INTRODUCTION

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Streptococcus pyogenes, the group A streptococcus (GAS), is a common and important gram-positive pathogen responsible for a range of human diseases, such as the self-limiting diseases impetigo and pharyngitis and the invasive, life-threatening diseases streptococcal toxic shock syndrome and necrotizing fasciitis (7, 42). Even after bacteria are cleared from the host, sequelae, such as glomerulonephritis, rheumatic fever, and rheumatic heart disease, sometimes follow GAS infections. Reports of invasive streptococcal disease have increased in recent years, prompting greater public awareness of the dangers associated with this organism (32, 37). This disease is especially menacing because it has a rapid onset and a high mortality rate and often occurs in otherwise healthy adults.

Unlike other pathogens, for which a given strain or serotype is responsible for a specific type of disease caused by that organism, GAS bacteria do not appear to be so highly specialized with regard to the types of disease they cause. Although there are associations between GAS strains of a given M type and invasive disease (9, 20, 21, 34, 38, 40), many strains cause both life-threatening and self-limiting infections (20, 25, 27). This implies that a given strain of GAS encodes the factors necessary for the establishment of more than one kind of disease and that differential expression of these factors, presumably in response to the host environment, determines the course of infection. Definition of the virulence factors that determine the course of disease is crucial to understanding the development of invasive streptococcal disease. While some invasive infections of deep tissues are associated with a breach of the skin barrier by a puncture, cut, or burn, invasive disease also occurs after blunt trauma and in the absence of identified tissue insult (37, 43). This suggests that in addition to being opportunistic invaders, GAS bacteria are intrinsically capable of breaching host tissue barriers.

Several streptococcal proteins may be involved in the breakdown of host tissues. Among these, Plr and streptokinase have been implicated in tissue destruction through activation of the host protein plasmin (8, 28). Tissue damage specifically associated with the formation of necrotic skin lesions in mice depends on the activity of the cytolysin streptolysin S (Sls) (6). GAS also encodes a second hemolysin, streptolysin O (Slo), which has well-documented cell- and tissue-destructive activity (1). Slo is a secreted protein produced by nearly all clinical GAS isolates tested (1, 26, 36) and is the best characterized of a family of oxygen-sensitive, thiol-activated toxins that includes such established virulence factors as perfringolysin (theta toxin), pneumolysin, and listeriolysin O (2, 5, 10). Slo interacts with cholesterol in target cells to form multisubunit pores and is active on a number of eukaryotic cell types, including erythrocytes, leukocytes, macrophages, platelets, and various cell culture lines (16, 23).

Purified Slo is a very potent toxin and is lethal within minutes when injected intravenously into mice or rabbits (4, 18, 41). At sublethal doses, Slo causes dermal necrotic lesions, venous congestion, and increased vascular permeability as well as various neurological abnormalities before death (11, 15, 29). When used to infect keratinocyte monolayers, Slo mutants show an altered cytokine induction profile compared to that for a wild-type strain (30), suggesting that a subset of the host cell response to GAS infection occurs in response to Slo.

The many biological effects of Slo on the host suggest that it may act at several stages in the infectious process. However, although the effects of purified Slo are well characterized, its role in virulence for GAS infection in vivo has not been addressed directly. We investigated the role of Slo in invasive streptococcal disease using the mouse model described by Schrager et al. (33). In this model, mice are inoculated subcutaneously with a suspension of GAS. Disease progression begins with the formation of a large, raised abscess at the site of inoculation within 24 h postinfection. The abscess next develops a necrotic center, then a sunken necrotic lesion, usually within 24 to 36 h. After formation of the necrotic lesion, mice either develop bacteremia and die or else begin to clear the infection and recover fully within 10 days. Our examination of two slo mutants of GAS in this model demonstrates that Slo is important for streptococcal virulence and suggests roles for Slo in the process of invasive disease.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. S. pyogenes strain AM3 (24) and its derivatives were grown in Todd-Hewitt broth supplemented with 0.2% yeast extract (ThyB) at 37°C. Plate cultures were grown on Todd-Hewitt yeast medium containing 1.5% agar (ThyA) at 37°C. Antibiotics were used at the following concentrations: streptomycin at 1,000 µg/ml, erythromycin at 0.5 µg/ml for S. pyogenes and at 500 µg/ml for Escherichia coli, and kanamycin at 300 µg/ml for S. pyogenes and at 50 µg/ml for E. coli.

Construction of slo mutant strains JRS809 and JRS821. An slo insertion mutant, JRS809, was generated in strain AM3 by insertion of the nonreplicating plasmid pSLO2 as described by Ruiz et al. (30). This plasmid contains an internal fragment of the slo gene. A single recombination event in this region integrates the plasmid within slo, resulting in two incomplete copies of the gene. Inactivation of slo was confirmed by kanamycin resistance and by a lack of Slo-specific hemolytic activity.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Construction of the Slo deletion mutant JRS821 by allele replacement. (A) The wild-type allele from strain AM3 was replaced with slo.300 carried on plasmid pSLOD2 as described in Materials and Methods. The arrows indicate primers used for PCR amplification. (B) PCR amplification of slo and slo.300 with primers 5SLO and 3SLO. These primers are common to both strains, but the product generated from strain JRS821 is 303 nt smaller than that from AM3, reflecting the deletion.

Hemolysis assays. Cell culture supernatants were prepared from 10-ml overnight cultures of the appropriate strains by centrifugation for 10 min at 7,500 × g and filtration of the supernatant through a 0.25-µm-pore-size filter. This cleared supernatant was then incubated for 10 min at room temperature with 10 mM dithiothreitol. Two 0.5-ml aliquots from each sample were placed in 1.5-ml sterile Eppendorf tubes, and 25 µg of cholesterol (Sigma) was added to one sample. Following a 30-min incubation at 37°C, 0.25 ml of a 2% suspension of bovine erythrocytes in phosphate-buffered saline (PBS) (31) was added to each sample, mixed by inversion, and incubated for 30 min at 37°C. Finally, 0.5 ml of PBS was added to each sample, and unlysed erythrocytes were spun out of solution for 1 min at 200 × g in a microcentrifuge. Supernatants were removed, and the release of hemoglobin was assessed by reading the absorbance at 541 nm. Erythrocytes were incubated in water as a positive control for lysis (100% lysis), and fresh ThyB was used as a negative control. Controls were treated exactly as experimental samples, except that medium rather than PBS was added to the water sample in the final step. At least three separate hemolysis assays were performed for each sample.

Reversion assays. Revertants to slo⁺ were identified by the loss of kanamycin resistance. Dilutions of overnight cultures grown in the absence of kanamycin were plated on ThyA with streptomycin (ThyA+Sm) and grown overnight. These colonies were then replica plated onto ThyA with kanamycin (ThyA+Km) and ThyA+Sm and incubated overnight. Clones that appeared to be kanamycin sensitive were picked from the original plate and spotted onto ThyA+Km and ThyA to confirm the loss of the marker. The reversion frequency (R.F.) was defined as the percent revertant bacteria divided by the number of total bacteria examined.

Inoculation of mice. Overnight cultures of the appropriate strains were used to inoculate 50 ml of ThyB in sterile Klett flasks. Cultures were incubated at 37°C until late log phase (Klett units, ~40), and bacteria were isolated by centrifugation for 10 min at 7,500 × g at 4°C. The cleared supernatant was decanted, bacterial pellets were suspended in the remaining liquid, and the cell suspensions were placed on ice. Bacteria were counted in a Petroff-Hauser chamber, and the suspension was adjusted to ~10⁹ cells/ml with saline. Inoculation of the mice was performed by the method of Schrager et al. (33). Briefly, 5- to 6-week-old female CD1 mice (Charles River Laboratories) were anesthetized by inhalation of Metofane (Schering-Plough, Bloomfield, N.J.), and ~2 cm² of hair was removed from the left flank with Nair (Carter products, Cranbury, N.J.). One hundred microliters of the adjusted cell suspension was injected subcutaneously with a tuberculin syringe in the center of the bare region. Mice were monitored twice daily and were euthanized by CO₂ inhalation when they became hunched, lethargic, and had paralysis in the hind limbs. Mice that did not show signs of illness were euthanized at the end of the 10-day trial. Seven independent experiments involving infections with the wild-type strain AM3 were performed on 76 mice, and five separate experiments involving JRS821 infection were performed on 57 mice. Results for strain JRS809 represent a single experiment with 11 mice.

Culturing of lesions and spleen. Lesion sites were wiped with alcohol pads and swabbed beneath the scab with a sterile urethro-genital calcium alginate swab. The swab was used to inoculate 1 ml of ThyB+Sm and cultured overnight at 37°C. Spleen cultures were obtained by aseptically removing spleens to Dounce tissue homogenizers containing 0.5 ml of ThyB+Sm, homogenizing the tissue, and plating directly on ThyA+Sm plates. Samples of bacteria isolated from both lesions and spleens were screened for Slo-specific hemolytic activity.

Statistical analysis. Survival data were analyzed by Kaplan-Meier product-limit estimates of survival functions and were tested for significance by log rank tests and the Yates correction for the log rank test (13).

	RESULTS

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Construction of an slo strain, JRS809, by insertion mutation. The slo mutation in strain AM3 was originally constructed as an insertion mutation, in which the nonreplicating plasmid pSLO2 (30) was integrated by homologous recombination into the slo locus to produce the strain JRS809. Integration of pSLO2 into slo was shown by resistance to kanamycin and by a loss of hemolytic activity. Cell culture supernatants from AM3 and JRS809 were analyzed for Slo-specific hemolytic activity by incubation with bovine erythrocytes in the presence or absence of cholesterol, an Slo-specific inhibitor. Hemolytic activity was detected in the supernatant of AM3 but not in that of JRS809. Hemolytic activity from the wild-type strain was reduced to background levels by the addition of cholesterol, confirming that it was due to Slo rather than to contaminating Sls (data not shown). We observed no difference between the growth rates of AM3 and JRS809 in broth cultures (data not shown).

Disease progression and reversion of the slo mutation following infection with JRS809. Mice were injected subcutaneously with ~2 × 10⁷ CFU of AM3 or JRS809. The infection progressed essentially as described by Ashbaugh et al. (3) for both strains. Within 24 h, all mice developed an abscess at the site of infection, and then a necrotic center developed, and this necrotic center finally became a sunken necrotic lesion, usually by day 3. Most of the mice died within 6 days postinfection, but mice that survived for 6 days generally survived through the end of the 10-day experiment. The surviving mice gained weight, and their lesions began to heal. There were no readily observable differences in the time it took to develop lesions or in lesion morphology between mice infected with AM3 and those infected with JRS809. Furthermore, there was no significant difference in mortality rates between the two sets of mice (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Change in body weight of mice injected with strain AM3, JRS809, or sterile saline. The average weight of mice at each day postinfection is shown in comparison to the average original weight. Bars represent the standard error in weight at each day.

Construction of JRS821 by gene replacement. To distinguish the effects of revertant bacteria from those of the slo mutant, we constructed a stable slo mutation for further examination in the mouse model. Plasmid pSLOD2 was used to replace the wild-type slo allele with slo.300, resulting in a 303-nt in-frame deletion as described in Materials and Methods (Fig. 1A). Replacement of the wild-type slo with slo.300 was confirmed by PCR. The slo allele from AM3 yielded a product that was 303 bp larger than the product from JRS821 (Fig. 1B), reflecting the internal deletion in JRS821. Hemolysis assays were performed as described for JRS809 to confirm that JRS821 lacked Slo-specific hemolytic activity. As with JRS809, there was no detectable difference in the growth rate of JRS821 from that of AM3 in broth culture (data not shown).

Effect of Slo on virulence of GAS in murine model of skin infection. Strain JRS821 was used to infect mice as described for the JRS809 mutant. Mice were injected subcutaneously with ~10⁷ to 10⁸ CFU of AM3 or JRS821 and were monitored twice daily for a period of 10 days. Mice were euthanized as they became moribund, and their wounds and spleens were cultured for the presence of streptomycin-resistant bacteria. Mice infected with the wild type and those infected with slo GAS showed no gross differences in the size and shape of necrotic lesions formed.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Kaplan-Meyer survival curves for mice injected subcutaneously with the wild-type GAS strain AM3 or the slo strain JRS821 (P < 0.05).

Hemolysis phenotype of bacteria isolated from infected mice. Although strain JRS821 carried a gene replacement that should not have been capable of reverting to slo⁺, hemolysis assays were performed to confirm that bacteria isolated from mice retained the expected hemolytic phenotype. Pooled bacteria isolated from spleens of mice infected with AM3 or JRS821 were cultured overnight in ThyB+Sm, and cell culture supernatants were analyzed for Slo-specific lysis of bovine erythrocytes. As expected, bacteria isolated from mice infected with the wild-type strain showed secreted hemolytic activity, while those isolated from the JRS821-infected mice did not (Fig. 4).

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Hemolysis phenotype of bacteria isolated from mice infected with the wild-type (wt) GAS strain AM3 or the slo strain JRS821. Isolates from mice infected with each strain were pooled and assayed for Slo-specific hemolysis as described in Materials and Methods.

	DISCUSSION

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Slo is ubiquitous among GAS strains. Slo, in contrast to many other candidate virulence factors, is expressed by nearly all GAS strains, and the amino acid sequence is conserved. Among virulence factors that appear to be present in all GAS strains, several have been found to be serologically variable. The classical example is the M protein, an important virulence factor whose antigenic diversity serves as the basis for classification of GAS strains (22, 35). More recently, sic, the streptococcal inhibitor of complement, has been found to be even more variable in sequence among GAS isolates than the M protein (17, 39). In these cases, it seems that the sequence of the protein is not essential for its pathogenic function. In contrast, the amino acid sequence of Slo appears to be conserved, since antibodies to Slo react with all strains tested and some even cross-react with the related sulfhydryl-activated toxins from other bacteria (2, 15). It seems likely that the need to preserve its cytolytic activity has served to prevent antigenic variation of Slo. This suggests that Slo activity is important for GAS in vivo and led us to expect that an Slo mutant would be likely to be attenuated for virulence in an animal model of infection.

Slo is important for GAS virulence in invasive disease. We have shown Slo to be important for GAS invasive disease in a murine model of skin infection. Our results demonstrate that mice infected with a stable slo deletion mutant have a significantly higher rate of survival than mice infected with wild-type bacteria (Fig. 4). By 6 days after infection, more than 65% of mice infected with the slo mutant were alive, compared to only 30% of mice infected with wild-type GAS. Nearly half (46%) of the mice infected with the mutant strain survived to the end of the 10-day experiment, compared to only 24% of the mice infected with the wild-type strain. Half (52%) of the mice that survived infection with the slo mutant had positive spleen cultures, indicating that the infection had disseminated in these apparently healthy animals. This demonstrates that dissemination of the slo mutant does not necessarily cause disease.

Role of Slo in invasive disease. Slo has many effects on various host systems, so it is likely that Slo has more than one role for invasive disease. However, our results suggest that Slo is not important for initiating infection, because mice developed necrotic lesions at the site of the injection regardless of the infecting strain. We did not detect any differences in the time to development of a necrotic lesion, or in the morphology of the lesions formed in mice infected with the slo mutants, compared to the wild type. In dramatic contrast, mutants deficient in production of Sls, a different streptococcal hemolysin, are unable to cause necrotic lesions in mice (6). Slo and Sls have been characterized extensively in vitro and shown to have very different properties. Slo is a secreted, immunogenic, oxygen-labile protein that is specifically inhibited by free cholesterol. Sls is membrane associated, oxygen stable, nonimmunogenic, and insensitive to cholesterol. Thus, it is not surprising that the two proteins, which would be expected to be active in different host environments, would have different roles in the infection process.

Degree of attenuation of GAS Slo mutants. Although slo GAS bacteria were less virulent than the wild-type strain, this difference was less dramatic than that observed for some other virulence factors that have been examined using this model of GAS infection. Mutants deficient in production of the M protein or hyaluronic acid capsule were strongly attenuated (less than 10% mortality) for virulence in this model of invasive disease (3). The role of streptococcal cysteine protease has also been examined in this murine model using two different M3 strains of GAS. The results varied from no observable effect to a very clear attenuation of virulence (10 to 80% mortality) (3, 24), suggesting that cysteine protease is important for virulence in some strains of GAS.

Conclusion. We have shown Slo to be important for full virulence in a mouse model of invasive GAS disease. Contrary to what one might expect, the cytolytic activity of Slo was not necessary for the formation of the necrotic lesion, nor was it necessary for the spread of GAS from the lesion. However, once the bacteria had disseminated, Slo was an important factor in the ability to cause systemic disease. Thus, the important activity of Slo does not appear to be in causing localized tissue damage following subcutaneous inoculation, a process for which Sls appears to be required.