Thiol-activated Toxin Research Articles

Complement C3aR/C5aR-binding protein Suilysin of Streptococcus suis contributes to monocyte chemotaxis

Streptococcus suis is an emerging swine and human pathogen causing severe infections and sudden death. During infection, complement C3a and C5a were reported to induce immune cells towards infection and injury sites via their corresponding receptors C3aR and C5aR. However, how S. suis evade immune surveillance mediated by C3aR and C5aR remains unclear. In this study, we analyze and construct an S. suis bacterial two-hybrid prey library containing 39 LPXTG motif anchored proteins and 18 secreted proteins. Two highly possible C3aR-binding proteins: thiol-activated toxin Suilysin, putative RTX family exoprotein A gene and three highly possible C5aR-binding proteins: thiol-activated toxin Suilysin, putative 5′-nucleotidase and subtilisin-like serine protease are identified through bacterial two-hybrid assay. Far-western blot assay confirms that a cholesterol-binding cytolysin Suilysin can interact with both C3aR and C5aR. Chemotaxis assays demonstrate that recombinant and natural Suilysin can inhibit monocyte chemotaxis mediated by C3a and C5a. These findings enlarge our knowledge of suilysin biological significance and provide a new perspective on S. suis complement evasion.

Cholesterol and the activity of bacterial toxins

Cholesterol may affect the activity of microbial toxins in a direct, specific way, or it may exert indirect effects because of its role in membrane fluidity, membrane line tension, and in the stabilization of rafts in the cytoplasmic membrane. The thiol-activated toxins of gram-positive bacteria, and the cytolysin of Vibrio cholerae are presented as examples of specific toxin-cholesterol interaction. Several mechanisms of indirect effects of cholesterol are discussed using examples such as Staphylococcus aureusα-hemolysin, aerolysin, and diphtheria toxin.

Cholesterol and the activity of bacterial toxins

Cholesterol may affect the activity of microbial toxins in a direct, specific way, or it may exert indirect effects because of its role in membrane fluidity, membrane line tension, and in the stabilization of rafts in the cytoplasmic membrane. The thiol-activated toxins of gram-positive bacteria, and the cytolysin of Vibrio cholerae are presented as examples of specific toxin–cholesterol interaction. Several mechanisms of indirect effects of cholesterol are discussed using examples such as Staphylococcus aureus α-hemolysin, aerolysin, and diphtheria toxin.

Distribution and genetic diversity of suilysin in Streptococcus suis isolated from different diseases of pigs and characterization of the genetic basis of suilysin absence.

Streptococcus suis is an economically important pathogen of pigs responsible for a variety of diseases including meningitis, septicemia, arthritis, and pneumonia, although little is known about the mechanisms of pathogenesis or virulence factors associated with this organism. Here, we report on the distribution and genetic diversity of the putative virulence factor suilysin, a member of the thiol-activated toxin family of gram-positive bacteria. On the basis of PCR analysis of over 300 isolates of S. suis, the suilysin-encoding gene, sly, was detected in 69.4% of isolates. However, sly was present in a considerably higher proportion of isolates obtained from cases of meningitis, septicemia, and arthritis (>80%) and isolates obtained from asymptomatic tonsillar carriage (>90%) than lung isolates associated with pneumonia (44%). With the exception of serotypes 1, 14, and 1/14, there was no strong correlation between the presence of suilysin and serotype. Analysis of the genetic diversity of suilysin by restriction fragment length polymorphism and sequence analysis found that the suilysin gene, where present, is highly conserved with a maximum of 1.79% diversity at the nucleotide level seen between sly alleles. Assays of hemolytic activity and hybridization analysis provided no evidence for a second member of the thiol-activated toxin family in S. suis. Inverse PCR was used to characterize regions flanking sly, which in turn allowed the first characterization of the equivalent region in a strain lacking sly. Sequence comparison of these regions from sly-positive (P1/7) and sly-negative (DH5) strains indicated that two alternative arrangements are both flanked by genes with highest similarity to haloacid dehalogenase-like hydrolases (5' end) and putative N-acetylmannosamine-6-phosphate epimerases (3' end). However, sly appears to be completely absent from the alternative arrangement, and a gene of unknown function is located in the equivalent position. Finally, PCR analysis of multiple sly-positive and -negative strains indicated that these two alternative genetic arrangements are conserved among many S. suis isolates.

Streptolysin O: the C-terminal, tryptophan-rich domain carries functional sites for both membrane binding and self-interaction but not for stable oligomerization

Streptolysin O belongs to the class of thiol-activated toxins, which are single chain, four-domain proteins that bind to membranes containing cholesterol and then assemble to form large oligomeric pores. Membrane binding involves a conserved tryptophan-rich sequence motif located within the C-terminally located domain 4. In contrast, sites involved in oligomerization and pore formation have been assigned to domains 1 and 3, respectively. We here examined the functional properties of domain 4, which was recombinantly expressed with an N-terminal histidine tag for purification and an additional cysteine residue for covalent labeling. The fluorescently labeled fragment readily bound to membranes, but it did not form oligomers nor lyse cell membranes. Moreover, the labeled fragment did not detectably become incorporated into hybrid oligomers when combined with lytically active full-length toxin. However, when present in large excess over the active toxin, the domain 4 fragment effected reduction of hemolytic activity and of functional pore size, which indicates interference with oligomerization of the lytically active species. Our findings support the notion that domain 4 of the streptolysin O molecule may fold autonomously, is essential for membrane binding and is capable not of irreversible but of reversible association with the entire toxin molecule.

Production of monoclonal antibodies to Listeria monocytogenes and their application to determine the virulence of isolates from channel catfish.

We produced monoclonal antibodies (MAbs) to the extracellular proteins of Listeria monocytogenes EGD grown in Chelex-treated improved minimal medium. Ten of the positive hybridomas generated were chosen for further characterization. Seven of the MAbs reacted with a protein having a molecular mass of 60 kDa. These MAbs inhibited listeriolysin (LLO)-mediated hemolysis, and two of them were specific for LLO and none of the other thiol-activated toxins tested. In an enzyme-linked immunosorbent assay and Western blot analysis, five of the anti-LLO MAbs reacted with ivanolysin from Listeria ivanovii. Three of the 10 MAbs reacted with a 29-kDa protein on Western blots and neutralized the phosphatidylcholine-specific phospholipase C (PC-PLC) activity of L. monocytogenes. These three anti-PC-PLC MAbs did not react with phospholipases from five different gram-positive bacteria. However, the anti-PC-PLC MAbs recognized a 27-kDa extracellular protein from L. ivanovii and neutralized sphingomyelinase activity in a hemolysis test that demonstrates the antigenic relatedness of listerial phospholipases. These data indicate that listerial thiol-activated toxins possess species-specific epitopes and share group-specific epitopes. This is the first description of MAbs that neutralize listerial PC-PLC, and the data suggest that there is antigenic similarity between L. monocytogenes PC-PLC and L. ivanovii sphingomyelinase. The reactions of the MAbs with catfish isolates of L. monocytogenes suggested that some of the isolates examined lack the LLO and/or PC-PLC required for pathogenicity. The MAbs described here differentiated some catfish isolates from previously described type strain-pathogenic isolates and could be useful for detecting and determining the virulence of L. monocytogenes in food and clinical samples and for detecting L. ivanovii in veterinary clinical samples.

Self-interaction of pneumolysin, the pore-forming protein toxin of Streptococcus pneumoniae

The pathogenically important cholesterol-binding pore-forming bacterial “thiol-activated” toxins (TATs) are commonly believed to be monomeric in solution and to undergo a transition on membrane binding mediated by cholesterol to an oligomeric pore. We present evidence, gained through the application of a number of biochemical and biophysical techniques with associated modelling, that the TAT from Streptococcus pneumoniae, pneumolysin, is in fact able to self-associate in solution to form the same oligomeric structures. The weak interaction leading to solution oligomerization is manifested at low concentrations in a dimeric toxin form. The inhibition of toxin self-interaction by derivatization of the single cysteine residue in pneumolysin with the thiol-active agent dithio (bis)nitrobenzoic acid indicates that self-interaction is mediated by the fourth domain of the protein, which has a fold similar to other proteins known to self-associate. This interaction is thought to have implications for the understanding of mechanisms of pore formation and complement activation by pneumolysin.

Streptolysin O: a proposed model of allosteric interaction between a pore-forming protein and its target lipid bilayer.

Streptolysin O, a polypeptide of 571 amino acids, belongs to the family of thiol-activated toxins that permeabilize animal cell membranes. The protein binds as a monomer to membrane cholesterol. Binding involves a conserved region close to the C-terminus and triggers subsequent polymerization into large arc- and ring-shaped structures surrounding pores of up to 30 nm. Besides the C-terminus, a distantly located region spanning residues 213-305 is involved in oligomerization and in membrane insertion. Here, we searched for conformational effects of monomer binding to the latter functionally important region. To this end, single cysteine substitution mutants were produced and derivatized with the polarity-sensitive fluorophore acrylodan. Fluorimetric measurements revealed that binding of the monomer to membranes is accompanied by distinct environmental changes at amino acid residues 218, 248, 266, and 277. Conspicuously, the environment of residues 218 and 266 became more hydrophilic, suggesting movement of these residues out of hydrophobic protein pockets. Upon oligomerization, further alterations in all side-chain environments were observed. The membrane-bound monomer thus differs in conformation from both the monomer in solution and the subunit of the oligomer. The putative binding site of the molecule is linked to remote domains involved in oligomerization and membrane insertion in an apparently allosteric fashion. It is proposed that allostery is responsible for restricting oligomerization to the membrane-bound state of the toxin.

Thiol-activated cytolysins

A significant part of the pathogenesis of bacterial diseases is explained by the production of toxins, over a third of which act by damaging membranes of target cells. Included with these membrane-damaging toxins is a large group of immunologically-related proteins known as the thiol-activated toxins. These toxins lyse or modulate the function of all cells containing cholesterol in their membranes: a property irreversibly inhibited by cholesterol. Some of the thiol-activated toxins are also antibody-binding proteins and can activate the classical complement pathway. A distinctive feature of the thiol-activated toxins is the formation of extremely large arc- and ring-shaped structures in target membranes. These structures are believed to represent the lesions within cell membranes. In the last decade there have been tremendous advances in our knowledge of these toxins. This review describes our current understanding of the structure and mode of action of the thiol-activated toxins. It also summarizes the evidence from in vitro and in vivo studies that they contribute significantly to the pathogenesis of disease caused by several Gram-positive bacteria.

Modeling the bacterial protein toxin, pneumolysin, in its monomeric and oligomeric form.

Pneumolysin is a member of the family of related bacterial thiol-activated toxins, which share structural similarities and a proposed common cytolytic mechanism. Currently the molecular mechanism of membrane damage caused by these toxins remains a matter of controversy. A prerequisite for defining this mechanism is a detailed knowledge of the monomeric and oligomeric pneumolysin structures. We present for the first time details of the monomeric structure of a thiol-activated toxin, pneumolysin. Electron microscope images of metal-shadowed pneumolysin monomers show an asymmetric molecule composed of four domains. We have studied the conformation of pneumolysin monomer by low resolution hydrodynamic bead modeling procedures. The bead model dimensions and shape are derived solely from the electron micrographs. The bead model has been evaluated in terms of the predicted solution properties, which in turn have been compared to the experimental values of the sedimentation coefficient, s(20,w)0, obtained by analytical ultracentrifugation and the intrinsic viscosity, [eta]. Pneumolysin oligomers, observed as ring- and arc-shaped structures, were also examined by electron microscopy. Metal shadowing and negative staining methods were used to establish the overall dimensions of the oligomer and were used to produce a morphological model for the oligomer, incorporating monomer subunits based on the hydrodynamic bead model.

A role in cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae

Cell binding of pneumolysin to target cells is an important step in the lysis of cells by this toxin. We sought to locate the cell-binding region of pneumolysin. Deletion of the six C-terminal amino acids decreased cell-binding activity by 98%. Furthermore mutagenesis of an amino acid near the C-terminus decreased the cell-binding activity of full-length pneumolysin by 90%. The C-terminus of pneumolysin has an important role in cell-binding activity.

A role in cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae.

Cell binding of pneumolysin to target cells is an important step in the lysis of cells by this toxin. We sought to locate the cell-binding region of pneumolysin. Deletion of the six C-terminal amino acids decreased cell-binding activity by 98%. Furthermore, mutagenesis of an amino acid near the C-terminus decreased the cell-binding activity of full-length pneumolysin by 90%. The C-terminus of pneumolysin has an important role in cell-binding activity.

Identification, purification, and characterization of a thiol-activated hemolysin (suilysin) of Streptococcus suis.

The present report describes the identification, purification, and characterization of a hemolysin produced by Streptococcus suis type 2. The hemolysin was purified from the culture supernatant by using different filtration steps, Superose-12 column chromatography, and selective (NH4)2SO4 precipitation. The purified hemolysin, designated suilysin, had an apparent molecular mass of 54,000 Da and exhibited a specific activity of 0.7 x 10(6) hemolytic units per mg. Suilysin appeared to belong to a family of toxins known as the thiol-activated toxins, with which it had several characteristics in common: loss of activity upon oxidation, reactivation upon reduction, and inhibition of activity by small amounts of cholesterol. The N-terminal amino acid sequence of suilysin showed many similarities with parts of the deduced N-terminal amino acid sequences of perfringolysin O, streptolysin O, listeriolysin O, alveolysin, and pneumolysin. Mice immunized with a vaccine containing purified suilysin appeared to be completely protected against a lethal S. suis type 2 challenge, indicating that suilysin is an important factor and that the neutralization of this single factor is sufficient to protect mice against the detrimental effects of an S. suis type 2 infection. Most of the different (serotype) strains appeared to secrete hemolytic activity which was biochemically and immunologically indistinguishable from suilysin into the culture supernatant in vitro, indicating that suilysin might be a cross-protection factor.

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 was 84%. The final product had a specific activity of 620 000 U/mg with an overall 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.

Production and characterization of neutralizing and nonneutralizing monoclonal antibodies against listeriolysin O

Listeriolysin O (LLO) is a thiol-activated toxin secreted by the facultative intracellular pathogen Listeria monocytogenes. LLO is essential for the survival of the bacterium in the infected cell because it promotes lysis of the phagosome membrane and escape of the bacterium into the cytosol. LLO was used as an antigen for the production of nine monoclonal antibodies (MAbs) in mice. Three of these could inhibit the hemolytic activity of LLO. One of them inhibited binding of LLO to erythrocyte membranes. The two other antibodies blocked the activity of LLO at a step subsequent to membrane binding. Only two of the nine MAbs recognized three other purified SH-activated toxins, streptolysin O, alveolysin, and pneumolysin. Western blot (immunoblot) analysis of culture supernatants of Listeria ivanovii and Listeria seeligeri, two hemolytic species of the genus Listeria, revealed that two MAbs recognized ivanolysin and seeligerolysin. The latter was also recognized by two other MAbs, including one of the neutralizing antibodies. MAbs raised against a peptide, ECTG LAWEWWR, present in all thiol-activated toxins sequenced to date, recognized all toxins and were not neutralizing. Taken together, these results demonstrate the existence of regions important for hemolytic activity that are unique to hemolysins of the genus Listeria and show that regions outside the conserved peptide are important for activity of LLO.

Pneumolysin Induces the Salient Histologic Features of Pneumococcal Infection in the Rat Lung In Vivo

Streptococcus pneumoniae infections are common, but how they cause host tissue injury and death is incompletely understood. Immunization with pneumolysin, a thiol-activated toxin produced by the pneumococcus, partially protects animals during subsequent infection. The mechanism by which pneumolysin contributes to disease is not known. The aim of the present investigation was to determine the histologic changes induced by recombinant pneumolysin in the rat lung and to compare them with the changes induced by live organisms. Injection of either toxin (200 or 800 ng) or bacteria into the apical lobe bronchus was associated with the development of a severe lobar pneumonia restricted to the apical lobe. The changes induced by the toxin were greater at the higher concentration, and changes were most severe in those animals in which there was partial ligation of the apical lobe bronchus. The pneumonitis was less severe following injection of a modified toxin with decreased hemolytic activity, generated by site-directed mutagenesis of the cloned pneumolysin gene, indicating that this property of the toxin was important in generating pulmonary inflammation. There was still considerable pneumonitis after injection of a modified toxin with decreased capacity to activate complement.

Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O

Listeriolysin O (LLO), a major virulence factor of the intracellular bacterium Listeria monocytogenes, shares with other known 'thiol-activated toxins' a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule. Single amino acid substitutions were created in this region to study the role of cysteine and tryptophan residues in the lytic activity of LLO as well as in the virulence of the bacterium. Transformation of a transposon-induced non-haemolytic mutant with plasmids carrying the mutated genes allowed allele exchange and transfer of mutations on to the chromosome by in vivo recombination. The mutant strains secreted a full-length 59 kilodalton LLO. A decrease of 25% in the haemolytic activity in culture supernatants was observed in the case of mutation Cys-484 to Ala and of 80% for mutation Cys-484 to Ser. Mutations Trp-491 and Trp-492 to Ala decreased activity by, respectively, 95% and 99.9%. LLOs produced by the mutants, as the wild type, were active at low pH, inhibited by cholesterol, and able to bind to cell membranes. A close relationship was found between virulence of mutants in the mouse model and haemolytic activity in their culture supernatants. These results demonstrate that the thiol group of Cys-484 is not essential for either haemolytic activity in vitro or virulence in vivo. In contrast, Trp-492 appears to be required for both haemolytic activity and virulence. The finding that the nearly non-haemolytic mutant Trp-492-Ala persisted in the spleen for several days after inoculation indicates that mutagenesis of a virulence determinant can attenuate virulence and provides a novel approach to the development of live vaccine strains.

Alveolysin, the thiol-activated toxin of Bacillus alvei, is homologous to listeriolysin O, perfringolysin O, pneumolysin, and streptolysin O and contains a single cysteine

The gene coding for alveolysin, the thiol-activated toxin produced by Bacillus alvei, has been cloned by means of an oligonucleotide based on the known N-terminal sequence of the secreted protein. The complete nucleotide sequence of the gene has been determined. The deduced amino acid sequence of alveolysin shows that alveolysin shares homologies with listeriolysin O, perfringolysin O, pneumolysin, and streptolysin O. Alveolysin, like the other members of the family, contains a single cysteine in the conserved peptide sequence ECTGLA WEWWR.

Purification and characterization of two Listeria ivanovii cytolysins, a sphingomyelinase C and a thiol-activated toxin (ivanolysin O)

The strong bizonal hemolysis on blood agar and the positive CAMP reaction with Rhodococcus equi denotes the production of two different cytolytic factors by Listeria ivanovii. One was characterized as a thiol-activated (SH) cytolysin of 61 kilodaltons and was termed ivanolysin O (ILO) since data suggested that it is different from listeriolysin O, the SH-cytolysin produced by Listeria monocytogenes. The other is a 27-kilodalton hemolytic sphingomyelinase C that was found to be the cytolytic factor responsible for the halo of incomplete hemolysis synergistically enhanced by R. equi exosubstances. When thiol-disulfide exchange affinity chromatography and gel filtration were applied to the purification of ILO from concentrated L. ivanovii culture supernatants, the copurification of the two cytolysins was observed. This phenomenon seems to be due to the formation of intermolecular disulfide bonds between ILO and the sphingomyelinase, since the latter was found to contain free SH groups, not essential for the activity. These SH groups could react with the single cysteine residue characteristically present in the SH-cytolysins, forming a dimeric cytolytic complex. The purification of ILO was achieved by a further gel filtration with a reducing agent (dithiothreitol) in the eluent. A method for the purification of the sphingomyelinase based on selective sequestration of ILO from the L. ivanovii concentrated culture supernatant by the SH cytolysin target molecule cholesterol and thiol-disulfide affinity chromatography is described.

Preliminary evidence that different domains are involved in cytolytic activity and receptor (cholesterol) binding in listeriolysin O, theListeria monocytogenesthiol-activated toxin

The inactive truncated 52 Kilodaltons (kDa) Listeriolysin O (LLO) produced by a transposon Tn1545-induced Listeria monocytogenes non-hemolytic/avirulent mutant previously described (Gaillard et al. (1986) Infect. Immun. 52, 50-55), that lacks a 48 aminoacid fragment at the C-terminal end including the single cysteine residue essential for activity (Mengaud et al. (1988) Infect. Immun. 56, 766-772), bound to the SH-cytolysin membrane receptor cholesterol, as did the active 60 kDa toxin. These results indicate that the missing fragment is a functionally important region needed in the 60 kDa LLO to cause membrane-disruption but not to bind to cholesterol, which strongly suggests that in LLO (and presumably in the other SH-cytolysins, in accordance with their structural and functional homologies) different domains are involved in cytolytic activity and cholesterol binding. The cysteine residue contained in the missing fragment, therefore, would not be essential for cholesterol binding, as is currently thought, rather it seems to be essential specifically for cell lysis.