Antibodies against a Synthetic Peptide of SagA Neutralize the Cytolytic Activity of Streptolysin S from Group A Streptococci

ABSTRACT Virtually all group A streptococci (GAS) produce streptolysin S (SLS), a cytolytic toxin that is responsible for the beta-hemolysis surrounding colonies of the organisms grown on blood agar. SLS is an important virulence determinant of GAS, and recent studies have identified a nine-gene locus that is responsible for synthesis and transport of the toxin. SLS is not immunogenic; thus, no neutralizing antibodies are evoked during the course of natural infection. In the present study, we show that a synthetic peptide containing amino acid residues 10 to 30 of the putative SLS (SagA) propeptide [SLS(10-30)] coupled to keyhole limpet hemocyanin evoked antibodies in rabbits that completely neutralized the hemolytic activity of the toxin in vitro. Inhibition of hemolysis was reversed by preincubation of the immune serum with soluble, unconjugated peptide, indicating the specificity of the antibodies. In addition, antibodies that were affinity purified over an SLS(10-30) peptide column completely inhibited SLS-mediated hemolysis. The SLS(10-30) antisera did not opsonize group A streptococci; however, when combined with type-specific M protein antisera, the SLS antibodies significantly enhanced phagocytosis mediated by M protein antibodies. Thus, we have shown for the first time that it is possible to raise neutralizing antibodies against one of the most potent bacterial cytolytic toxins known. Our data also provide convincing evidence that the sagA gene actually encodes the SLS peptide of GAS. The synthetic peptide may prove to be an important component of vaccines designed to prevent GAS infections.

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Cytotoxic Effects of Streptolysin O and Streptolysin S Enhance the Virulence of Poorly Encapsulated Group A Streptococci

Infection and Immunity ◽

10.1128/iai.71.1.446-455.2003 ◽

2003 ◽

Vol 71 (1) ◽

pp. 446-455 ◽

Cited By ~ 85

Author(s):

Gabriele Sierig ◽

Colette Cywes ◽

Michael R. Wessels ◽

Cameron D. Ashbaugh

Keyword(s):

Bacterial Resistance ◽

Cell Injury ◽

Natural Infection ◽

Group A Streptococci ◽

Cytotoxic Effects ◽

In Vivo Models ◽

Streptolysin S ◽

Streptolysin O ◽

Group A

ABSTRACT Although the toxicity of streptolysin O (SLO) and streptolysin S (SLS) in purified group A streptococci (GAS) has been established, the effect of these molecules in natural infection is not well understood. To identify whether biologically relevant concentrations of SLO and SLS were cytotoxic to epithelial and phagocytic cells that the bacteria would typically encounter during human infection and to characterize the influence of cell injury on bacterial pathogenesis, we derived GAS strains deficient in SLO or SLS in the background of an invasive GAS M3 isolate and determined their virulence in in vitro and in vivo models of human disease. Whereas bacterial production of SLO resulted in lysis of both human keratinocytes and polymorphonuclear leukocytes, GAS expression of SLS was associated only with keratinocyte injury. Expression of SLO but not SLS impaired polymorphonuclear leukocyte killing of GAS in vitro, but this effect could only be demonstrated in the background of acapsular organisms. In mouse invasive soft-tissue infection, neither SLO or SLS expression significantly influenced mouse survival. By contrast, in a mouse model of bacterial sepsis after intraperitoneal inoculation of GAS, SLO expression enhanced the virulence of both encapsulated and acapsular GAS, whereas SLS expression increased the virulence only of acapsular GAS. We conclude that the cytotoxic effects of SLO protect GAS from phagocytic killing and enhance bacterial virulence, particularly of strains that may be relatively deficient in hyaluronic acid capsule. Compared to SLO, SLS in this strain background has a more modest influence on GAS pathogenicity and the effect does not appear to involve bacterial resistance to phagocytosis.

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STUDIES ON THE PATHOGENICITY OF GROUP A STREPTOCOCCI

Journal of Experimental Medicine ◽

10.1084/jem.110.4.617 ◽

1959 ◽

Vol 110 (4) ◽

pp. 617-628 ◽

Cited By ~ 36

Author(s):

Marie Judith Foley ◽

W. Barry Wood

Keyword(s):

Hyaluronic Acid ◽

Virulent Strain ◽

Critical Role ◽

M Protein ◽

Group A Streptococci ◽

Streptococcal Infections ◽

Full Complement ◽

Group A ◽

Hyaluronic Acid Capsule

A quantitative study of the combined antiphagocytic effects of the M protein and the hyaluronic acid capsules of four strains of Group A streptococci revealed the following facts relating to their intraperitoneal virulence in mice and rats: 1. The most virulent strain, S23M (matt), produced both a large hyaluronic acid capsule and a full complement of M protein, the combined effects of which rendered the organism highly resistant to surface phagocytosis. 2. The slightly less virulent strain, T14/46 (matt virulent) was somewhat more susceptible to surface phagocytosis owing to the fact that its smaller capsule was less antiphagocytic than that of the S23M organism. 3. The glossy variant of the S23 strain (S23G), which ranked third in virulence, was still more susceptible to surface phagocytosis because of its lack of detectable M substance. Its large hyaluronic acid capsule, however, was capable of protecting it against phagocytosis on glass. 4. The least virulent strain, T14 (matt avirulent), was the most susceptible of all to phagocytosis. Though it possessed both M substance and capsule, which together prevented its phagocytosis on glass, each of them was shown to be quantitatively and functionally deficient as compared to Strain S23M. The differences in phagocytability, which appear to be directly related to the pathogenicity of the organisms, could be adequately demonstrated in vitro only by phagocytic tests designed to measure surface phagocytosis in the absence of opsonins. This fact is in keeping with the observation, previously reported, that surface phagocytosis plays a critical role in the defense of the host, particularly during the earliest stages of experimental streptococcal infections. Its possible relation to suppuration during the later stages of infection is also discussed.

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STUDIES ON STREPTOCOCCUS PYOGENES

Journal of Experimental Medicine ◽

10.1084/jem.109.6.589 ◽

1959 ◽

Vol 109 (6) ◽

pp. 589-600 ◽

Cited By ~ 2

Author(s):

Hutton D. Slade ◽

Yoshitami Kimura

Keyword(s):

Rabbit Antiserum ◽

M Protein ◽

Rabbit Serum ◽

Chemical Fractionation ◽

Group A Streptococci ◽

Heterologous Antiserum ◽

Group A ◽

Normal Rat ◽

Group B

Heat-killed cells of Group A streptococci caused death of the adrenalectomized rat. While the adrenalectomized rat readily succumbed to intraperitoneal infection with living cells, death was due primarily to toxicity. The normal rat was highly resistant under either condition. For studies on the toxic materials, the cells of numerous serological types of group A streptococci, and of a Group B and a Group D streptococcus, were extracted with 0.1 N HCl at 100°C. or by sonic oscillation. The extracts, containing macromolecular components, were subjected to chemical fractionation and purification. C substance and M protein of Group A streptococci released from the cell by sonic oscillation were toxic to the adrenalectomized rat in quantities of 1 mg./100 gm. rat. Death usually occurred within 2 hours. On the other hand, C substance and M protein released from the cell with HCl at 100°C. were relatively non-toxic to the adrenalectomized rat. The sonic-extracted C substance of streptococcal Groups B, C, and D was also toxic. The toxic property of the C and M preparations was neutralized in vitro in each case by group and type-specific rabbit antiserum. Heterologous antiserum was without effect. Adrenalectomized rats which received homologous antiserum 18 hours before challenge were also resistant to the toxicity of the C and M preparations. Trypsin destroyed the toxic effect of the M protein preparations and was without effect on the toxicity of the C substance. The R antigen and a nucleoprotein component of Group A streptococci, preparations of protein from Groups B and D streptococci, and coagulase from Staphylococcus aureus were all found to be essentially non-toxicic for the adrenalectomized rat. Large quantities of peptone, crystalline albumin, and rabbit serum were also without effect.

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Multivalent Group A Streptococcal Vaccine Elicits Bactericidal Antibodies against Variant M Subtypes

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.7.833-836.2005 ◽

2005 ◽

Vol 12 (7) ◽

pp. 833-836 ◽

Cited By ~ 33

Author(s):

James B. Dale ◽

Thomas Penfound ◽

Edna Y. Chiang ◽

Valerie Long ◽

Stanford T. Shulman ◽

...

Keyword(s):

Wide Spectrum ◽

Epidemiologic Studies ◽

M Protein ◽

Group A Streptococci ◽

Protein Vaccine ◽

Amino Terminal ◽

M Proteins ◽

Group A ◽

Clinical Illness

ABSTRACT Group A streptococci cause a wide spectrum of clinical illness. One of several strategies for vaccine prevention of these infections is based on the type-specific M protein epitopes. A multivalent M protein-based vaccine containing type-specific determinants from 26 different M serotypes is now in clinical trials. Recent epidemiologic studies have shown that, within some serotypes, the amino-terminal M protein sequence may show natural variation, giving rise to subtypes. This raises the possibility that vaccine-induced antibodies against the parent type may not be as effective in promoting bactericidal killing of variant subtypes. In the present study we used rabbit antisera against the 26-valent M protein-based vaccine in bactericidal tests against M1, M3, and M5 streptococci, which were represented by multiple subtypes. We show that the vaccine antibodies effectively promoted in vitro bactericidal activity despite the fact that the M proteins contained naturally occurring variant sequences in the regions corresponding to the vaccine sequence. Our results show that the variant M proteins generally do not result in significant differences in opsonization promoted by rabbit antisera raised against the 26-valent vaccine, suggesting that a multivalent M protein vaccine may not permit variant subtypes of group A streptococci to escape in a highly immunized population.

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STUDIES OF PHAGOCYTOSIS OF GROUP A STREPTOCOCCI BY POLYMORPHONUCLEAR LEUCOCYTES IN VITRO

Journal of Experimental Medicine ◽

10.1084/jem.111.3.309 ◽

1960 ◽

Vol 111 (3) ◽

pp. 309-322 ◽

Cited By ~ 38

Author(s):

James G. Hirsch ◽

Alice B. Church

Keyword(s):

Hyaluronic Acid ◽

M Protein ◽

Rabbit Serum ◽

Polymorphonuclear Leucocytes ◽

Group A Streptococci ◽

Test Conditions ◽

Human Sera ◽

Group A ◽

Hyaluronic Acid Capsule

Studies have been made on phagocytosis and killing of Group A streptococci during mixing with suspensions of leucocytes in vitro. Under appropriate test conditions an anti-phagocytic effect can be demonstrated for the streptococcal hyaluronic acid capsule as well as for its M protein. The results obtained suggest an explanation for the suitability of human, but not rabbit, blood for opsonophagocytic tests designed to measure type-specific streptococcal antibodies. Human sera contain a factor which counteracts the anti-phagocytic effects of streptococcal hyaluronic acid capsules, and hence human blood serves well for detection of antibodies which combine with the only other phagocytosis-resisting component of this microorganism, namely M protein. In contrast, rabbit sera contain none of this factor, and addition of antibody to M protein to phagocytic test systems employing rabbit serum does not necessarily render the streptococci susceptible to engulfment by white cells, since the hyaluronic acid capsule may continue to interfere with phagocytosis. The nature of the human serum factor which opsonizes encapsulated streptococci is unknown. It does not appear to be an antibody or an enzyme capable of depolymerizing hyaluronic acid.

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