Staphylococcal Hemolysins Research Articles

Forssman and the staphylococcal hemolysins.

Forssman was a Swedish pathologist and microbiologist who, in the 1920s and 1930s conducted a long series of experiments that led to unique insights into surface antigens of blood cells, as well as added to the discrimination of toxins produced by staphylococci that lyse red blood cells. This review takes offset in the studies published by Forssman in APMIS addressing the hemolytic properties of staphylococcal toxins displayed against erythrocytes of animal and human origin. In light of current knowledge, we will discuss the insights we now have and how they may pave the way for curing infections with pathogenic staphylococci, including Staphylococcus aureus.

Chimeric mutants of staphylococcal hemolysin, which act as both one-component and two-component hemolysin, created by grafting the stem domain.

Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane β-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component α-hemolysin (α-HL) and two-component γ-hemolysin (γ-HL). The α-HL forms a homo-heptamer, whereas γ-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a β-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the β-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT β-barrels for use in further promising applications.

Direct and synergistic hemolysis caused by Staphylococcus phenol-soluble modulins: implications for diagnosis and pathogenesis

Phenol-soluble modulins are secreted staphylococcal peptides with an amphipathic α-helical structure. Some PSMs are strongly cytolytic toward human neutrophils and represent major virulence determinants during Staphylococcus aureus skin and blood infection. However, capacities of PSMs to lyse human erythrocytes have not been investigated. Here, we demonstrate that many S. aureus and Staphylococcus epidermidis PSMs lyse human erythrocytes. Furthermore, synergism with S. aureus β-toxin considerably increased the hemolytic capacities of several PSMs. This synergism may be of key importance in PSM and β-toxin-producing S. aureus or in mixed-strain or -species infections with PSM and β-toxin producers. Of specific interest, several PSMs, in particular PSMα peptides, contributed to a considerable extent to synergistic hemolysis with β-toxin or when using the β-toxin-producing strain RN4220 in CAMP assays. Thus, CAMP-type assays should not be used to detect or quantify S. aureus δ-toxin production, but may be used for an overall assessment of Agr functionality. Our study suggests an additional role of PSMs in staphylococcal pathogenesis and demonstrates that the repertoire of staphylococcal hemolysins is not limited to S. aureus and is much larger and diverse than previously thought.

Invasion of human keratinocytes by Staphylococcus aureus and intracellular bacterial persistence represent haemolysin-independent virulence mechanisms that are followed by features of necrotic and apoptotic keratinocyte cell death.

Colonization of human skin by Staphylococcus aureus is a characteristic feature of several inflammatory skin diseases, which is often followed by tissue invasion and severe cell damage. A crucial role has been attributed to staphylococcal haemolysins in the cytotoxicity to epidermal structures. To investigate haemolysin-independent virulence to human keratinocytes. The stable alpha-haemolysin, beta-haemolysin double-negative S. aureus mutant DU 5720 was compared with the fully virulent parent strain 8325-4 and with its isogenic fibronectin-binding protein A/B-negative variant DU 5883 in an invasion model. This assay showed dose-dependent internalization of all the strains investigated by human HaCaT keratinocytes, with reduced internalization of DU 5883. Transmission electron microscopy revealed adhesion of staphylococci to cellular pilus-like extrusions, followed by the embedding of the bacteria in cellular grooves. Following attachment to the keratinocytes the staphylococci were engulfed into vesicles within the cytoplasm where some bacteria persisted for 24-48 h. Addition of cytochalasin D strongly reduced the bacterial uptake, suggesting an active keratinocyte process. Bacterial invasion was followed by severe keratinocyte cell damage showing the morphological changes of cytotoxic and, to a lesser extent, apoptotic cell death as determined by the trypan blue exclusion test and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay. The highest levels of lethal cytotoxicity were observed in haemolysin-producing strains, whereas the induction of apoptosis seemed to depend on internalization. Staphylococcal invasion of human keratinocytes represents a potent staphylococcal virulence factor, which, independently of alpha- and beta-haemolysins, leads to necrotic and apoptotic cell damage.

Exotoxins of Staphylococcus aureus.

This article reviews the literature regarding the structure and function of two types of exotoxins expressed by Staphylococcus aureus, pyrogenic toxin superantigens (PTSAgs) and hemolysins. The molecular basis of PTSAg toxicity is presented in the context of two diseases known to be caused by these exotoxins: toxic shock syndrome and staphylococcal food poisoning. The family of staphylococcal PTSAgs presently includes toxic shock syndrome toxin-1 (TSST-1) and most of the staphylococcal enterotoxins (SEs) (SEA, SEB, SEC, SED, SEE, SEG, and SEH). As the name implies, the PTSAgs are multifunctional proteins that invariably exhibit lethal activity, pyrogenicity, superantigenicity, and the capacity to induce lethal hypersensitivity to endotoxin. Other properties exhibited by one or more staphylococcal PTSAgs include emetic activity (SEs) and penetration across mucosal barriers (TSST-1). A detailed review of the molecular mechanisms underlying the toxicity of the staphylococcal hemolysins is also presented.

Exotoxins of Staphylococcus aureus

This article reviews the literature regarding the structure and function of two types of exotoxins expressed by Staphylococcus aureus, pyrogenic toxin superantigens (PTSAgs) and hemolysins. The molecular basis of PTSAg toxicity is presented in the context of two diseases known to be caused by these exotoxins: toxic shock syndrome and staphylococcal food poisoning. The family of staphylococcal PTSAgs presently includes toxic shock syndrome toxin-1 (TSST-1) and most of the staphylococcal enterotoxins (SEs) (SEA, SEB, SEC, SED, SEE, SEG, and SEH). As the name implies, the PTSAgs are multifunctional proteins that invariably exhibit lethal activity, pyrogenicity, superantigenicity, and the capacity to induce lethal hypersensitivity to endotoxin. Other properties exhibited by one or more staphylococcal PTSAgs include emetic activity (SEs) and penetration across mucosal barriers (TSST-1). A detailed review of the molecular mechanisms underlying the toxicity of the staphylococcal hemolysins is also presented.

Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to staphylococcal hemolysins.

Enterocin L50 (EntL50), initially referred to as pediocin L50 (L. M. Cintas, J. M. Rodríguez, M. F. Fernández, K. Sletten, I. F. Nes, P. E. Hernández, and H. Holo, Appl. Environ. Microbiol. 61:2643-2648, 1995), is a plasmid-encoded broad-spectrum bacteriocin produced by Enterococcus faecium L50. It has previously been purified from the culture supernatant and partly sequenced by Edman degradation. In the present work, the nucleotide sequence of the EntL50 locus was determined, and several putative open reading frames (ORFs) were identified. Unexpectedly, two ORFs were found to encode EntL50-like peptides. These peptides, termed enterocin L50A (EntL50A) and enterocin L50B (EntL50B), have 72% sequence identity and consist of 44 and 43 amino acids, respectively. Interestingly, a comparison of the deduced sequences of EntL50A and EntL50B with the corresponding sequences obtained by Edman degradation shows that these bacteriocins, in contrast to other peptide bacteriocins, are secreted without an N-terminal leader sequence or signal peptide. Expression in vivo and in vitro transcription/translation experiments demonstrated that entL50A and entL50B are the only genes required to obtain antimicrobial activity, strongly indicating that their bacteriocin products are not posttranslationally modified. Both bacteriocins possess antimicrobial activity on their own, with EntL50A being the most active. In addition, when the two bacteriocins were combined, a considerable synergism was observed, especially with some indicator strains. Even though the enterocins in some respects are similar to class II bacteriocins, several conserved features common to class II bacteriocins are absent from the EntL50 system. The enterocins have more in common with members of a small group of cytolytic peptides secreted by certain staphylococci. We therefore propose that the enterocins L50A and L50B and the staphylococcal cytolysins together constitute a new family of peptide toxins, unrelated to class II bacteriocins, which possess bactericidal and/or hemolytic activity.

Antagonistic Effect of Staphylococcus hyicus and Staphylococcus chromogenes Exhibited on Staphylococcal Delta Hemolysin

Skalka B.: Antagonistic Effect of Staphylococcus hyicus and Staphylococcus chromo­ genes Exhibited on Staphylococcal Delta Hemolysin. Acta vet. Bmo, 60,1991: 61-69. Staphylococcus hyicus and Staphylococcus chromo genes are able to inhibit hemolytic effect of the staphylococcal delta hemolysin on agar media supplemented with human or sheep erythrocytes. This inhibition takes place even when the effect of delta hemo­ lysin on agars with sheep erythrocytes is potentiated with staphylococcal beta hemo­ lysin. Although less markedly expressed, similar antagonistic effect on staphylococcal delta hemolysin is also found in' a majority of strains Staphylococcus sciuri, Staphylo­ coccus lentus and some Micrococcus spp. Other species of coagulase-negative staphy­ lococci have no antagonistic effect on delta hemolysin. A technique of a combined test of hemolytic interactions (CTHI) for a simultaneous assay of hemolytic antagonism and synergism was proposed, which can be used to differentiate S. hyicus and S. chro­ mogenes from other novobiocin-sensitive staphylococcal species. Staphylococcus hyicus, Staphylococcus chromo genes, staphylococcal delta hemolysin, anta­ gonistic effect, combined test of hemolytic interactions (CTHI) In our previous paper (Skalka 1988) we described hemolytic activity of S. hyicus and S. chro­ :mogenes on blood agars with rabbit or human erythrocytes. Hemolytic effects of these two staphy­ lococcal species on agar media supplemented with erythrocytes of various animal origin differed from the data on sensitivity of various erythrocytes to staphylococcal hemolysins alpha, beta, ga­ mmaand delta (Dolman 1932; Glenny and Stevens 1935; Smith and Price 1938; Williams and Harper 1947; Marks and Vaughan 1950; Jackson 1962; Jeljaszewicz 1972; Wiseman 1975). In the paper mentioned we were not even able to confirm the descriptions of delta hemolysin production by certain strains of the two staphylococcal species (Hebert and Hancock 1985; Lammler and Blobel 1987; Watts and Owens 1987) or the information on their ability to pro­ ·duce gamma hemolysin (Goodfellow et al. 1987). The discrepancy between our findings and the descriptions of other authors cited stimulated our continuous study of hemolytic manifestations of the two above-mentioned staphylococcal :1lpecies, which is the subject-matter of the present paper.

Comparison of the exotoxins of four strains of Corynebacterium pseudotuberculosis

The activity of exotoxins produced by four strains of Corynebacterium pseudotuberculosis were compared by their ability to kill white mice, their haemolytic activity, staphylococcal haemolysin-inhibiting effect and activity in an enzyme-linked immunosorbent assay (ELISA). Exotoxins with a haemolytic titre of 1 in 256 or more killed all mice and had the most inhibitory effect (1 in 64 or more) on staphylococcal haemolysin. The haemolytic test and staphylococcal haemolysin inhibitory test of exotoxin activities were highly correlated (P less than 0.001) with the mouse toxicity of the four exotoxins examined. No significant correlation was evident between the ELISA and relative toxicity of the exotoxins.

Staphylococcal haemolysin production

Staphylococcal haemolysin production

Staphylococcal enterotoxins fail to disrupt membrane integrity or synthetic functions of Henle 407 intestinal cells.

The potential cytotoxic activity of purified staphylococcal enterotoxins for mammalian cells was evaluated. The effects of staphylococcal enterotoxins A (SEA) and B (SEB) on cell membrane integrity as measured by leakage of labeled cytoplasmic constituents ([3H]uridine), amino acid transport (lysine and aminoisobutyric acid), and macromolecular synthesis (protein, ribonucleic acid, and deoxyribonucleic acid) was evaluated for a human intestinal epithelial cell (Henle 407). No evidence of cytotoxicity by any of these criteria could be detected for cell monolayers incubated with SEA for periods of between 30 min and 24 h. Purified staphylococcal hemolysins (alpha- and delta-toxins) were shown to exert cytotoxicity by the leakage and amino acid uptake assays. In efforts to detect synergistic effects between enterotoxin and the staphylococcal cytotoxins, membrane functions were evaluated after sequential or combined treatment with enterotoxin and alpha-toxin or with enterotoxin and delta-toxin. In no instance could a contribution to cytotoxicity by the staphylococcal enterotoxin be detected. That the assays were sufficiently sensitive to detect synergistic effects was shown by the greater than additive effects achieved with a combination of alpha- and delta-toxins. The data, contrary to previous reports, showed that staphylococcal enterotoxins did not behave as bacterial cytotoxins.

The action of formaldehyde on staphylococcal delta haemolysin.

Native staphylococcal delta haemolysin was poorly immunogenic in the mouse. After treatment with formaldehyde at pH 5 or pH 7.5, haemolytic activity was rapidly lost but antigenic reactivity retained and, at pH 5, immunogenicity was enhanced. Treatment of the haemolysin with formaldehyde at pH 9.5 reduced haemolytic activity by 97% but did not enhance immunogenicity. There was no evidence of polymerisation of formaldehyde-treated delta haemolysin, and the enhanced immunogenicity was considered to be partly due to a reduced affinity for phospholipids.

Immunogenicity and molecular characterisation of staphylococcal delta haemolysin.

In rabbit antisera to staphylococcal delta haemolysin the haemolysin-neutralising activity was associated with the IgG fraction as well as with the alpha and beta lipoproteins. On an equal-weight basis, the neutralising capacity of specific antibody was not significantly greater than that of serum alpha or beta lipoproteins. Anti-alpha haemolysin was not detected in any of the anti-delta-haemolysin antisera. Amino-acid analysis of delta haemolysin showed that lysine, aspartic acid, threonine and isoleucine were the predominant amino acids present; histidine, arginine, proline, cysteine and tyrosine were absent. The molecular weight of delta-haemolysin in phosphate-buffered saline was approximately 210 000.

Effects of staphylococcal alpha-, beta-, delta-, and gamma-hemolysins on human diploid fibroblasts and HeLa cells: evaluation of a new quantitative as say for measuring cell damage.

Human diploid embryonic lung fibroblasts and HeLa cells were cultivated in Eagle minimaĺ essential medium supplemented with 10% calf serum. Monolayer cultures were labeled with (3)H-uridine and treated with highly purified staphylococcal alpha-, beta-, delta-, or gamma-hemolysin. The release of soluble radioactive substances into the medium was used as an indicator of damage to the cell membrane after treatment with each hemolysin. The assay method described is simple, sensitive, and rapid. It allows quantitative estimation of changes in membrane permeability to be detected before a morphological damage is observed microscopically. Upon incubation for up to 30 min with highly purified staphylococcal hemolysins, only delta-hemolysin caused release of a significant amount of tritiated substances from fibroblasts. Such leakage occurred immediately after addition of delta-lysin and was independent of temperature. With minor exceptions, this was similar to the release of isotopes after treatment of the cells with the nonionic detergent Triton X-100. Treatment of fibroblasts with combinations of two or three of these toxins gave neither a synergistic nor an antagonistic effect. Evidence is presented which indicates that delta-hemolysin is the only important fibroblast damaging activity in crude preparations of extracellular proteins of four strains of S. aureus, whereas HeLa cells are susceptible also to purified alpha-toxin.

Identification of penicillin-stimulated staphylococcal haemolysin as alpha-lysin.

Cultivation of S. aureus in the presence of 0.03 IU benzyl‐penicillin stimulated the formation of a haemolysin which was suggested to be alpha‐lysin. Its molecular weight was estimated to be 30000 by molecular sieve chromatography. It was mainly active on rabbit erythrocytes and was neutralized by alphalysin antibody. Furthermore it gave a reaction of identity in comparative immunoelectrophoresis with an are which was shown to represent alpha‐lysin.

Some biological properties of purified staphylococcal haemolysins: T. Wadström and R. Möllby, Toxicon, 10, 511. (Department of Bacteriology, Karolinska Institutet, S-104 01, Stockholm 60, Sweden)

Some biological properties of purified staphylococcal haemolysins: T. Wadström and R. Möllby, Toxicon, 10, 511. (Department of Bacteriology, Karolinska Institutet, S-104 01, Stockholm 60, Sweden)

Some biological properties of purified staphylococcal haemolysins

Staphylococcal alpha-, beta-, gamma and delta-haemolysin were extensively purified and several criteria of homogeniety for each were investigated before biological studies were started. The haemolytic spectrum, the lethal effect and the cytotoxicity of each lysin were studied, and several other properties were also compared. The LD 50-dose of beta-lysin was 10–100 μg for mice and rabbits, which means that this lysin, on the basis of weight, is about as potent a lethal toxin as alpha-lysin, while the LD 50-dose for gamma-lysin was about 1 mg and for delta-lysin about 125 mg. For the first time beta- and gamma-lysin were shown to be cytotoxic for mammalian cells (HeLa-cells and human fibroblasts) and gamma-lysin was shown to be lethal for mice and rabbits. Human thrombocytes and their α-granules were rapidly lysed by beta-lysin, while human leucocytes and bacterial protoplasts were resistant. Beta-lysin possesses sphingomyelinase activity. The susceptibility to lysis of different cells by this enzyme also seems to be compatible with the different phospholipid composition of these cell membranes. The importance to use purified toxins fulfilling several criteria of purity for biological studies will be discussed.

Effect of antibiotics on staphylococcal hemolysin production.

Thirty-seven per cent of 126 strains of Staphylococcus aureus, when tested on sheep blood-agar with sensitivity discs containing cephalothin, carbenicillin, oxacillin, penicillin, and cycloserine, produced rings of beta-hemolysis surrounding zones of inhibition of bacterial growth. Each strain capable of producing a ring of beta-hemolysis did so with at least two of the mentioned antibiotics. None of the other 15 antibiotics tested was associated with a ring of beta-hemolysis surrounding any of the zones of inhibition. It appears, therefore, that the beta-hemolysis observed was produced by certain strains of S. aureus only in association with certain antibiotics.

Effect of Antibiotics on Staphylococcal Hemolysin Production

Thirty-seven per cent of 126 strains of Staphylococcus aureus, when tested on sheep blood-agar with sensitivity discs containing cephalothin, carbenicillin, oxacillin, penicillin, and cycloserine, produced rings of beta-hemolysis surrounding zones of inhibition of bacterial growth. Each strain capable of producing a ring of beta-hemolysis did so with at least two of the mentioned antibiotics. None of the other 15 antibiotics tested was associated with a ring of beta-hemolysis surrounding any of the zones of inhibition. It appears, therefore, that the beta-hemolysis observed was produced by certain strains of S. aureus only in association with certain antibiotics.

Identification of staphylococcal hemolysins by an electrophoretic localization technique.

A technique for identifying and characterizing staphylococcal hemolysins by first separating them electrophoretically in barbital-buffered agar gel (pH 8.4) at 5 ma/cm for 2 hr and then determining their hemolytic activities by exposing them to human, horse, rabbit, and sheep erythrocytes is described. The alpha-hemolysin produced by a White variant of the Wood 46 strain of Staphylococcus aureus migrated 18 mm towards the cathode, and it lysed horse, rabbit, and sheep erythrocytes, whereas a Clear variant of the Wood 46 strain of S. aureus produced a lysin which migrated similarly to the alpha-hemolysin but lysed only rabbit cells. This latter lysin was tentatively named alpha(1)-lysin. This strain of S. aureus also produced beta-hemolysin which migrated 36 mm towards the cathode and lysed sheep cells. beta-Hemolysin produced by some strains of S. aureus showed considerable tailing during electrophoresis, whereas beta-hemolysin produced by other strains of S. aureus migrated as a well-defined peak. A lysin migrating 11 mm towards the anode was probably delta-lysin. It was, however, not produced in sufficient concentration when the cultures were grown in semisolid medium.