Virulence studies on chromosomal α‐toxin and Θ‐toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α‐toxin in Clostridium perfringens‐mediated gas gangrene

Awad, Milena M.; Bryant, Amy E.; Stevens, Dennis L.; Rood, Julian I.

doi:10.1111/j.1365-2958.1995.tb02234.x

Cited by 298 publications

(225 citation statements)

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“…SNTX was concentrated to 5-10 mg/mL for crystallography. The activity of the purified SNTX was confirmed by a doubling dilution hemolysin assay using 1% (vol/vol) rat erythrocytes as described previously (41). The titer was defined as the reciprocal of the last well that showed complete hemolysis [log 2 (hemolytic activity)] and shown to be 8.25 ± 0.4 (compared with crude venom: 5.8 ± 0.3).…”

Section: Methodsmentioning

confidence: 99%

Stonefish toxin defines an ancient branch of the perforin-like superfamily

Ellisdon

Reboul

Panjikar

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack ComplexPerforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.H uman envenoming by the tropical stonefish (Synanceia horrida and related species) results in extreme pain, edema, hypotension, respiratory distress, and on rare occasions, death (1). The lethal factor in stonefish venom is an ∼150-kDa protein termed stonustoxin (SNTX), an unusual example of a vertebrate cytolytic protein complex (2). SNTX is a soluble heterodimeric assembly of two closely related proteins termed SNTX-α and -β that share sequence identity of ∼50% (3). With the exception of a C-terminal PRY SPla and the RYanodine Receptor (PRYSPRY) domain in each protein (4), SNTX shares no obvious sequence similarity to any structurally or functionally characterized molecule. SNTX induces species-specific hemolytic activity (2) by an apparent pore-forming mechanism (5). It induces platelet aggregation (6), and like the closely related Trachynilysin (from Synanceia trachynis), SNTX exhibits activity suggesting that it may function as a neurotoxin (7,8).Because eukaryote pore-forming toxins are relatively rare, we reasoned that SNTX might represent a new exemplar of a vertebrate pore-forming protein. Previous studies had shown that it was possible to purify and crystallize SNTX (9); however, no structure has been reported to date. Accordingly, to address the structural basis for SNTX activity, we determined its X-ray crystal structure.

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Section: Methodsmentioning

confidence: 99%

Stonefish toxin defines an ancient branch of the perforin-like superfamily

Ellisdon

Reboul

Panjikar

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Perfringolysin O activity was determined by measuring the hemolysis of horse erythrocytes. Four-hour C. perfringens cultures and supernatants were obtained as described previously (1). Hemolysin assays were performed by a doubling dilution assay, as described previously (43).…”

Section: Methodsmentioning

confidence: 99%

“…It is characterized by its ability to produce many extracellular toxins and enzymes (33), including alpha-toxin (phospholipase C) and theta-toxin (perfringolysin O), which have been shown to act synergistically in gas gangrene (1,2,12,44). The production of these toxins, as well as collagenase (kappa-toxin), sialidase (19), and alpha-clostripain (40), is regulated by a two-component signal transduction system that comprises the VirS sensor histidine kinase and the VirR response regulator.…”

mentioning

confidence: 99%

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

et al. 2004

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The transcriptional regulation of toxin production in the gram-positive anaerobe Clostridium perfringens involves a two-component signal transduction system that comprises the VirS sensor histidine kinase and its cognate response regulator, VirR. Previous studies showed that VirR binds independently to a pair of imperfect direct repeats, now designated VirR box 1 and VirR box 2, located immediately upstream of the promoter of the pfoA gene, which encodes the cholesterol-dependent cytolysin, perfringolysin O. For this study, we introduced mutated VirR boxes into a C. perfringens pfoA mutant and found that both VirR boxes are essential for transcriptional activation. Furthermore, the spacing between the VirR boxes and the distance between the VirR boxes and the ؊35 region are shown to be critical for perfringolysin O production. Other VirR boxes that were previously identified from the strain 13 genome sequence were also analyzed, with perfringolysin O production used as a reporter system. The results showed that placement of the different VirR boxes at the same position upstream of the pfoA promoter yields different levels of perfringolysin O activity. In all of these constructs, VirR was still capable of binding to the target DNA, indicating that DNA binding alone is not sufficient for transcriptional activation. Finally, we show that the C. perfringens RNA polymerase binds more efficiently to the pfoA promoter in the presence of VirR, indicating that interactions must occur between these proteins. We propose that these interactions are required for VirR-mediated transcriptional activation.

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“…The culture supernatant was passed through a 0.45 mm filter (Millipore) and used for assays to determine the activities of alpha-, kappa-and theta-toxins. The assay for alpha-toxin activity was performed as described previously (Awad et al, 1995), and the number of units of activity was determined by reference to control assays, which used a standard phospholipase C preparation (Sigma). The theta-toxin and kappa-toxin activities were determined as described elsewhere (Shimizu et al, 1994).…”

Section: Toxin Assaysmentioning

confidence: 99%

The luxS gene is involved in cell–cell signalling for toxin production in Clostridium perfringens

Ohtani

Hayashi

Shimizu

2002

Molecular Microbiology

159

112

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Summary A Gram‐positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. C. perfringens possesses a homologue of the luxS gene that is reported to be responsible for the production of autoinducer 2 (AI‐2), which participates in quorum sensing in bacteria. The luxS mutant was constructed using C. perfringens strain 13, and the role of the luxS gene in toxin production was examined. The cell‐free culture supernatant from wild‐type strain 13 greatly stimulated the luminescence of Vibrio harveyi BB170, whereas that from the luxS mutant caused no significant stimulation, indicating that the luxS gene is necessary for AI‐2 production in C. perfringens. The luxS mutant showed a reduced level of production of alpha‐, kappa‐ and theta‐toxins. In the luxS mutant, the transcription of the theta‐toxin gene (pfoA) was lower at mid‐exponential growth phase, whereas alpha‐ and kappa‐toxin gene transcription was not significantly affected. The production of toxins in the luxS mutant was stimulated by the addition of the culture supernatant from the wild‐type cells, possibly because of the presence of AI‐2. Moreover, the expression of the pfoA gene in the luxS mutant was apparently activated when the mutant cells were cultured in the presence of culture supernatants from the wild‐type C. perfringens, Escherichia coli DH5α carrying the luxS gene of C. perfringens. A deletion analysis of the luxS operon showed that the luxS gene alone is responsible for cell–cell signalling, and that the metB or cysK genes located upstream of luxS are not involved in regulating toxin production. Our results indicate that cell–cell signalling by AI‐2 plays an important role in the regulation of toxin production in C. perfringens.

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Virulence studies on chromosomal α‐toxin and Θ‐toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α‐toxin in Clostridium perfringens‐mediated gas gangrene

Cited by 298 publications

References 48 publications

Stonefish toxin defines an ancient branch of the perforin-like superfamily

Stonefish toxin defines an ancient branch of the perforin-like superfamily

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

The luxS gene is involved in cell–cell signalling for toxin production in Clostridium perfringens

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