Research ArticleANTIMICROBIALS

New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability

See allHide authors and affiliations

Science Translational Medicine  06 Sep 2017:
Vol. 9, Issue 406, eaah6813
DOI: 10.1126/scitranslmed.aah6813
  • Fig. 1. Mutations in slpA confer Av-CD291.2 resistance.

    (A) Alignment of the slpA sequence (nucleotides 268 to 294) from R20291, FM2.5, FM2.6, FM2.5RW, and FM2.6RW. A nucleotide insertion at position 283 of FM2.5 slpA results in a frameshift and premature stop codon (blue). A nucleotide substitution at position 280 of FM2.6 slpA results in a nonsense mutation (red). To allow differentiation from the wild-type sequence, we introduced two synonymous mutations into slpA in FM2.5RW and FM2.6RW (green). (B) SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of S-layer extracts from R20291, FM2.5, FM2.6, FM2.5RW, and FM2.6RW. The positions of the LMW and HMW SLPs and minor cell wall proteins Cwp2 and Cwp6 are indicated. (C and D) The impact of Av-CD291.2 on exponentially growing R20291, FM2.5, FM2.5RW, and FM2.6RW was monitored by measuring the OD600nm. Av-D291.2 addition is indicated with an arrow. Experiments were carried out in triplicate on biological duplicates. Means and SDs are shown. (E and F) SDS-PAGE analysis and Av-CD291.2 sensitivity of FM2.5 complemented with slpA alleles from multiple SLCTs after induction with anhydrotetracycline (20 ng/ml). R20291 and FM2.5RW are included as controls. A zone of clearance in the agar lawn indicates killing.

  • Fig. 2. Avidocin-CD sensitivity correlates with SLCT.

    (A) SDS-PAGE analysis of SLPs extracted from a panel of strains representing the 11 most commonly isolated SLCTs. (B) Spot bioassays with eight Avidocin-CDs on the C. difficile strains used in (A), as well as FM2.5 alone (−) and FM2.5 complemented with slpA alleles from 10 SLCTs after induction with anhydrotetracycline (20 ng/ml). The zone of clearance caused by each Avidocin-CD is shown along with SLCT. H, H2/6.

  • Fig. 3. Phenotypic characterization of FM2.5.

    (A and B) Cultures of R20291, FM2.5, and FM2.5RW were challenged with lysozyme (500 μg/ml) (A) or LL-37 (5 μg/ml) (B) in exponential phase after 2.5 hours (indicated with arrows). Untreated control cultures were grown in parallel. Experiments were carried out in triplicate on biological duplicates. Means and SDs are shown. (C) Sporulation of R20291, FM2.5, and FM2.5RW after 5 days. Spore CFUs were determined after a standard 65°C heat treatment for 30 min or a harsher 75°C heat treatment for 30 min. Heat-resistant spore CFUs are expressed as a percentage of total viable CFUs (spores and vegetative cells). Experiments were carried out in triplicate on biological duplicates. Means and SDs are shown. *P < 0.01, determined using two-tailed t tests with Welch’s correction. (D) Germination of R20291, FM2.5, and FM2.5RW spores. Synchronous germination of purified spores was induced with the bile salt taurocholate. Germination initiation was monitored by measuring the resulting decrease in OD600nm.

  • Fig. 4. In vivo analysis of slpA mutant in the Syrian Golden hamster.

    (A) Times to experimental end point of animals infected with R20291 (black line), FM2.5 (dark blue line), and FM2.5RW (light blue line), respectively. Each line represents six animals. (B) Total CFUs and spore CFUs (after heat treatment at 56°C for 20 min) were determined for lumen-associated (LA) and tissue-associated (TA) bacteria recovered from cecum (CE) and colon (COL) of infected animals and quantified at experimental end point (R20291 and FM2.5RW) or at 14 days after infection (FM2.5). Means and SEs are shown. The horizontal dashed line indicates the limit of detection. None of the observed differences are statistically significant. (C and D) Relative toxin activity of filtered gut samples on HT-29 (toxin A) and Vero cells (toxin B), respectively. Values represent the reciprocal of the first dilution in which cell morphology was indistinguishable from untreated wells. Samples were taken at experimental end point (R20291 and FM2.5RW) or at 14 days after infection (FM2.5). *P < 0.05 and **P < 0.01, determined using a two-tailed nonparametric Mann-Whitney test; NS, not significant.

  • Fig. 5. Toxin production and release in vitro.

    (A) In vitro cell lysate and culture supernatant samples from R20291, FM2.5, and FM2.5RW cultures were normalized to an equivalent optical density and separated on 6% SDS polyacrylamide gels. Toxin B was detected by Western immunoblot using an anti–toxin B monoclonal antibody. Samples were taken at the indicated time points. (B) To determine whether Avidocin-CD killing released intracellular toxin, R20291 was incubated for an hour either with Av-CD291.2 at the indicated ratio of agent to cells or Av-CD684.1, which does not kill strain R20291, at a 500:1 ratio (IS), or left untreated (UT). After treatment, viable bacteria were enumerated (bar graph), and the percentage killed relative to the untreated control was determined (numbers above each bar). The number of spores present in the untreated sample was determined after heat treatment at 65°C for 30 min to kill vegetative cells (HT). (C) After Avidocin-CD treatment, released toxin B in culture supernatants (ExC) was detected by Western immunoblot using an anti–toxin B monoclonal antibody. As a positive control for toxin release, R20291 was treated with the phiCD27L bacteriophage endolysin (41). The amount of remaining intracellular toxin B (IntC) was determined by lysing cells with CD27L endolysin and detection by Western immunoblot as before. A fresh sample of untreated R20291 was lysed with CD27L endolysin to show normal intracellular toxin quantities (EL). The original uncropped images for each Western immunoblot can be found in fig. S9.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/406/eaah6813/DC1

    Materials and Methods

    Fig. S1. Characterization of growth.

    Fig. S2. Restoration of wild-type slpA to the chromosome of FM2.5 and FM2.6.

    Fig. S3. Avidocin-CD sensitivity correlates with SLCT.

    Fig. S4. C. difficile strain sensitivity patterns to Avidocin-CDs and Diffocin-4.

    Fig. S5. Comparison of C. difficile bacteriophage host range versus Avidocin-CD sensitivity.

    Fig. S6. Clustal Omega alignment of SlpA sequences from strains 630 and M68.

    Fig. S7. Characterization of sporulation.

    Fig. S8. Spore morphology and thermal sensitivity.

    Fig. S9. Expression of toxin B in vitro.

    Fig. S10. Schematic diagram summarizing the roles of S-layer in C. difficile biology and pathophysiology.

    Fig. S11. Schematic describing Avidocin-CD construction.

    Table S1. Newly identified C. difficile phages.

    Table S2. Primers used in this study.

    Table S3. GenBank accession identifiers.

    Table S4. Primary data.

    Reference (42)

  • Supplementary Material for:

    New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability

    Joseph A. Kirk, Dana Gebhart, Anthony M. Buckley, Stephen Lok, Dean Scholl, Gillian R. Douce, Gregory R. Govoni,* Robert P. Fagan*

    *Corresponding author. Email: ggovons{at}gmail.com (G.R.G.); r.fagan{at}sheffield.ac.uk (R.P.F.)

    Published 6 September 2017, Sci. Transl. Med. 9, eaah6813 (2017)
    DOI: 10.1126/scitranslmed.aah6813

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Characterization of growth.
    • Fig. S2. Restoration of wild-type slpA to the chromosome of FM2.5 and FM2.6.
    • Fig. S3. Avidocin-CD sensitivity correlates with SLCT.
    • Fig. S4. C. difficile strain sensitivity patterns to Avidocin-CDs and Diffocin-4.
    • Fig. S5. Comparison of C. difficile bacteriophage host range versus Avidocin-CD sensitivity.
    • Fig. S6. Clustal Omega alignment of SlpA sequences from strains 630 and M68.
    • Fig. S7. Characterization of sporulation.
    • Fig. S8. Spore morphology and thermal sensitivity.
    • Fig. S9. Expression of toxin B in vitro.
    • Fig. S10. Schematic diagram summarizing the roles of S-layer in C. difficile biology and pathophysiology.
    • Fig. S11. Schematic describing Avidocin-CD construction.
    • Table S1. Newly identified C. difficile phages.
    • Table S2. Primers used in this study.
    • Table S3. GenBank accession identifiers.
    • Legend for table S4
    • Reference (42)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4 (Microsoft Excel format). Primary data.

    [Download Table S4]

Stay Connected to Science Translational Medicine

Navigate This Article