Live Cell Microscopy and Flow Cytometry to Study Streptolysin S-Mediated Erythrocyte Hemolysis.

The ability to induce hemolysis, the rupturing of erythrocytes with the consequent release of their intracellular contents, is a phenotypic hallmark of a number of microbial toxins. Streptococcus pyogenes or Group A Streptococcus (GAS) is a human pathogen responsible for a wide range of diseases from mild pharyngitis to severe conditions such as toxic shock syndrome. GAS produces a powerful hemolytic toxin called streptolysin S (SLS). Herein, we describe a procedure for the preparation of SLS toxin and the use of two complementary approaches, live microscopy and flow cytometry, to study the effects of the SLS toxin on erythrocytes. In addition to providing insights into SLS-mediated hemolysis, these assays have the potential to be modified for the study of other hemolytic toxins and compounds.

Human L-ficolin, a recognition molecule of the lectin activation pathway of complement, activates complement by binding to pneumolysin, the major toxin of Streptococcus pneumoniae.

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q(-/-) mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.

Time course of virulence factors produced by group A streptococcus during a food-borne epidemic.

We studied the protein amount and activity of the major virulence factors hemolysin, cysteine protease streptococcal pyrogenic exotoxin B (SpeB), and NAD glycohydrolase (NADase), which are produced by Streptococcus pyogenes type T-25, with a food poisoning outbreak. The three virulence factors were analyzed by activity and amount of protein using supernatants at 2-30 h of culture. All these virulence factors were confirmed by their activity. Streptolysin O (SLO), SpeB, and NADase were immunochemically confirmed at protein level by Western blot analysis. Two hemolytic forms (70 and 60 kDa) of SLO were identified. SpeB was detected as a 44-kDa precursor form and a 30-kDa mature form. NADase was 50 kDa. SLO protein peaked at 8 h of culture, which corresponded with the hemolytic activity peak. Conversion from precursor to SpeB protein peaked at 14 h of culture. The conversion peak corresponded to the activity expression time. Also, mature SpeB protein peaked at 24 h of culture and corresponded to SpeB activity peak. Electrophoretic analysis clarified the relationship between SLO protein and SpeB protein, although amounts of SLO and SpeB have been reported to be inversely proportional to activity. NADase protein peaked at 12 h of culture, but protein level did not correspond to the peak. Because the NADase protein peak was closer to SpeB activity than SLO protein, our results suggested NADase protein was degraded at 12 h of culture. The time course production of these virulence factors is discussed.

Changes in astrocyte shape induced by sublytic concentrations of the cholesterol-dependent cytolysin pneumolysin still require pore-forming capacity.

Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin's pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20-40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin's lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.

Detection of pbp2b and ermB genes in clinical isolates of Streptococcus pneumoniae.

BACKGROUND: Streptococcus pneumoniae is a major human pathogen. The emergence of penicillin resistant strains since the 1970s has been life threatening and the evolution of the bacteria have enabled itself to develop resistance to many other antibiotics such as the macrolides and the fluoroquinolones. This study aims to characterize S. pneumoniae isolates for the presence of penicillin and macrolide resistance genes. METHODOLOGY: One hundred and twenty clinical isolates of S. pneumoniae were obtained from patients of University Malaya Medical Centre (UMMC). The strains were screened using a multiplex real-time PCR method for the presence of alterations in the genes encoding the penicillin binding proteins: pbp2b, macrolide resistance determinant ermB and the pneumolysin gene, ply. Dual-labelled Taqman probes were used in the real-time detection method comprising three different genes labeled with individual fluorophores at different wavelengths. One hundred and twenty isolates from bacterial cultures and isolates directly from blood cultures samples were analyzed using this assay. RESULTS: A multiplex PCR comprising the antibiotic resistance genes, ermB and and pneumolysin gene (ply), a S. pneumoniae species specific gene, was developed to characterize strains of S. pneumoniae. Out of the 120 pneumococcal isolates, 58 strains were categorized as Penicillin Sensitive Streptococcus pneumoniae (PSSP), 36 as Penicillin Intermediate Streptococcus pneumoniae (PISP) and 26 as Penicillin Resistant Streptococcus pneumoniae (PRSP). All the 58 PSSP strains harboured the pbp2b gene while the 36 PISP and 26 PRSP strains did not harbour this gene, thus suggesting reduced susceptibility to penicillin. Resistance to erythromycin was observed in 47 of the pneumococcal strains while 15 and 58 were intermediate and sensitive to this drug respectively. Susceptibility testing to other beta-lactams (CTX and CRO) also showed reduced susceptibility among the strains within the PISP and PRSP groups but most PSSP strains were sensitive to other antibiotics. CONCLUSION: The characterization of pneumococcal isolates for penicillin and erythromycin resistance genes could be useful to predict the susceptibility of these isolates to other antibiotics, especially beta-lactams drugs. We have developed an assay with a shorter turnaround time to determine the species and resistance profile of Streptococcus pneumoniae with respect to penicillin and macrolides using the Real Time PCR format with fluorescent labeled Taqman probes, hence facilitating earlier and more definitive antimicrobial therapy which may lead to better patient management.

Purification of recombinant histidine-tag streptolysin O using immobilized metal affinity expanded bed adsorption (IMA-EBA).

In this report, we describe the recombinant SLO expression as a fusion protein with a C-terminal hexahistidine tag and its purification using immobilized metal affinity expanded bed adsorption (STREAMLINE(trade mark) Chelating). In order to facilitate downstream processing of the purification, an efficient fermentation process was developed focusing on the achievement of high yields of soluble protein. The purification strategy resulted in a 40% recovery of active recombinant SLO and the protein was purified eight-fold. SDS-PAGE and Western-blot analysis of the purified protein revealed the presence of a 75 Mr form, which was the estimated relative Mass of the recombinant SLO.

Construction and expression of recombinant streptolysin-o and preevaluation of its use in immunoassays.

Commercially available immunoassays for assessment of anti-streptolysin-O antibodies use native streptolysin-O obtained by a complex process. We prepared a biologically active recombinant streptolysin-O with higher yield and a simpler purification process. An enzyme-linked immunosorbent assay developed with this recombinant showed good correlation with a commercial test, suggesting that it could be suitable for immunoassays.

Expression of recombinant Streptolysin O and specific antibody production.

Streptolysin O (SLO), an oxygen-labile cytolysin, is the cholesterol-binding exotoxin of hemolytic streptococci. Besides microbiological and pathological interests, this cytolysin has been used as a tool for permeabilization of biomembranes. SLO serves as a diagnostic reagent for determination of anti-SLO antibody titer in streptococcal infection. Availability of highly purified SLO, however, has been limited by low yield in streptococcal culture and purification process. Present subcloning of mature-type full-length SLO gene into an expression vector having strictly controllable araBAD promoter enabled efficient production of the cytolysin. Further, anti-SLO antibody with high specificity was obtained by immunizing with purified SLO protein.

Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae.

The vaccine potential of a combination of three pneumococcal virulence proteins was evaluated in an active-immunization-intraperitoneal-challenge model in BALB/c mice, using very high challenge doses of Streptococcus pneumoniae. The proteins evaluated were a genetic toxoid derivative of pneumolysin (PdB), pneumococcal surface protein A (PspA), and a 37-kDa metal-binding lipoprotein referred to as PsaA. Mice immunized with individual proteins or combinations thereof were challenged with high doses of virulent type 2 or type 4 pneumococci. The median survival times for mice immunized with combinations of proteins, particularly PdB and PspA, were significantly longer than those for mice immunized with any of the antigens alone. A similar effect was seen in a passive protection model. Thus, combinations of pneumococcal proteins may provide the best non-serotype-dependent protection against S. pneumoniae.

[Production and partial purification of the main hemolysin (pneumolysin) of Streptococcus pneumoniae]. / Producción y purificación parcial de la hemolisina principal (Neumolisina) de Streptococcus pneumoniae.

The capacity of 36 Cuban strains of Streptococcus pneumoniae to produce pneumolysin was studied, and 94.4% of them were determined to be producers of that enzyme. One of the best producers was cultured at a great scale and the pneumolysin found in the supernatan was partially purified through an ion-exchange chromatography in mono-Q column. This method made it possible to recover the enzyme whose purity level increased by 4.39 with 100% output.

Characterization of the haemolytic activity of Streptococcus equi.

The haemolytic activity of Streptococcus equi, the cause of equine strangles, was characterized. Production of haemolysin in Todd Hewitt broth was dependent on an equine serum supplement and the logarithmic phase of growth after which activity declined sharply. RNA core also induced haemolysin production from cells harvested at the end of the logarithmic phase of growth. Haemolysis was not affected by cholesterol, was only slightly increased in reducing conditions and was completely inactivated by trypan blue, identifying the haemolytic activity as streptolysin S-like (SLS-like). Purification by hydroxyapatite and Sephacryl column chromatography yielded proteins of molecular weights of approximately 6000 and 17 000-22 000 Da with a 64-fold increase in specific activity. Low molecular weight proteins from the RNA core were still present in the purified toxin. Two non-haemolytic mutants were derived by conjugation with an Enterococcus faecalis-carrying transposon Tn916. Southern blots of HindIII digests of DNA revealed that one of the mutants contained three transposon insertions and the other just one. A lambda phage library of S. equi contained plaques whose haemolytic activity was enhanced by reducing conditions and inhibited by cholesterol, suggesting a streptolysin O-like (SLO-like) activity. However, haemolysin in culture sonicates of host E. coli in which the lambda phage insert was subcloned into plasmid (pUC18), was not affected by these conditions. Seven isolates of S. equi in medium without SLS-like inducers showed no SLO-like activity and no evidence for an SLO-like toxin could be found by immunoblotting with pneumolysin antiserum and monoclonal antibodies or by polymerase chain reaction with primers derived from sequences conserved between the SLO genes of Lancefield group A, C and G streptococci. S. equi does not appear to possess a streptolysin O but does make a streptolysin S-like toxin whose production can be interrupted at just one genetic locus.

Cysteine-specific radioiodination of proteins with fluorescein maleimide.

A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label is then removed by gel-permeation chromatography. The procedure avoids exposition of the protein to oxidative conditions and does not require purification of the labeled carrier reagent. Suitability of the method for a given protein can be evaluated spectrophotometrically without employing radioactivity. It can be applied under denaturing conditions and may be particularly useful with mutant proteins carrying engineered single cysteine residues at sites that are not functionally critical.

Structural and functional characterisation of two proteolytic fragments of the bacterial protein toxin, pneumolysin.

Proteolytic cleavage of the bacterial protein toxin pneumolysin with protease K creates two fragments of 37 and 15 kDa. This paper describes the purification of these two fragments and their subsequent physical and biological characterisation. The larger fragment is directly involved in the cytolytic mechanism of this pore-forming protein, via membrane binding and self-association. The smaller fragment lacks ordered structure or discernible activity.

[Properties of extracellular products produced by group A streptococci isolated from patient with streptococcal toxic shock syndrome].

Extracellular products of group A streptococci isolated from patients with streptococcal toxic shock syndrome (STSS) examined. The outline of discussion of the 4 products are as follows; Products were streptolysin O (SLO), proteinase, erythrogenic toxin and DNase. SLO showed a large amount of products more than proteinase, erythrogenic toxin and DNase. It should not be concluded that there is a large amount of SLO in the isolated strains from patients with STSS. SLO produced by group A streptococci isolated from the patient with STSS has an isoelectric point (pI) of 6.0 and a molecular weight of 64,000 and shows a hemolytic activity in the presence of 2 mercaptoethanol (2-ME). Furthermore, the hemolytic activities of all components were inhibited by gamma-globulin and cholesterol. These results indicated that all component with hemolytic activity are SLO. These data suggest that STSS does not make up a single syndrome but, rather, that the multiple STSS clinical criteria probably reflects a different characteristic of individual S. pyogenes isolates.

Membrane-penetrating domain of streptolysin O identified by cysteine scanning mutagenesis.

Streptolysin O (SLO), a polypeptide of 571 amino acids, belongs to a family of highly homologous toxins that bind to cell membranes containing cholesterol and then polymerize to form large transmembrane pores. A conserved region close to the C terminus contains the single cysteine residue of SLO and has been implicated in membrane binding, which has been the only clear assignment of function to a part of the sequence. We have used a cysteine-less active mutant of SLO to introduce single cysteine residues at 19 positions distributed throughout the sequence. The cysteines were derivatized with the polarity-sensitive fluorophore acrylodan, and the fluorescence emission of the label was examined at the different stages of SLO pore assembly. With several mutants, oligomerization on membranes was accompanied by emission blue-shifts, indicating movement of the label into a more hydrophobic environment. These effects were essentially confined to the range of amino acids 213-305. With oligomeric mutants L274C, S286C, and S305C, additional environmental alterations were induced when different nondenaturing detergents were used to dislodge the membrane lipids from the oligomers. The corresponding amino acid residues thus insert into the lipid bilayer during pore formation. Conversely, the spectra of oligomeric mutants A213C and T245C were not affected by detergents. Devoid of contact with the lipid bilayer, these amino acid residues probably participate in the interaction of SLO molecules within the oligomer.

Sequence variation in the Streptococcus pneumoniae pneumolysin gene affecting haemolytic activity and electrophoretic mobility of the toxin.

When 30 clinical isolates of Streptococcus pneumoniae, representing 16 capsular serotypes, were analysed by Western blot for production of the haemolytic toxin pneumolysin (Ply), all strains produced an immunoreactive band of similar intensity. However, six isolates of serotype 8 and two of type 7F expressed Ply whose mobility on SDS-PAGE was anomalously slow. Culture lysates from these strains also had low haemolytic activities compared with those for clinical isolates of other serotypes, suggesting the possibility of mutations affecting specific activity. Genes encoding Ply from one type 8 isolate and one type 7F isolate were cloned into Escherichia coli and sequenced. Compared with the published sequence for Ply, the deduced amino acid sequence for the type 8 Ply variant contained three amino acid substitutions, and the type 7F variant four amino acid substitutions. Both variants also had Val270 and Lys271 deleted. The variant Ply proteins were purified from recombinant E. coli expressing the cloned genes, and shown to have substantially reduced specific haemolytic activities [6.8 x 10(4) haemolytic units (HU)/mg and 2.3 x 10(4) HU/mg for type 8 Ply and type 7F Ply respectively] compared with Ply itself (1.2 x 10(6) HU/mg). Studies with chimeric toxin gene constructs indicated that both the reduced haemolytic specific activity and the anomalous electrophoretic mobility of the variant Plys were attributable to a single amino acid substitution (Thr172-->Ile).

Practical considerations regarding the use of streptolysin-O as a permeabilising agent for cells in the investigation of exocytosis.

Streptolysin-O is widely used in cell biological investigations in order to make large (>12 nm) pores in the plasma membrane and so to render the cytosol directly accessible to experimental manipulation. We have compared the effect of streptolysin-O commercially formulated (Murex Diagnostics) as a diagnostic reagent in pathology with two pure reagents (a conventional purified protein, and a recombinant protein generated in E.coli) on exocytotic secretion from mast cells. For mast cells permeabilised by streptolysin obtained from the commercial source, exocytosis (of beta-D-N-acetylglucosaminidase) is dependent on provision of both Ca(2)+ and a guanine nucleotide. In contrast, for cells permeabilised by either of the two pure proteins, a substantial extent of Ca(2)+-independent exocytosis can be elicited. When the Murex material is subject to dialysis or ultrafiltration, some secretion can be induced in the absence of Ca(2)+, indicating a modulatory function of the low mol wt additives of formulation, mainly phosphate and cysteine. However, Ca(2)+-independent exocytosis is still manifest when the pure proteins are reconstituted with ultrafiltrates from the Murex material. These observations indicate that reagents used to permeabilise cells should be characterised thoroughly and used with great care. Confirmation that the cytolytic activity of the Murex material derives from a cholesterol directed factor was demonstrated by inhibition of exocytosis when red blood cell derived (and hence cholesterol containing) sonicated liposomes were provided.

Hydrophobic interaction chromatography for the purification of cytolytic bacterial toxins.

The usefulness of hydrophobic interaction chromatography for the simple purification of cytolytic bacterial toxins was studied. Conditions are described for different hydrophobic interaction chromatographic media for purifying with high yields two different kinds of such haemolysins, the thiol-activated toxin listeriolysin O from Listeria monocytogenes and alpha-toxin from Staphylococcus aureus. For listeriolysin O, purification on butyl-Sepharose was followed by gel filtration chromatography. From butyl-Sepharose the recovery of 22%. Alpha-toxin was obtained by a single purification step from alkyl-Superose with 80% recovery and a specific activity of 29,000 U/mg. On sodium dodecyl sulphate polyacrylamide gel electrophoresis purified listeriolysin O and alpha-toxin showed a single band. Another thiol-activated toxin, streptolysin O from group A streptococci, showed a recovery of 38% from butyl-Sepharose. The results suggest the feasibility of using hydrophobic interaction chromatography, particularly with columns of weak hydrophobicity, for the purification of bacterial haemolysins in high yield.

Purification and characterization of streptolysin O secreted by Streptococcus equisimilis (group C).

Streptolysin O (SLO) was purified from culture supernatants of group C streptococci. The final product was either the complete, native molecule (SLOn [pI, 6.0]) with the N-terminal sequence (Asp)-Ser-Asn-Lys-Gln-Asn-Thr-Ala-Asn-Thr-Glu-Thr- or a large fragment (SLOf [pI, 7.3]) with the N-terminal sequence Ala-Pro-Lys-Glu-Met-Pro-Leu-Glu-Ser-Ala-Glu-Lys-Glu-Glu-Lys-.

Purification and characterization of streptolysin O from Streptococcus pyogenes.

1. Streptolysin O, an exotoxin produced by group A beta-hemolytic streptococci, has been purified from Streptococcus pyogenes culture supernatants. 2. The isolation and purification procedure involved ammonium sulphate and polyethylene glycol precipitations, and ion-exchange chromatographies on CM-Sepharose and Mono Q. 3. The proposed procedure introduces two ion-exchange chromatography steps making the purification process simpler and, especially, more reproducible than other described protocols. 4. The purified streptolysin O was hemolytically active, had a specific activity of 415,000 HU/mg, an optimum pH of 7.0, a relative molecular mass of 60,100 and an isoelectric pH of 7.5.