can be altered without affecting the toxicity of the molecule, its lipid component cannot. This lipid-toxicity re-
lationship is strikingly similar to another anionic polymer, lipopolysaccharide from gram-negative bacteria. Earlier, others demonstrated (i) the involvement of Lp-TA in the binding of a group A streptococcus to cells scraped from the hand, skin, and buccal mucosa and (ii) the major role of the fatty acid component of this polymer for this affinity (1, 11, 18, 19). This study confirms and expands upon these findings. This anionic polymer from the L-form and coccus not only prevented the binding of S. pyogenes to growing human kidney cells, as already indicated, but it also destroyed monolayers of these and human heart and liver cells. Finally, the importance of the fatty acid component of Lp-TA was confirmed by the failure of deacylated Lp-TA to prevent this binding (1, 11), indicating the necessity of fatty acids not only for binding but also for the destruction of monolayers of various human cell lines in tissue culture as well. The complete destruction of heart, kidney, and liver monolayers by the viable L-form required 3 to 4 days of incubation. However, heatkilled inocula required 7 days for comparable destruction to occur. If this detrimental effect is due to Lp-TA, one possible explanation might be membrane conformational changes after heat inactivation, resulting in the masking of Lp-TA on the membrane surface. This, in effect, would decrease the amount of exposed Lp-TA and extend the time necessary for destruction to occur. Lp-TA is involved in streptococcal binding to body tissues (see below), and others have shown the adherence of S. pyogenes to be impaired by pretreatment with heat at 560C (6). The number of this coccus (and L-form) binding to growing kidney cell monolayers was less than that shown by Alkan et al. (1) for human oral epithelial cells obtained from body scrapings by pharyngeal and pyoderma strains of the group A cocci. However, it is equal to that shown by others for S. pyogenes and nasal mucosal cells (2). Such differences are probably related to tissue streptococcal specificities. A marked specificity for the binding of certain bacteria to nasal mucosal cells has been documented (2). This initial demonstration of the binding of an L-form to monolayers of human tissue cells is noteworthy. It confirms our earlier electron microscope studies in which L-forms were seen on, as well as within, infected human heart cells (13). Also, it shows that the rigid cell wall is not necessary for binding to occur in vitro. As noted earlier, the L-form contains much less Lp-TA than does the parental coccus, but both organisms bind equally to human kidney cells. Also, it is known that Lp-TA is a membrane compo-
264
DEVUONO AND PANOS
nent of a group A coccus and that it can penetrate the pores of the cell wall and appear as a surface component. Thus, the same small amount of Lp-TA exposed on the surface of this coccus and its L-form would account for equal binding by both organisms. If so, this would suggest that only a minimal amount of Lp-TA is necessary or available for streptococcal attachment to a host. M protein has been implicated in the binding of cocci to host cells (5). Recently, however, others have shown that binding of cocci to oral epithelial cells also occurred with S. pyogenes lacking M protein (1, 4). Also, these authors suggested that "Lp-TA is more centrally involved than M protein in binding streptococci to skin and mucosal surfaces" (1). The binding of this isotonic L-form lacking M protein to host cells and the inhibition of coccal binding by exogenously supplied Lp-TA tend to confirm this belief. Finally, studies have indicated that fimbriae in gram-positive bacteria are involved in binding bacteria to mammalian cell membranes (4, 10). Also, the binding substance on the fimbriae of the group A cocci has been shown to be Lp-TA (1). To date, such fimbriae have not been reported on the surface of an L-form of a gram-positive bacterium. The amount of Lp-TA necessary for the destruction of these tissue cell monolayers in vitro might seem excessive. Although direct evidence is not at hand on the excretion of Lp-TA by the group A cocci or their L-forms, other streptococci are known that do excrete large amounts of this polymer into the medium (16). A similar excretion of Lp-TA by the group A cocci, perhaps during adverse conditions or with antibiotic therapy, could result in sufficient accumulations by tissues with a predilection for S. pyogenes for destruction in vivo similar to that in vitro. Also, such accumulations might be foci for initiating hypersensitivity-type reactions (glomerulonephritis?) during pathogenesis. In this regard, it has recently been shown that large amounts of lipids are released by S. pyogenes with the use of antibiotics (12). The role of membrane Lp-TA in such nonsuppurative streptococcal sequelae as rheumatic fever and glomerulonephritis is unknown, but the fact that this membrane component is involved in the binding of S. pyogenes and its L-form to human cells and in the rapid destruction of these cells is noteworthy. Also, the finding that an organism without a rigid cell wall can bind to and eventually destroy growing human cells only enhances the potential role of the bacterial Lform in pathogenesis. ACKNOWLEDGMENTS This investigation was supported by Public Health Service
INFECT. IMMUN. research grant AI-11161 from the National Institute of Allergy and Infectious Diseases and, in part, by biomedical research support grant RR5414 from the Division of Research Resources, National Institutes of Health. The assistance of 0. Leon is gratefully acknowledged. LITERATURE CITED 1. Alkan, M., I. Ofek, and E. H. Beachey. 1977. Adherence of pharyngeal and skin strains of group A streptococci to human skin and oral epithelial cells. Infect. Immun. 18:555-557. 2. Aly, R., H. I. Shinefield, W. G. Strauss, and H. I. Maibach. 1977. Bacterial adherence to nasal mucosal cells. Infect. Immun. 17:546-549. 3. Beachey, E. H., T. M. Chiang, I. Ofek, and A. H. Kang. 1977. Interaction of lipoteichoic acid of group A streptococci with human platelets. Infect. Immun. 16:649-654. 4. Beachey, E., and L. Ofek. 1976. Epithelial cell binding of group A streptococci by lipoteichoic acid on fimbriae denuded of M protein. J. Exp. Med. 143:759-771. 5. Ellen, R. P., and R. J. Gibbons. 1972. M protein-associated adherence of Streptococcus pyogenes to epithelial surfaces: prerequisite for virulence. Infect. Immun. 5:826-830. 6. Ellen, R. P., and R. J. Gibbons. 1974. Parameters affecting the adherence and tissue tropisms of Streptococcus pyogenes. Infect. Immun. 9:85-91. 7. Fernandes, P. B., and C. Panos. 1976. Persistence, pathogenesis, and morphology of an L-form of Streptococcus pyogenes adapted to physiological isotonic conditions when in immunosuppressed mice. Infect. Immun. 14:1228-1240. 8. Ganfield, M.-C. W., and R. A. Pieringer. 1975. Phosphatidylkojibiosyl diglyceride: the covalently linked lipid constituent of the membrane lipoteichoic acid from Streptococcus faecalis (faecium) ATCC 9790. J. Biol. Chem. 250:702-709. 9. Gibbons, R. J., and J. van Houte. 1971. Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. Infect. Immun. 3:567-573. 10. Gibbons, R. J., and J. van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29:19-44. 11. Hausmann, E., 0. Luderitz, K. Knox, and N. Weinfeld. 1975. Structural requirements for bone resorption by endotoxin and lipoteichoic acid. J. Dent. Res. 54(Special Issue B):B94-B99. 12. Horne, D., R. Hakenbeck, and A. Tomasz. 1977. Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J. Bacteriol. 132:704-717. 13. Leon, O., and C. Panos. 1976. Adaptation of an osmotically fragile L-form of Streptococcus pyogenes to physiological osmotic conditions and its ability to destroy human heart cells in tissue culture. Infect. Immun. 13:252-262. 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 15. Marecki, N., F. Becker, 0. G. Baca, and D. Paretsky. 1978. Changes in liver and L-cell plasma membranes during infection with Coxiella burnetii. Infect. Immun. 19:272-280. 16. Markham, J. L., K. W. Knox, A. J. Wicken, and M. J. Hewett. 1975. Formation of extracellular lipoteichoic acid by oral streptococci and lactobacilli. Infect. Immun. 12:378-386. 17. Merchant, D. J., R. H. Kahn, and W. H. Murphy. 1964. Handbook of cell and organ culture. Burgess Publishing Co., Minneapolis, Minn. 18. Ofek, I., E. H. Beachey, F. Eyal, and J. C. Morrison. 1977. Postnatal development of binding of streptococci and lipoteichoic acid by oral mucosal cells of humans.
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COCCAL L-FORM AND HUMAN CELLS
J. Infect. Dis. 135:267-274. 19. 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. 20. Panos, C. 1978. Effects of an L-form of Streptococcus pyogenes on cultured cells and in immunosuppressed mice, p. 408-411. In D. Schlessinger (ed.), Microbiology-1978. American Society for Microbiology, Washington, D.C. 21. Panos, C., G. Fagan, and C. G. Zarkadas. 1972. Comparative electrophoretic and amino acid analyses of isolated membranes from Streptococcus pyogenes and stabilized L-form. J. Bacteriol. 112:285-290. 22. Panos, C., and S. Rottem. 1970. Incorporation and elongation of fatty acid isomers by Mycoplasma laidlawii A. Biochemistry 9:407-412. 23. Phillips, H. J., and J. E. Terryberry. 1957. Counting actively metabolizing tissue cultured cells. Exp. Cell
Res. 13:341-347. 24. Rottem, S., and S. Razin. 1967. Electrophoretic patterns of membrane proteins of mycoplasma. J. Bacteriol. 94:359-364. 25. Slabyj, B. M., and C. Panos. 1973. Teichoic acid of a stabilized L-form of Streptococcus pyogenes. J. Bacteriol. 114:934-942. 26. 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. 27. Waltersdorff, 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. 28. Wicken, A. J., and K. W. Knox. 1970. Studies on the group F antigen of lactobacilli: isolation of a teichoic acid-lipid complex from Lactobacillus fermenti NCTC 6991. J. Gen. Microbiol. 60:293-301.
265
INFECTION AND IMMUNITY, Oct. 1978, p. 255-265 0019-9567/78/0022-0255$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Effect of L-Form Streptococcus pyogenes and of Lipoteichoic Acid on Human Cells in Tissue Culture JOANNA DEVUONO AND CHARLES PANOS* Department ofMicrobiology, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania 19107 Received for publication 2 August 1978
These studies showed the destruction of growing primary and established human cell lines with a predilection for the group A streptococci by an L-form of Streptococcus pyogenes adapted to grow in isotonic media. Also, this L-form was detected by fluorescent antibody for longer periods of time than by viable count in infected but recovered tissue culture monolayers. Additional studies with human heart cells showed changes in their protein profile and fatty acid content (but not composition) after L-form infection. This report is the first to show that the morphological changes and death of human kidney cells by this viable L-form were mimicked by the structurally different lipoteichoic acids from this organism and its parental streptococcus. These lipoteichoic acids were also equally effective in preventing attachment of S. pyogenes to human cell monolayers, but their deacylation obviated these two activities. Finally, the attachment of the isotonic L-form, as well as the parental streptococcus, to growing human kidney cells suggested that a rigid cell wall is not a prerequisite for host attachment in vitro. This laboratory was the first to document the destructive effect of an L-form of Streptococcus pyogenes, which was able to grow in ordinary, physiologically osmolar media (i.e., isotonic Lform), on human heart cells in tissue culture (13). Also, we quantitated and characterized the teichoic acid and the lipoteichoic acid (Lp-TA) from the membrane of this streptococcus and its L-form (25, 26). Pertinent findings were that: (i) the teichoic acid moiety of Lp-TA from the Lform is only one-half as long and lacks D-alanine as compared with that from the parental coccus; (ii) teichoic acid from this coccus and L-form contains D-glucose, but the L-form has only onesixth the teichoic acid or Lp-TA content of the parental coccus; and (iii) the lipid of Lp-TA from both organisms is unusual, it being a phosphoglycolipid, glycerophosphoryl diglucosyl diglyceride. Much work has been done involving Lp-TA in the binding or adherence of group A streptococci to human platelets and cells scraped from body tissues (8-11). Also, data have been obtained indicating that group A streptococci bind to epithelial cells because of their content of LpTA rather than M protein (9, 12) and that this polymer induces nephrocalcinosis in rabbit kidneys (27). Until recently (20), nothing was known on the destructive nature of this anionic polymer for growing human cells in tissue culture. Likewise, no information was available on the continued ability of an L-form of a bacterial
pathogen to bind to human cells. This report examines the destructive effect of an isotonic Lform of S. pyogenes on growing tissues with a predilection for the group A streptococci. It also compares this destruction with that caused by the Lp-TA from this organism and its parental coccus. In addition, the inhibition of streptococcal binding by Lp--TA from S. pyogenes and its L-form and the ability of these two organisms to bind to human kidney cells are compared. (Parts of this investigation were presented in preliminary form at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, La., 8-13 May 1977, and elsewhere
[20].) MATERLALS AND METHODS Bacteria, media, and viable counts. S. pyogenes type 12, its stabilized but osmotically fragile L-form, and this L-form adapted to grow in physiological isotonic medium (i.e., the isotonic L-form) were used (13). Usually, from 20 to 90 liters of culture (in 10-liter
batches) was incubated at 370C for 18 h and harvested by centrifugation (10,000 x g, 40C). The streptococcus was cultured in regular brucella broth (Pfizer Diagnostics, Brooklyn, N.Y.) with 8 g of bovine serum albumin per liter (fraction V; Armour Pharmaceutical Co., Chicago, Ill.). The isotonic and osmotically fragile L-forms were grown in the same medium but with the addition of 0.35 or 3.0% (wt/vol) NaCl, respectively. Viable counts were performed in duplicate by addition of 1.5% agar to the media described above. Extraction and purification of Lp-TA. S. py255
256
DEVUONO AND PANOS
ogenes and its osmotically fragile L-form were washed with saline and 3.0% (wt/vol) NaCl, respectively, and extracted with phenol at 40C, and the crude Lp-TA was purified as done previously (25, 28). Deacylation of Lp-TA was performed as detailed by Ganfield and Pieringer (8). The Lp-TA from either source contained from 2 to 5% protein, and each was devoid of nucleic acids. Tissue culture cells, medium, and infectivity studies. Because of the relationship between streptococcal infection and acute glomerulonephritis, human embryonic kidney cells (Grand Island Biological Co., Grand Island, N.Y.) were used. The same Girardi human heart cells which we used earlier (13) and Chang human liver cells (CCL 27 and CCL 13, respectively; American Type Culture Collection Repository, Rockville, Md.) were also employed. Cells were grown in flat 75-cm2, 250-ml plastic bottles (Corning Glass Works, Corning, N.Y.) with 25 ml of Eagle base medium (10x) diluted 1:10 plus 10% (vol/vol) inactivated fetal calf serum (Grand Island Biological Co.), 0.29 mg of glutamine per ml, and the final pH adjusted to 7.5 with sodium bicarbonate. Cells were also grown in multi-well plates (16-mm diameter per well; Costar; Rochester Scientific Co., Rochester, N.Y.) with 1 to 2 ml of medium per well. Heart, liver, and kidney control cells were subcultured at 5- to 7-day intervals with a split ratio of 1:3 after trypsinization (0.25% solution). Viable cell counts were determined by the dye exclusion method with Erythrocin B and a hemocytometer (23). Cells were prepared for photography by fixing with methanol and staining with the May-Gruenwald and Giemsa blood stains. To assess cytological damage, 1-day-old tissue cultures were refed and infected with an 18-h culture of the isotonic L-form and incubated as detailed by us earlier (13). The infectivity ratios of L-form to tissue culture cells used were 100:1 and 40:1 (13). Finally, identical infectivity ratios with heat-killed (1 h, 70'C) L-form cells were also employed. Morphological changes of viable and heatkilled L-form-infected tissue cultures were followed by light microscopy in situ (inverted microscope, x100) and by fixation with methanol and staining with the May-Gruenwald and Giemsa blood stains. Destruction of kidney cells by Lp-TA. One-dayold human kidney cells were subjected to various concentrations (see below) of Lp-TA and chemically deacylated Lp-TA. Lp-TA from the coccus and L-form was used. Medium (1 ml) was decanted from tissue culture cells growing in wells and replaced with fresh medium containing dissolved Lp-TA or deacylated LpTA after the pH was adjusted to 7.2 with sodium bicarbonate. Resulting morphological changes of kidney cells after these additions were noted as described above after 3 days of incubation. Microbial binding assay. The ability of S. pyogenes and its isotonic L-form to bind to human embryonic kidney cells was determined as previously described (9, 17), with modifications. Streptococcal and L-form cells from overnight cultures were suspended in phosphate-buffered saline (PBS), pH 7.2 (17), to a concentration of approximately 2 X 108 colony-forming units per ml. Human embryonic kidney cells were counted with a hemocytometer as described above and seeded into Leighton tubes. Each
INFECT. IMMUN. Leighton tube contained approximately 2 x 106 tissue culture cells per ml in a total volume of 1 ml. After overnight incubation, kidney cells were then washed twice with PBS, 1 ml of streptococcal or L-form suspension was added, and each tube was incubated at 370C for 0.5 h with constant end-over-end agitation on a rotary apparatus (1 rpm). After washing kidney cells twice with PBS to remove unattached streptococcal or L-form cells, microslides from each Leighton tube were fixed, stained with the Giemsa stain, and examined by bright-field microscopy (x400, x1,000). Control cells were incubated with PBS but without organisms. The ratio of tissue cells to organisms employed was critical if nonspecific binding of organisms to the microslide surface was to be avoided. This modified binding assay permitted, for the first time, use of monolayers of growing host cells instead of tissue scrapings for assessing the binding capabilities of this coccus and its isotonic L-form. Quantitation of organisms attached to kidney cells was made by counting individual host cells (at least 10) from enlarged photographs of growing monolayers (similar to Fig. 5) and expressing the results as number of organisms per kidney cell. Inhibition of streptococcal binding to human kidney cells by b-form Lp-TA. To test for inhibitory effects on binding by Lp-TA before and after deacylation, human embryonic kidney cells were seeded into Leighton tubes as described above and incubated at 37°C overnight. Tissue culture cells were then washed twice with PBS, and 1 ml of a PBS solution containing 1 mg of Lp-TA or deacylated LpTA per ml (adjusted to pH 7.2 with sodium bicarbonate) was added. After incubation and washing of kidney cells as described above to remove excess Lp-TA, 1 ml of streptococcal suspension (approximately 2 x 108 colony-forming units per ml) was added, and the mixture was incubated (0.5 h at 37°C) with agitation on the rotating apparatus as described above. Controls included experiments without Lp-TA or deacylated Lp-TA. After washing kidney cells twice to remove unattached streptococci, microslides were fixed and stained with the Giemsa stain and examined by brightfield microscopy (x400). L-form detection of infected cells by fluorescent antibody. Antibody to the L-form was prepared, and the indirect fluorescent antibody technique described by us elsewhere was utilized (7). Polyacrylamide disc gel electrophoresis. Gels with 7.5% (wt/vol) acrylamide, 35% (vol/vol) acetic acid, and 5 M urea in glass tubes (0.6 by 7.0 cm; Canalco Inc., Rockville, Md.) were prepared as described previously (21, 24), including solubilization of L-form and heart cells before and after infection with phenol-acetic acid-water. For these experiments, heart cell controls and cells 4 days after infection (infectivity ratio, 100:1) were removed by scraping with a rubber policeman in lieu of trypsin, washed with PBS, and lyophilized. Each gel was loaded with from 0.3 to 0.4 mg of protein and run for 165 min. A current of 5 mA/gel was used, and all gels were stained with amido black. Destaining was done nonelectrically with acetic acid (7%, vol/vol). Fatty acid studies. The same infected heart cells utilized for polyacrylamide disc gel electrophoresis
COCCAL L-FORM AND HUMAN CELLS
VOL. 22, 1978 were used for fatty acid studies. Methods for their extraction and quantitation and identification of their fatty acids by highly resolving capillary column gas chromatography have been detailed previously (22). Protein determinations. The method of Lowry et al. (14) was used for protein determinations, with bovine serum albumin as the standard.
257
RESULTS L-form infectivity and detection. Figure 1 illustrates a primary kidney cell line before and on day 4 after infection with the intact L-form. In addition to the confluent layer becoming scattered after infection, other effects noted were
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258
INFECT. IMMUN.
DEVUONO AND PANOS
In addition to the morphological differences observed.with the primary kidney cell line, these infected cells displayed a cytoplasm which appeared to be more scattered or diffuse than defined, as compared with infected kidney cells (Fig. 1). Approximately one-half of all kidney and liver
the appearance of enlarged, heavily staining nuclei, the cytoplasm becoming spindle shaped, the presence of giant cells (at least two times normal size), and the dinucleation of approximately 6% of the infected cells. Figure 2 shows the effect of L-form infectivity on an established cell line of human liver cells.
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VOL. 22, 1978
cells inoculated with heat-killed L-form cells equivalent to an infectivity ratio of 100:1 showed cell damage characterized by vacuolated cytoplasm, enlarged nuclei, and granulation within the cytoplasm after 7 days of incubation. Dinucleated and giant cells were also observed. Refeeding alone of such cells did not result in recovery, but recovery was achieved after trypsinization plus refeeding (see below). To detect or isolate the L-form in infected liver and kidney cells for an extended time period, an infectivity ratio of 40:1 was used. This was necessary to decrease or minimize the rapid destruction of the host cells observed with the higher (100:1) infectivity ratio. Such infected cultures showed the same type of morphological changes indicated above, but after 10 to 14 days (instead of 4 days), and the effects were not as prevalent. In contrast to experiments with the higher infectivity ratio, host cells returned to a normal morphology after at least two trypsinizations and two refeedings (i.e., about 1 month). In these long-term experiments, viable L-form cells could be recovered at 10 to 12 days after infection in quantitatively similar fashion to those from infected human heart cells detailed by us earlier (13). In contrast to the parental coccus and as in previous studies (13), this Lform did not grow (i.e., divide) in tissue culture medium with these host cells, and the final viable count obtained never reached the inoculum size used. When fluorescent antibody was used, the L-form was detected up to 1 month after the time possible by viable count in infected cells that had regained their typical morphology after trypsin treatment and multiple refeedings. Detection was not possible after 3 months by fluorescent antibody. Positive preparations appeared similar to those from the organs of immunosuppressed mice infected with the L-form as photographed by us earlier (7). Biochemical changes of heart cells after infection. Our earlier morphological study had established the destructive nature of this isotonic L-form for human heart cells in vitro (13), but no information was at hand concerning biochemical changes. The polyacrylamide disc gel electrophoresis results with human heart cells after L-form infection are illustrated in Fig. 3. Gels A and B are L-form and heart cell controls, respectively. In addition to the L-form-infected heart cell (gel C) being a composite of the controls, a major difference in its protein profile is an overtly intensified, rapidly migrating band not predominant in either control. Other more subtle quantitative differences are also apparent in this gel relative to the controls. On a dry weight basis, the fatty acid content
COCCAL L-FORM AND HUMAN CELLS
259
of these infected heart cells increased by 62% (Table 1), whereas their total protein content decreased by only 16% (i.e., 62% and 52% for control and infected cells, respectively). The
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FIG. 3. Polyacrylamide disc gel electrophoresis of human heart cells before and after L-form infection. (A) Heart cell control; (B) L-form control; (C) heart cells at 4 days after infection with the isotonic Lform. Direction of migration was downward. TABLE 1. Major fatty acid composition of human heart cells before and 4 days after infection with the isotonic L-form % of total fatty acids'
Fatty acid
Before infection
After infec-
tionh
Myristic 3.77 4.32 Palmitic 30.57 32.76 Palmitoleic 6.33 8.58 Stearic 15.66 13.91 Oleic 29.79 27.19 cis-Vaccenic 10.11 10.93 Linoleic 3.78 2.31 Palmitic + oleic 60.36 59.95 % Total fatty acids 5.3 8.6 aAverage of two determinations. Components below 2% are omitted. h Infectivity ratio, 100:1.
260
DEVUONO AND PANOS
sizes of a heart cell and this L-form are markedly different (and similar to that for this L-form and kidney cell [Fig. 4]) but their fatty acid content is the same (each about 5% of its dry weight). Also, the viability of this L-form slowly decreases with time in heart cell tissue cultures (13). These facts and the fact that the fatty acid composition (but not content) of these heart cells remained unchanged after infection (Table 1) suggest that the fatty acids of the L-form inoculum are not solely responsible for the increased fatty acid content of these infected heart cells. Palmitic and oleic acids continued to account for over one-half of the long-chain fatty acids of this human cell line after microbial infection (Table 1). Destruction of human cell monolayers by Lp-TA. The destructive effect upon human heart (13), kidney, and liver cells by the heatkilled L-form suggested a toxic cellular component. Therefore, purified Lp-TA from the parental coccus and its osmotically fragile L-form was tested for its destructive effect on human kidney cell monolayers. Results with the Lp-TA's from the coccus and L-form were identical. Maximum destruction was achieved with 250 tg of Lp-TA per ml of tissue culture medium, with proportionally less destruction occurring with lesser amounts (e.g., 142 jg of Lp-TA resulted in 50% destruction of cells after 3 days of incubation).
INFECT. IMMUN.
The lowest amount of Lp-TA causing no discernible effect was 75 tig per ml of tissue culture medium. Complete destruction was characterized by less than 10% of the initial cell population still remaining attached and their atypical appearance being identical to that described earlier for kidney cells infected with viable L-form cells after 4 days of incubation, including the presence of dinucleated and giant cells (e.g., identical to Fig. 1B). Figures 5A and B illustrate at a higher magnification the effect of 250 tg of Lp-TA from either organism per ml on these human kidney cells after 3 days of incubation; characteristic spindle-shaped (Fig. 5A) and rounded-up cells with enlarged nuclei and a granular, vacuolated cytoplasm (Fig. 5B) are obvious. These remaining infected cells could not be rescued by trypsin treatment and refeedings as was done with kidney cells infected with heat-killed L-form preparations (see above). Conversely, with 50% destruction the changes were identical to those seen with the heat-killed inoculum (see above). There was no difference in the rate or amount of destruction observed when these experiments were repeated in an enriched (5%) CO2 atmosphere. Lp-TA from the streptococcus, after fatty acid and alanine removal, and deacylated Lp-TA from the L-form with its fatty acids removed failed to alter the morphology or growth rate of
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FIG. 4. An individual human kidney cell with the much smaller isotonic L-form attached. Arrows indicate areas of maximum attachment. Bar, 25 [im.
COCCAL L-FORM AND HUMAN CELLS
VOL. 22, 1978
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these three cell lines. A maximum concentration of 285 pg of deacylated Lp-TA per ml, from intact Lp-TA of the coccus or L-form, was inactive. Binding of the coccus and L-form to human kidney cells. Experiments by Ofek et al. (18) had shown Lp-TA to be involved in the
binding of group A streptococci to scrapings of buccal mucosal cells. Therefore, and in addition to its destructive capability, this anionic polymer from this coccus and L-form was also tested for its ability to inhibit streptococcal binding to a host cell with a predilection for S. pyogenes. Kidney cells first treated with coccal or L-form
262
INFECT. IMMUN.
DEVUONO AND PANOS
rapidly and in significant numbers (Fig. 6). Figure 4 also illustrates the binding of the much smaller L-form to an untreated kidney cell. This was confirmed by use of an oil immersion lens. Also, this procedure illustrates the new tensile
Lp-TA failed to bind S. pyogenes; an average of not more than 1 organism per tissue culture cell was observed. However, in the absence of pretreatment with Lp-TA from either organism, these cells did bind suspensions of S. pyogenes ax
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263
VOL. 22, 1978
COCCAL L-FORM AND HUMAN CELLS
strength of a membrane once responsible for the osmotic fragility of this intact organism (13). Finally, no marked quantitative difference was apparent between this coccus and its L-form in their affinities for human kidney cell monolayers (42 ± 6 streptococci bound per human kidney cell, compared with 50 ± 19 for the isotonic Lform). Pretreatment of kidney cells with deacylated Lp-TA (1 mg/ml), prepared from L-form Lp-TA, failed to prevent or lessen the binding of the coccus to kidney cells. DISCUSSION The death of human kidney and liver cells in vitro caused by the L-form shows this organism to be equally effective against established and primary cell lines with a predilection for the group A streptococci. A major difference between this and our earlier study with human heart cells (13) was that only these kidney and liver cells became enlarged and/or dinucleated after infection with the L-form. The significance of this at present is obscure. Earlier, we showed this L-form to be detectable in various organs from immunosuppressed mice by fluorescent antibody for a maximum of 17 days (7). Use of L-form-infected kidney and liver monolayers with fluorescent antibody now extends this time of detection beyond 1 month. Before this, viable L-forms had only been recovered after from 10 to 14 days from infected human heart cells and organs of immunosuppressed mice (7, 13). The altered protein profile of human heart cells infected with the L-form is in accord with the most recent findings of others showing protein alterations in liver and L-cell plasma membranes during infection with Coxiella burnetii (15). Such changes might also be related to the increased fatty acid content of these infected heart cells (altered regulatory mechanism[s]?). Nevertheless, these changes and our earlier electron microscope studies showing marked deterioration of heart cells after L-form infection (13) prove that supposedly innocuous wall-less organisms of pathogenic bacteria can induce morphological and biochemical changes within a susceptible host. This report is the first to show the marked destructive capability of Lp-TA for human cells in tissue culture. Also, it is clear that loss of Dalanine and a significantly shorter glycerol-phosphate chain length do not affect the toxicity of Lp-TA, whereas chemically deacylated Lp-TA from both organisms does. Therefore, although the teichoic acid moiety of Lp-TA can be altered without affecting the toxicity of the molecule, its lipid component cannot. This lipid-toxicity re-
lationship is strikingly similar to another anionic polymer, lipopolysaccharide from gram-negative bacteria. Earlier, others demonstrated (i) the involvement of Lp-TA in the binding of a group A streptococcus to cells scraped from the hand, skin, and buccal mucosa and (ii) the major role of the fatty acid component of this polymer for this affinity (1, 11, 18, 19). This study confirms and expands upon these findings. This anionic polymer from the L-form and coccus not only prevented the binding of S. pyogenes to growing human kidney cells, as already indicated, but it also destroyed monolayers of these and human heart and liver cells. Finally, the importance of the fatty acid component of Lp-TA was confirmed by the failure of deacylated Lp-TA to prevent this binding (1, 11), indicating the necessity of fatty acids not only for binding but also for the destruction of monolayers of various human cell lines in tissue culture as well. The complete destruction of heart, kidney, and liver monolayers by the viable L-form required 3 to 4 days of incubation. However, heatkilled inocula required 7 days for comparable destruction to occur. If this detrimental effect is due to Lp-TA, one possible explanation might be membrane conformational changes after heat inactivation, resulting in the masking of Lp-TA on the membrane surface. This, in effect, would decrease the amount of exposed Lp-TA and extend the time necessary for destruction to occur. Lp-TA is involved in streptococcal binding to body tissues (see below), and others have shown the adherence of S. pyogenes to be impaired by pretreatment with heat at 560C (6). The number of this coccus (and L-form) binding to growing kidney cell monolayers was less than that shown by Alkan et al. (1) for human oral epithelial cells obtained from body scrapings by pharyngeal and pyoderma strains of the group A cocci. However, it is equal to that shown by others for S. pyogenes and nasal mucosal cells (2). Such differences are probably related to tissue streptococcal specificities. A marked specificity for the binding of certain bacteria to nasal mucosal cells has been documented (2). This initial demonstration of the binding of an L-form to monolayers of human tissue cells is noteworthy. It confirms our earlier electron microscope studies in which L-forms were seen on, as well as within, infected human heart cells (13). Also, it shows that the rigid cell wall is not necessary for binding to occur in vitro. As noted earlier, the L-form contains much less Lp-TA than does the parental coccus, but both organisms bind equally to human kidney cells. Also, it is known that Lp-TA is a membrane compo-
264
DEVUONO AND PANOS
nent of a group A coccus and that it can penetrate the pores of the cell wall and appear as a surface component. Thus, the same small amount of Lp-TA exposed on the surface of this coccus and its L-form would account for equal binding by both organisms. If so, this would suggest that only a minimal amount of Lp-TA is necessary or available for streptococcal attachment to a host. M protein has been implicated in the binding of cocci to host cells (5). Recently, however, others have shown that binding of cocci to oral epithelial cells also occurred with S. pyogenes lacking M protein (1, 4). Also, these authors suggested that "Lp-TA is more centrally involved than M protein in binding streptococci to skin and mucosal surfaces" (1). The binding of this isotonic L-form lacking M protein to host cells and the inhibition of coccal binding by exogenously supplied Lp-TA tend to confirm this belief. Finally, studies have indicated that fimbriae in gram-positive bacteria are involved in binding bacteria to mammalian cell membranes (4, 10). Also, the binding substance on the fimbriae of the group A cocci has been shown to be Lp-TA (1). To date, such fimbriae have not been reported on the surface of an L-form of a gram-positive bacterium. The amount of Lp-TA necessary for the destruction of these tissue cell monolayers in vitro might seem excessive. Although direct evidence is not at hand on the excretion of Lp-TA by the group A cocci or their L-forms, other streptococci are known that do excrete large amounts of this polymer into the medium (16). A similar excretion of Lp-TA by the group A cocci, perhaps during adverse conditions or with antibiotic therapy, could result in sufficient accumulations by tissues with a predilection for S. pyogenes for destruction in vivo similar to that in vitro. Also, such accumulations might be foci for initiating hypersensitivity-type reactions (glomerulonephritis?) during pathogenesis. In this regard, it has recently been shown that large amounts of lipids are released by S. pyogenes with the use of antibiotics (12). The role of membrane Lp-TA in such nonsuppurative streptococcal sequelae as rheumatic fever and glomerulonephritis is unknown, but the fact that this membrane component is involved in the binding of S. pyogenes and its L-form to human cells and in the rapid destruction of these cells is noteworthy. Also, the finding that an organism without a rigid cell wall can bind to and eventually destroy growing human cells only enhances the potential role of the bacterial Lform in pathogenesis. ACKNOWLEDGMENTS This investigation was supported by Public Health Service
INFECT. IMMUN. research grant AI-11161 from the National Institute of Allergy and Infectious Diseases and, in part, by biomedical research support grant RR5414 from the Division of Research Resources, National Institutes of Health. The assistance of 0. Leon is gratefully acknowledged. LITERATURE CITED 1. Alkan, M., I. Ofek, and E. H. Beachey. 1977. Adherence of pharyngeal and skin strains of group A streptococci to human skin and oral epithelial cells. Infect. Immun. 18:555-557. 2. Aly, R., H. I. Shinefield, W. G. Strauss, and H. I. Maibach. 1977. Bacterial adherence to nasal mucosal cells. Infect. Immun. 17:546-549. 3. Beachey, E. H., T. M. Chiang, I. Ofek, and A. H. Kang. 1977. Interaction of lipoteichoic acid of group A streptococci with human platelets. Infect. Immun. 16:649-654. 4. Beachey, E., and L. Ofek. 1976. Epithelial cell binding of group A streptococci by lipoteichoic acid on fimbriae denuded of M protein. J. Exp. Med. 143:759-771. 5. Ellen, R. P., and R. J. Gibbons. 1972. M protein-associated adherence of Streptococcus pyogenes to epithelial surfaces: prerequisite for virulence. Infect. Immun. 5:826-830. 6. Ellen, R. P., and R. J. Gibbons. 1974. Parameters affecting the adherence and tissue tropisms of Streptococcus pyogenes. Infect. Immun. 9:85-91. 7. Fernandes, P. B., and C. Panos. 1976. Persistence, pathogenesis, and morphology of an L-form of Streptococcus pyogenes adapted to physiological isotonic conditions when in immunosuppressed mice. Infect. Immun. 14:1228-1240. 8. Ganfield, M.-C. W., and R. A. Pieringer. 1975. Phosphatidylkojibiosyl diglyceride: the covalently linked lipid constituent of the membrane lipoteichoic acid from Streptococcus faecalis (faecium) ATCC 9790. J. Biol. Chem. 250:702-709. 9. Gibbons, R. J., and J. van Houte. 1971. Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. Infect. Immun. 3:567-573. 10. Gibbons, R. J., and J. van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29:19-44. 11. Hausmann, E., 0. Luderitz, K. Knox, and N. Weinfeld. 1975. Structural requirements for bone resorption by endotoxin and lipoteichoic acid. J. Dent. Res. 54(Special Issue B):B94-B99. 12. Horne, D., R. Hakenbeck, and A. Tomasz. 1977. Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J. Bacteriol. 132:704-717. 13. Leon, O., and C. Panos. 1976. Adaptation of an osmotically fragile L-form of Streptococcus pyogenes to physiological osmotic conditions and its ability to destroy human heart cells in tissue culture. Infect. Immun. 13:252-262. 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 15. Marecki, N., F. Becker, 0. G. Baca, and D. Paretsky. 1978. Changes in liver and L-cell plasma membranes during infection with Coxiella burnetii. Infect. Immun. 19:272-280. 16. Markham, J. L., K. W. Knox, A. J. Wicken, and M. J. Hewett. 1975. Formation of extracellular lipoteichoic acid by oral streptococci and lactobacilli. Infect. Immun. 12:378-386. 17. Merchant, D. J., R. H. Kahn, and W. H. Murphy. 1964. Handbook of cell and organ culture. Burgess Publishing Co., Minneapolis, Minn. 18. Ofek, I., E. H. Beachey, F. Eyal, and J. C. Morrison. 1977. Postnatal development of binding of streptococci and lipoteichoic acid by oral mucosal cells of humans.
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COCCAL L-FORM AND HUMAN CELLS
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Res. 13:341-347. 24. Rottem, S., and S. Razin. 1967. Electrophoretic patterns of membrane proteins of mycoplasma. J. Bacteriol. 94:359-364. 25. Slabyj, B. M., and C. Panos. 1973. Teichoic acid of a stabilized L-form of Streptococcus pyogenes. J. Bacteriol. 114:934-942. 26. 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. 27. Waltersdorff, 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. 28. Wicken, A. J., and K. W. Knox. 1970. Studies on the group F antigen of lactobacilli: isolation of a teichoic acid-lipid complex from Lactobacillus fermenti NCTC 6991. J. Gen. Microbiol. 60:293-301.
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