[9] Production, purification, and assay of streptolysin S

Alouf, Joseph E.; Loridan, Catherine

doi:10.1016/s0076-6879(88)65012-9

Cited by 24 publications

(19 citation statements)

References 6 publications

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“…Although additional granule marker assays and ultrastructural studies would be needed for confirmation, our findings suggest that this leucotoxin is unlikely to act in a manner similar to that of streptolysin S (Densen & Mandell, 1980;Alouf, 1980;Alouf & Loridan, 1988) or staphylococcal alpha toxin (Densen & Mandell, 1980), which are thought to stimulate massive degranulation or directly lyse granules as part of their activity. Staphylococcal leucocidin is also thought to induce degranulation (Densen & Mandell, 1980) but requires two protein components for activity which is lost when these are separated during purification (Noda et al, 1980;Noda & Kato, 1988); a similar requirement might explain the difficulty of purifying P. haemolytica leucotoxin in an active form.…”

Section: Discussionmentioning

confidence: 80%

Time and temperature dependence of granulocyte damage by leucotoxic supernatants from Pasteurella haemolytica A1

Styrt¹,

Walker²,

Dahl³

et al. 1990

Journal of General Microbiology

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Bacterial exotoxins may contribute to the pathogenic potential of micro-organisms through interactions wjth cells of the host defence system as well as by directly damaging host tissue. The present studies were designed to explore mechanisms of interaction between bovine granulocytes and the leucotoxin produced by PasteurefZa haemolytica, a major cause of bovine respiratory disease. Leucotoxin-containing supernatant from P. haemofytica A1 caused rapid cell death in isolated bovine granulocytes that was close to half-maximal by 5 min and nearly 90 % complete after 30 min at 37 "C. Maintaining granulocytes at ice-water temperature markedly attenuated or prevented the toxic effect; furthermore, if exposed to supernatants at ice-water temperature and then washed, most cells remained viable even after rewarming to room temperature. However, even a very brief exposure (about 5 s) at 37 "C led to extensive cell death even after immediate cold dilution and washing. Granule enzymes such as arylsulphatase were released far more slowly than cytosol contents. Leucotoxin purified by column chromatography showed temperature dependence and divergence between cytosol and granule marker release similar to those observed with the crude supernatant preparation. These findings indicate that irreversible interaction between P. haemolytica leucotoxin and bovine granulocytes is initiated very rapidly at 37 "C but markedly impeded at low temperature, while granule enzyme release follows cytosol marker release over a much longer period. The results suggest either a requirement for target cell metabolic activity to initiate toxin effects or a temperature-dependent receptor conformation, with granule enzyme release following as a secondary consequence of granulocyte death.

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Section: Discussionmentioning

confidence: 80%

Time and temperature dependence of granulocyte damage by leucotoxic supernatants from Pasteurella haemolytica A1

Styrt¹,

Walker²,

Dahl³

et al. 1990

Journal of General Microbiology

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“…The presence of such unusual residues may help explain why amino acid analyses of partially purified SLS preparations (2,28) have differed from the sequence predicted for the SagA propeptide (e.g., absence of cysteine). Previous attempts at sequencing SLS peptide by Edman degradations were unsuccessful (2), which may reflect cyclical thioether bridge structures or N-terminal blockage by a 2-oxobutyryl group as seen in other bacteriocins (35).…”

Section: Discussionmentioning

confidence: 99%

Genetic Locus for Streptolysin S Production by Group A Streptococcus

et al. 2000

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Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis. Streptolysin S (SLS) is the cytolytic factor that creates the zone of betahemolysis surrounding GAS colonies grown on blood agar. We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S. D. Betschel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671-1679, 1998). Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project (www .genome.ou.edu/strep.html), we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production. The sag locus is conserved among GAS strains regardless of M protein type. Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype. Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon. A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant. Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis. We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins.Group A streptococcus (GAS), specifically Streptococcus pyogenes, is a common cause of pharyngitis, impetigo, and many other human respiratory tract and soft tissue infections. Recently, there has been a dramatic increase in reports of severe invasive GAS infections, including necrotizing fasciitis and toxic shock syndrome (44). Although GAS produces a wide array of virulence factors, those responsible for the rapid bacterial spread and tissue injury seen with invasive GAS infections are unknown.A hallmark feature of GAS in the clinical laboratory is the zone of beta-hemolysis observed surrounding colonies grown on the surface of blood agar media. The factor responsible for the beta-hemolytic phenotype is streptolysin S (SLS), the oxygen-stable cytolytic toxin of GAS. Despite detailed investigations over several decades, the exact chemical nature of SLS has remained a great mystery of GAS biology. SLS can exist in intracellular, cell-surface-bound, and extracellular forms (16), but attempts at purification are complicated by instability of the cytolytic activity in the absence of high-molecular-weight carriers, such as yeast RNA core or albumin (47).We have adopted a molecular genetic approach towards the identification of SLS and the study of...

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“…Similarly, ⌬sagA mutants showed no detectable cholesterol-resistant hemolysin activity when aliquots of broth culture were tested in exponential and stationary phases (1). Cysteine proteinase-deficient mutants were generated by exchange of the wild-type speB gene with a speB fragment inactivated by insertion of an ⍀Km-2 interposon (kindly provided by Ulf Sjobring, Lund, Sweden) (34).…”

Section: Methodsmentioning

confidence: 99%

Contribution of CsrR-Regulated Virulence Factors to the Progress and Outcome of Murine Skin Infections byStreptococcus pyogenes

Engleberg

Heath²,

Vardaman³

et al. 2004

Infect Immun

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Streptococcus pyogenes with null mutations in the csrRS regulatory locus are highly virulent in mice due to derepression of hyaluronic acid capsule synthesis and exotoxins, e.g., streptolysin S (SLS) and pyrogenic exotoxin B (SpeB). We generated derivatives of a ⌬csrRS strain that also carry deletions in hasAB (leading to an acapsular phenotype) or in sagA (phenotypically SLS ؊ ) or an interruption of speB (SpeB ؊ ) to test the relative contributions of these factors to the development of necrotic skin lesions. Inoculation of 2 ؋ 10 6 to 4 ؋ 10 6 CFU of either acapsular or SLS ؊ strains into hairless mice resulted in lesions ϳ70% smaller than those of the ⌬csrRS parent strain. Elimination of SLS also reduced lethality from 100% to 0% at this inoculum (P < 10 ؊7 ; Fisher exact test). In contrast, SLS ؉ SpeB ؊ mutants yielded lesions that were only 41% smaller than the parent strain (t ‫؍‬ 2.2; P ‫؍‬ 0.04), but only 3 the 17 lesions had dermal sloughing (P ‫؍‬ 10 ؊5 ). The nonulcerative lesions associated with SpeB ؊ strains appeared pale with surrounding erythema. We conclude that capsule and SLS contribute to the subcutaneous spread of S. pyogenes and to a fatal outcome of infection. SpeB facilitates early dermal ulceration but has minor influence on lesion size and mortality. Large ulcerative lesions are observed only when both toxins are present.Enhanced virulence of Streptococcus pyogenes csrR mutants is associated with increased expression of the hyaluronic acid capsule and several exotoxins, most notably streptolysin S (SLS) and pyrogenic exotoxin B (SpeB, or cysteine proteinase) (12). In the hairless mouse model of streptococcal skin infection, csrR mutants produce rapidly expanding, necrotic lesions, whereas the CsrR ϩ wild-type M1 strain from which they are derived induces only small abscesses or self-limited erythema.There is little disagreement about the importance of the hyaluronic acid capsule in streptococcal pathogenesis; however, significant controversy exists concerning the importance of many of the other CsrRS-regulated gene products in virulence. Several research groups have characterized SLS as a key factor contributing to virulence (4,17,28). In contrast, discrepancies have been reported concerning the importance of cysteine proteinase in studies with speB mutants (2,3,21,24,32,34). We hypothesized that the incremental pathogenicity associated with the mutations in csrR depends on the expression of these regulated virulence factors. Accordingly, we characterized here the impact of these toxins on murine infection by deleting them individually or in combination in S. pyogenes strains lacking CsrR. We show an additive effect of hyaluronic acid capsule, SLS, and SpeB on mouse virulence, and we observe that these factors contribute in different ways to the character, extent, and lethality of the skin lesions. MATERIALS AND METHODSAll experiments used derivatives of a type M1 isolate, MGAS166 (27). Streptococci were stored at Ϫ70°C and passaged minimally before and after genetic manipulations. Tod...

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[9] Production, purification, and assay of streptolysin S

Cited by 24 publications

References 6 publications

Time and temperature dependence of granulocyte damage by leucotoxic supernatants from Pasteurella haemolytica A1

Time and temperature dependence of granulocyte damage by leucotoxic supernatants from Pasteurella haemolytica A1

Genetic Locus for Streptolysin S Production by Group A Streptococcus

Contribution of CsrR-Regulated Virulence Factors to the Progress and Outcome of Murine Skin Infections byStreptococcus pyogenes

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