Clostridiolysin S, a post-translationally modified biotoxin from Clostridium botulinum

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.

FIGURE 1.

Comparison of the streptolysin S-associated genetic cluster (Sag) with the SLS-like clostridiolysin S genetic cluster in Clostridium botulinum. A, the Sag cluster in GAS contains a SLS precursor sequence (SagA), which is modified by the SagBCD synthetase complex. SagE and -F are potentially involved in immunity function. SagG–I have homology to ABC transporters. The clostridiolysin S gene cluster contains genes similar to the Sag locus, in identical order, and is present in C. botulinum ATCC 3502, and C. sporogenes ATCC 15579. B, shown is the genetic complementation of Clos genes in GAS M1 Sag mutants. BSA-stabilized extracts were assayed for lytic activity. Bar 1, M1wtGAS; bar 2, M1 GAS ΔsagA and wtsagA; bar 3, M1 GAS ΔsagA and wtclosA; bar 4, M1 GAS ΔsagB and wtclosB; bar 5, M1 GAS ΔsagC and wtclosC; bar 6, M1 GAS ΔsagD and wtclosD; bar 7, M1 GAS ΔsagA and plasmid alone; bar 8, M1 GAS ΔsagB and plasmid alone; bar 9, M1 GAS ΔsagC and plasmid alone; and bar 10, M1 GAS ΔsagD and plasmid alone. Hemolytic activity was normalized against a Triton X-100 positive control. C, C. sporogenes exhibits a strong β-hemolytic phenotype when grown on blood agar plates. ΔclosA and ΔclosC C. sporogenes allelic exchange mutants lose the ability to lyse erythrocytes. Bacteria were grown for 36 h at 37 °C under anaerobic conditions.

FIGURE 2.

Hemolytic activity of clostridiolysin is detectable in vitro. A, in vitro reconstitution of ClosA hemolytic activity. Synthetase reactions using MBP-ClosA and SagB/ClosC/ClosD produce a hemolytic toxin. Hemolytic activity was normalized against a Triton X-100 positive control. Bar 1, wt SagA and SagBCD; bar 2, wt ClosA and SagB/ClosC/ClosD; bar 3, wt ClosA only; bar 4, SagB/ClosC/ClosD only; bar 5, wt ClosA and SagB/ClosC; bar 6, wt ClosA and ClosC/ClosD; bar 7, wt ClosA and SagB/ClosD; bar 8, SagA alone. B, in vitro reconstitution of ClosA hemolytic activity using CxxC mutants as wild-type ClosC surrogates. Bar 1, wt ClosA and SagB/ClosC/ClosD; bar 2, wt ClosA and SagB/ALEC/ClosD wt; bar 3, ClosA and SagB/CLEA/ClosD; bar 4, wt ClosA and SagB/APAA/ClosD wt; bar 5, ClosA and SagB/APAC/ClosD; bar 6, wt ClosA and SagB/CPAA/ClosD. Hemolytic activity was normalized against a Triton X-100 positive control. C, CD spectra of MBP-ClosC and MBP-tagged CxxC mutants. ■, CD spectra of MBP-ClosC (a negative minimum at 215 and 225 nm confirms α-helical integrity); □, CD for CPAA mutant; ▵, CD for CLEA mutant; ×, CD for APAC mutant; *, CD for APAA mutant; ○, CD for ALEC mutant. CD was repeated three times with different protein purifications and resulted in consistent results.

FIGURE 3.

Alignment of ClosC in C. botulinum ATCC 3503 and MoeB/ThiF and proposed mechanisms. A, mechanisms for the formation of thiazole or methyloxazole. B, sequence alignments of ClosC, MccB, E. coli MoeB, and E. coli ThiF. Blue highlighted boxes show conserved ATP binding residues determined by the crystal structures of ThiF and MoeB. Green highlighted boxes show the residues involved in zinc-tetrathiolate formation.

FIGURE 4.

Detection of heterocyclized ClosA peptides via nanocapillary LC-MS/MS method. Mass spectra of C-terminal peptides identified as being heterocyclized in the SLS and CLS in vitro systems. A detailed description of mass spectral annotations is supplied in the supplemental data.

FIGURE 5.

Hemolytic activity of MBP-ClosA T46A and structures of identified PTM peptides. A, synthetase reactions using MBP-ClosA T46A and SagB/ClosC/ClosD. Hemolytic activity was normalized against a Triton X-100 positive control. Lane 1, wt ClosA and SagB/ClosC/ClosD; lane 2, SagB/ClosC/ClosD only; lane 3, ClosA T46A and SagB/ClosC/ClosD. B, peptide 1, CLS peptide containing a methyloxazole at position Thr⁴⁶; peptide 2, SLS peptide containing two oxazoles at positions Ser⁴⁶ and Ser⁴⁸.