Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile

Abstract

The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-alpha and Trn-beta, that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [(13)C,(15)N] Trn-alpha and [(13)C,(15)N]Trn-beta were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the alpha-carbons of the modified residues, forming three sulfur to alpha-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S'-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to alpha-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora.

Fig. 1.

(A) Colony of B. thuringiensis DPC 6431 on the initial isolation plate showing inhibition in seeded overlay of C. difficile ATCC 43593. Colonies within the zone of inhibition are gut isolates, not resistant colonies of the target strain. (B) Inhibition of C. difficile ATCC 43593 by cell-free supernatant of B. thuringiensis DPC 6431, and (C) demonstration of its proteinaceous nature through the effect of Proteinase K. (D) Well diffusion assay of purified peptides showing the synergistic effect of peptides in equimolar concentrations over a range of concentrations. (E) RP-HPLC chromatogram of thuricin CD showing separation of Trn-α and Trn-β and their molecular masses.

Fig. 2.

Thuricin CD versus metronidazole in a human distal colon model. The effect of adding thuricin CD and metronidazole at equimolar concentrations (90 μM) at times 0, 8, and 16 h on survival of C. difficile ribotype 001 is shown.

Fig. 3.

(A) Organization of genes in putative thuricin CD operon with an alignment of the amino acid sequences of Trn-α and Trn-β. (B) Results from tandem MS analysis indicate that residues 21, 25, and 28 in both Trn-α and Trn-β are two mass units lighter than expected (predicted masses in black; exact masses in gray).

Fig. 4.

(A) The structural connectivity of Trn-α and Trn-β as suggested by NMR analysis, and (B) two ¹H-¹H strips from a ¹³C HSQC-NOESY showing NOE correlations between the Cys13 and Thr21 residues of Trn-β.