Abstract
Through elaboration of its botulinum toxins, Clostridium botulinum produces clinical syndromes of infant botulism, wound botulism, and other invasive infections. Using comparative genomic analysis, an orphan nine-gene cluster was identified in C. botulinum and the related foodborne pathogen Clostridium sporogenes that resembled the biosynthetic machinery for streptolysin S, a key virulence factor from group A Streptococcus responsible for its hallmark beta-hemolytic phenotype. Genetic complementation, in vitro reconstitution, mass spectral analysis, and plasmid intergrational mutagenesis demonstrate that the streptolysin S-like gene cluster from Clostridium sp. is responsible for the biogenesis of a novel post-translationally modified hemolytic toxin, clostridiolysin S.
Highlights
Covery of its biosynthetic gene cluster more than a decade ago [4] has guided investigations into its post-translational modification and likely heterocyclic nature [6, 7]
We demonstrate that the gene clusters in clostridia are functionally equivalent to the SLS biosynthetic pathway by complementing targeted GAS SLSoperon knock-outs with the corresponding Clostridium genes
These were compared with the hemolytic activity of wild-type GAS, ⌬sagA complemented with pDCermsagA, and ⌬sagA–D complemented with the pDCerm plasmid alone
Summary
Plasmid Integrational Mutagenesis of closA and closC—PCR was used to amplify the central regions of C. sporogenes closA and closC genes. Amplicons were excised and purified using the Qiagen QIAquick PCR purification kit. The purified amplicons and the pET28 vector containing an N-terminal maltose-binding protein (MBP) tag were sequentially digested by allowing the NotI digest to proceed overnight followed by a 2-h BamHI digestion at 37 °C (New England Biolabs). The digested amplicons and vector were separated by agarose gel electrophoresis, excised, and purified with a Qiaquick gel extraction kit. Single colonies of each transformant were picked and grown overnight in preparation for plasmid recovery. Colonies that contained the closA–D genes were grown and stored at Ϫ80 °C until used in the hemolytic/cytolyic assays. Protein Purification of MBP-ClosA–D and MBP-SagA–D— All MBP-tagged proteins used in the in vitro hemolytic assays were purified as described previously [6]. All proteins were immediately stored at Ϫ80 °C until further use
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