Oxygen-Stable Hemolysins of Group A Streptococci

EXPERIMENTAL AND MOLECULAR PATHOLOGY 4, 93-107 (1965)

Oxygen-Stable Hemolysins of Group A Streptococci

V. Effect on Rat Heart and Kidney Cells Grown

in Tissue Culture’

Z. MARCUS, A. M. DAVIES, AND I. GINSBURG"

Departments of Medical Ecology and of Experimental Medicine and Cancer Research,

The Hebrew University-Hadassah Medical School, Jerusalem, Israel

Received A’ovember 2, 1964

Most strains of group A streptococci possess a cell-bound hemolytic factor (CBH) which hemolyses red blood cells although no extracelluar hemolysin can be detected in supernates of such incubation mixtures (Ginsburg and Grossowicz, 1958). It has been recently postulated that CBH is the cell-bound form of streptolysin S (SLS) (Ginsburg and Grossowicz, 1958; Ginsburg et al., to be published) and both CBH and SLS cause cytopathogenic changes in Ehrlich ascites tumor cells and other mammalian cells in vitro (Ginsburg 19.59; Ginsburg and Grossowicz, 1960). In view of the possible role of SLS in the pathogenesis of post-streptococcal sequellae the effect of the oxygen-stable hemolysins was tested on rat heart and kidney cells grown in tissue culture.

MATERIALS AND METHODS

Streptococcal strains and culture media

Two strains were used: (a) strain S xJ type 3, which yields high amounts of streptolysin S; (b) strain CZ,,sU type 3, kindly supplied by Dr. A. W. Bernheimer, New York University, deficient in both SLS (Bernheimer, 1954) and CBH (Gins- burg and Grossowitz, 1958). The cells were cultivated in 200 ml lots of brain heart infusion broth (Difco) or in trypticase soy broth (BBL) and harvested from the stationary phase growth as previously described (Ginsburg and Harris, 1936 ; Gins- burg and Bentwich, 1964). They were washed with saline buffered with 0.051M phosphate (pH 7.2) resuspended in buffer and used to prepare hemolysins.

Preparation of hemolysins

1. RNA core hemolysin (SLS). Yeast RNA (N.B.C. Cleveland, Ohio) was di- gested with pancreatic ribonuclease (Worthington, Freehold, New Jersey) as described previously (Ginsburg and Harris, 1963). The RNA core obtained was further purified by ion-exchange chromatography on DEAE-Sephadex A-SO columns, and a fraction, eluted from the column with 0.75M KCl, was dialyzed against 0.011M phosphate (pH 7.2) and used to prepare hemolysin from resting streptococci. Strep-

1 Supported by research grants HE05739 from US. h’ational Institutes of Health and BSS-

CD-IS-2 from the U.S. Public Health Service. 4 Present address: Laboratory for Microbiology and Immunology, the Hebrew University-

Hadassah School of Dentistry, Jerusa!em.

93

94 Z. MARCUS, A. M. DAVIES, AND I. GINSBURG

tococci (lO”/ml) were incubated for 30 minutes at 37 ‘C with maltose (O.Olfif), MgSO.i.7H30 (O.OlJ4) and cysteine free base (O.OOOlM) to which 1 mg, 1 ml of RNA core fraction was added. After incubation the cells were removed by high- speed centrifugation and the clear supernate was filtered through a Millipore filter with a pore size of 0.2 p. The hemolysin (lO,OOO-20,000 hemolytic units/ml) was further concentration by precipitation with 8 volumes of methanol at 0°C and re- chromatographed on DEAE-Sephadex columns. Such hemolysin preparations did not show any precipitins in immunoelectrophoretic analysis against a pool of human gamma globulin known to contain at least 14 different antibodies to streptococcal antigens.

2. Cell-bound hemolysin. Washed streptococci (lO”/ml) were incubated for 10 minutes at 37°C with maltose, Mg++, and cysteine (see above). Such streptococcal cells possess high hemolytic activity but no extracellular hemolysin can be detected (Ginsburg and Grossowicz, 1958; Ginsburg et al., to be published).

Determination of hemolytic activity

One hemolytic unit of the RNA hemolysin or CBH was defined as the highest dilution of hemolysin (or cells) to produce 50% hemolysis of a 176 suspension of human red blood cells in a final volume of 1 ml, after 30 minutes of incubation at 37’C. No attempt is made here to correlate the hemolytic unit of CBH with RNA- hemolysin.

Inhibition of hemolytic activity

To inhibit SLS and CBH activity, 100 pg/ml of trypan blue, 1 mg/ml vegetable lecithin (WBC), or 10 pg/ml crystalline papain were incubated with the hemolysins before they were added to tissue cultures. Inhibition of the hemolysins was checked against red blood cells and to assure that streptolysin 0 was not present; 25 lq,“ml of cholesterol were added to reaction mixtures.

Tissue cultures of rat heart

Hearts of day-old rats were removed aseptically and immersed in cold Earle’s solution without Ca+ + and Mg+ +. The hearts were cut into 1 X l-mm pieces with sharp scissors and incubated on a magnetic stirrer with 0.25’j/o trypsin (Difco 1: 250) for 10 minutes at 37°C.

The procedure was repeated several times until all the tissue was disintegrated. The 2 X 10” cells in 1 ml of Eagle’s medium (with four-fold concentration of amino acids and supplemented with S-10% of inactivated calf serum) were put into a Leighton tube together with a 5 X lo-mm strip of cover glass. Cultures were grown at 37°C in an atmosphere of .5-S% CO- for 4-5 days by which time they had developed into confluent sheets. For studyin g the effect of the RNA hemolysin or CBH on the cells the culture was first washed twice with warm Eagle’s medium.

In some experiments the cover glass to which the cultures were attached was in- verted over a well slide with a plastic rim; this permitted the addition of the medium and hemolysin directly to the slides mounted on the warm stage microscope.

HEMOLYSINS OF GROUP A STREPTOCOCCI 95

Tissue cultures of rat kidney cells

Kidneys were removed from the same newborn rats and processed by the same methods described for the heart. One million cells per tube were grown in medium 199 with 20% calf serum, in an atmosphere of 558% CO-.

Photography

Photographs were taken through a Zeiss phase microscope at magnifications of 400 and 1000. In some studies the same microscopic fields were photographed at j-10 minute intervals while in others different microscopic fields were photographed.

RESULTS

EFFECT OF OXYGEN-STABLE HEMOLYSINS ON RAT HEART TISSUE CULTURES

Ccl/-bound hemolysin (CBH)

Six Leighton tubes containing 4- to S-day-old heart culture, twice washed with Earle’s medium, were inoculated with 0.1 ml of washed streptococci (IO”/ml) pos- sessing approximately 1500 HU of CBH and the volumes brought to 1 ml with Eagle’s medium containing 200 u/ml crystalline sodium penicillin G and 100 ltg/ml streptomycin sulfate (without serum). The tubes were then incubated at 37°C and at intervals a tube was removed and the cover slide containing the culture photographed directly.

Culture at zero time (Fig. A). The heart cells appear elongated. The nuclei contain 1-3 nucleoli, the cytoplasm is moderately granular and the nuclear membrane is not prominent. The cells are separated by a well discernible cell membrane and few streptococcal chains are seen in the preparation.

Culture at 10 minutes (Fig. B). The major changes seen are the appearance of large phase-dense cytoplasmic granules of varying size. Some areas of the cytoplasm contain several vacuoles which accumulate mostly around the nuclei. The nuclei appear swollen and the nuclear membrane is easily visible. Some streptococcal chains are seen adhering to the damaged cells.

Culture at 20-40 minutes. Granulation and vacuolization of the cells is more marked (Fig. C), with formation of blebs (Fig. D ) which increase in size (Fig. E).

Culture at 60 minutes (Fig. F). There is complete disintegration of most of the heart cells leaving some nuclei intact. Most cells have become detached from the glass and large numbers of streptococcal chains can be seen.

EFFECT OF TRYPAN BLUE ON THE CYTOTOXIC EFFECT OF CBH

I’reincubation of CBH with trypan blue (100 mg <‘ml) resulted in complete inhibition of cytotoxicity.

R-Y.4 hemolysin (streptolysins)

In this experiment the same microscope field of heart culture was photographed at intervals. 1000 H.U. of RNA hemolysin were added to the preparation.

Culture at zero time (Fig. G). Cells appear as a confluent sheet. Only moderate amounts of cytoplasmic granules are seen and the nuclei, containing 2-4 nucleoli,

96 z. MARCUS, A. M. DAVIES, AND I. GINSBURG

F ‘IO. 4. Four-day-old normal rat heart culture, zero time, phase contrast, X 400.

strc ?ptc )CO( :cal chains are seen in the preparation. I iIG B. The effects of CBH on rat heart cultures after 10 minutes (phase contrast X

.t few

400).


FIGS. C and D. The effects of CBH on rat heart cultures after 20-40 minutes (phase contrast

x 400).

98 2. MARCUS, A. M. DAVIES. AND I. GINSBURG

FIG. E. The effects of CBH on rat heart cultures after 20-40 minutes (phase contrast X 400) FIG. F. The effects of CBH on rat heart cultures after 60 minutes (phase contrast X 400)


do not show a conspicuous membrane. A few vacuoles are seen scattered in the cytoplasm of some cells.

Culture at 10 minutes (Fig. H). A large number of cytoplasmic granules have appeared and in some areas vacuoles are more prominent. The nuclear membranes are well marked.

Culture at 20 minutes (Fig. I). Granulation and vacuolization have increased both in size and extent. Signs of cellular disintegration are apparent and some cells show blebs emerging from the cellular membrane.

Culture at 30 minutes (Fig. J). Cell distintegration is more extensive and some of the cells have completely disappeared.

Culture at 60 minutes (Fig. K). Most of the cells are destroyed leaving a few intact nuclei and there is accumulation of cellular debris. Similar changes can be seen in Fig. L: taken from a different microscopic field.

EFFECT OF TRYPAN BLUE ON SLS ACTIVITY

As in the case of CBH? preincubation of RXA-hemolysin with trypan blue (100 lq/ml) completely abolished the cytopathogenic effect of SLS on the heart cultures.

EFFECT OF CELL-BOUND HEMOLYSIN (CBH) ON RAT KIDNEY TISSUE CULTURES

The effect of CBH (1,500 HU;ml) on rat kidney tissue cultures is very similar to the effect of CBH in rat tissue cultures.

Cuhres at zero time (Fig. &I). Rat kidney cells appear more elongated than the rat heart cell. A few granules are seen in the cytoplasm.

Cultures at 10 minutes (Fig. N). There are very slight changes in the cytoplasm, and the picture is very similar to Fig. M.

Czdtures at 20 minutes (Fig. 0). The cytoplasm of the kidney cells show some vacuolization. The nuclear membrane is easily visible, but the borders of the cells are less well marked.

Cultures at 80 minutes. Figure P shows typical signs of cellular damage; large vacuolization of the cytoplasm, swelling of the cells and the appearance of blebs.

Cultures at 120 minutes (Fig. Q). Most of the cells are destroyed and many of those remaining have peeled off the glass.

EFFECT OF RNA-HEMOLSYIN ON R,\T KIDNEY CELLS

Similar cytologic changes were induced in kidney cells by 4000 HU/ml of RNA- hemolysin. Smaller amounts of RXA-hemolysin were inactive under the conditions of the experiment.

EFFECT OF STRAIN C20:lU ON HEART AND KIDNEY CELLS

The strain C,,,,U, a mutant lacking RNA-hemolysin and CBH, has no effect on heart or kidney tissue cultures even after 3 hours incubation.

DISCUSSIOX

The oxygen-stable hemolysins of group A streptococci are markedly toxic for heart and kidney cells grown in culture. The cytological changes are characterized by swelling, by the appearance of phase-dense granules and by vacuolization. The


FIG. G. Five-day-old normal rat heart, zero time; cells appear as a confluent sheet (phase contrast X 400).

FIG. H. The same field after the addition of 1000 H.U./ml of RNA-hemolysin after 10

minutes.

HEMOLYSINS OF GROUP A STREPTOCOCCI

FIG. minute

FIG. minute

The same field after the addition of 1000 H.U.,‘ml of RNA-hemolysin

The same field after the addition of 1000 H.U./ml of RN.4-hemolpin

after

after


FIG .I< minul :es.

FIG L. field, 60

. The same field after the addition of 1000 H.U.,/ml of RNA-hemolysin a .fter 60

Rat heart culture after the addition of 1000 H.U./ml of RNA-hemolysin,

minutes (phase contrast Y, 400). ‘ent


FIG. M. Normal rat kidney culture at zero time (phase contrast X 403). FIG. N. The effect of CBH on rat kidney cultures after 10 minutes.

104 2. MARCUS, A. M. DAVIES, AND I. GINSBURG

FIG. 0. The effect of CBH on rat kidney cultures after 20 minutes.

FIG. P. The effect of CBH on rat kidney cultures after 80 minutes.


cells show blebbing and then complete disintegration. These cytopathic changes indicate alteration in the permeability and continuity of the cell membrane as in the case of the red blood cell (Ginsburg, 1959; Ginsburg and Grossowicz, 1960). These results also confirm the findings of Koshimura et al. (1955), Bernheimer and Schwartz (1960), Havas et al. (1963), and of Snyder and Hamilton (1963) on the cytotosic effects of streptococcal hemolysins on a variety of mammalian cells in vitro.

The effect of the RNA-hemolysin on the heart and kidney cells could be completely inhibited by antihemolytic agents (trypan blue, lecithin, papain), indicating

Frn. Q, The effect of CBH on rat kidney cultures after 120 minutes

that the hemolytic activity of the streptolysin was probably responsible for the cytopathic changes obtained. On the other hand, RNA extract obtained from a mutant lacking both RKA-hemolysin and CBH failed to damage the cells (see also Ginsburg and Grossowicz. 1960).

Bernheimer and Schwartz (1960) found that streptolysins S and 0 were the only streptococcal products capable of injurin, u leucocytes. In the case of the cytotoxic effect caused by washed streptococci it is more difficult to prove that CBH was the sole active agent against the cultured cells. The inhibition by trypan blue, however, and the lack of cytotoxicity of strain Cz,,:s U, known to be deficient in CBH, are highly suggestive that this is indeed the case.

Recently, the transfer of CBH to RBC was described (Ginsburg and Harris, 1962; Ginsburg and Harris, 1963). Under such conditions no extracellular hemolysin can be detected in supernates obtained from streptococci-red blood cell mixtures, yet complete hemolysis of the RBC occurred. It is assumed that some red cell components have a higher affinity for the hemolysin than do the streptococci and the hemolysin is transferred from one cell to another. A similar event may occur in the tissue cultures studied. Since the tissue cultures were washed with a


medium devoid of serum, it can be assumed that no serum hemolysin (Ginsburg and Harris, 1963) participated in the cytotoxic reaction.

Nor could the cytopathic effects be attributed to the proliferation of the streptococci, since the medium contained both penicillin and streptomycin in amounts sufficient to inhibit multiplication. The presence of cholesterol in the agents used to prepare RNA-hemolysin assured that streptolysin 0 was not present in detect- able amounts.

Little is known about the mechanics of lysis of mammalian cells by streptolysin. Hirsch et al. (1963) have demonstrated that rabbit leucocytes exposed to streptolysin S show an initial rapid and extensive lysis of cytoplasmic granules, followed by liquification of the cytoplasm and cell destruction. Weissmann et al. (1963) have also shown that streptolysin S and 0 added to granules of tissue homogenates (lysosomes) were able to release the lysosomal enzymes fi-glucuronidase and acid phosphatase and, to a lesser extent, a mitochondrial enzyme, malic dehydrogenase. Ehrlich ascites tumor cells injured by streptolysins 0 and S have been shown to be disintegrated by small amounts of proteolytic enzymes (streptococcal proteinase, papain, trypsin. and plasmin), which by themselves were not injurious to the cells (Ginsburg, 1959). Similarly, proteolytic enzymes enhanced the destruction of tumor cells injured by antibodies and complement (Ginsburg and Ram, 1960; Hirata, 1963).

Further studies (Elias et al., 1965) have shown that SLS activity is strongly inhibited by phospholipids obtained from ghosts of human red blood cells and that splitting of the phospholipids of the ghosts by phospholipase C markedly reduced the capacity of the treated ghosts to absorb SLS. These results indicate that some of the phospholipids of the cell membrane may be the binding sites for SLS. Whether or not SLS possesses enzymatic activity capable of splitting cell membrane components remains to be determined. It is probable therefore that SLS interacts with the cellular membrane causing permeability changes and as a result, autolytic mechanisms are activated which release hydrolytic enzymes from lysosomes.

Finally, the fact that SLS was found to be highly cytotoxic in viva, causing de- generation of parenchymateous organs (Barnard and Todd, 1940) and destroying the basement membrane of the kidney (Tan and Kaplan, 1963), points to the possible role of streptolysin S and other hemolysins in the pathogenesis of streptococcal infections and post streptococcal sequellae.

SUMMARY

The effects of streptolysin S and cell-bound hemolytic factor were tested on tissue cultures of rat heart and kidney. Cytopathogenic changes were observed, characterized by swelling,

cytoplasmic vacuolization and bleb formation and cells were disintegrated within 1-2 hours.

These changes were abolished by known inhibitors of streptolysin S.

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Oxygen-Stable Hemolysins of Group A Streptococci

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