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Cited by 185Clostridium difficile and Clostridium perfringens researchBacterial Genetics and BiotechnologyAntimicrobial Resistance in Staphylococcus

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High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile

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Dembek, Marcin, Barquist, Lars, Boinett, Christine J., Cain, Amy K., Mayho, Matthew, Lawley, Trevor D., Fairweather, Neil F., Fagan, Robert P.·2015·mBio
DOI 10.1128/mbio.02383-14PMC PMC4358009
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Abstract

UNLABELLED: Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. IMPORTANCE: Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection.

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Figures

FIG 1 Identification of sporulation-specific genes in C. difficile. (A and C) Insertion index distribution plots from the C. difficile 630Δerm (A) and R20291 (C) transposon libraries. The clear bimodal distribution in the R20291 plot (fitted gamma distributions in blue) allows differentiation of genes that do not tolerate transposon insertions in the sharp left-hand peak and genes containing insertions in the broad right-hand peak. (B and D) S-layer biogenesis locus. Each vertical line indicates the location of a unique transposon insertion, with height indicating the total number of Illumina reads at each insertion point. The secA2 and slpA genes show no transposon insertions, which suggests that they are required for growth in vitro. (E and F) Changes in prevalence of the mutants from the initial transposon library after overnight growth (E) or sporulation (F). Red lines show the cutoff criteria of 10% false discovery rate (horizontal) and a log2 fold change (Log2FC) of 2 (vertical).
FIG 1 Identification of sporulation-specific genes in C. difficile. (A and C) Insertion index distribution plots from the C. difficile 630Δerm (A) and R20291 (C) transposon libraries. The clear bimodal distribution in the R20291 plot (fitted gamma distributions in blue) allows differentiation of genes that do not tolerate transposon insertions in the sharp left-hand peak and genes containing insertions in the broad right-hand peak. (B and D) S-layer biogenesis locus. Each vertical line indicates the location of a unique transposon insertion, with height indicating the total number of Illumina reads at each insertion point. The secA2 and slpA genes show no transposon insertions, which suggests that they are required for growth in vitro. (E and F) Changes in prevalence of the mutants from the initial transposon library after overnight growth (E) or sporulation (F). Red lines show the cutoff criteria of 10% false discovery rate (horizontal) and a log2 fold change (Log2FC) of 2 (vertical).CC BY · PubMed Central
FIG 2 Sporulation efficiency of individual isogenic mutants. CD0106, CD0125, CD3494, and CD3567 deletion mutants were compared to strains complemented on the chromosome at the pyrE locus and the parental 630Δerm strain (wild type [WT]). Stationary-phase cultures in BHIS broth were incubated anaerobically for 5 days, and samples were taken at 24-h intervals for analysis. Total cell numbers were determined by counting the number of CFU on BHIS agar containing the germinant taurocholate. Spore numbers were determined by the same method following incubation at 70°C for 30 min. Experiments were carried out in triplicate on biological duplicate samples. The means ± standard deviations (error bars) are shown.
FIG 2 Sporulation efficiency of individual isogenic mutants. CD0106, CD0125, CD3494, and CD3567 deletion mutants were compared to strains complemented on the chromosome at the pyrE locus and the parental 630Δerm strain (wild type [WT]). Stationary-phase cultures in BHIS broth were incubated anaerobically for 5 days, and samples were taken at 24-h intervals for analysis. Total cell numbers were determined by counting the number of CFU on BHIS agar containing the germinant taurocholate. Spore numbers were determined by the same method following incubation at 70°C for 30 min. Experiments were carried out in triplicate on biological duplicate samples. The means ± standard deviations (error bars) are shown.CC BY · PubMed Central
FIG 3 Microscopic examination of deletion mutants and complemented strains. (A) Cultures were sporulated on SMC agar for 24 h, harvested in PBS, and fluorescently labeled with a membrane stain (FM4-64) and DNA stain (Hoechst 33258). Both the ΔCD0106 and ΔCD3494 mutants appeared to produce normal phase-bright spores. The ΔCD0125 mutant failed to produce visible mature spores, and sporulation appeared to halt at forespore formation (white arrowheads). The ΔCD3567 mutant also did not produce mature spores, and only small, phase-dark spores were observed (black arrowheads). Complementation of the ΔCD0125 and ΔCD3567 mutants restored normal spore production. (B and C) Higher magnification micrographs of ΔCD0125 and ΔCD3567 mutants showing severe sporulation defects.
FIG 3 Microscopic examination of deletion mutants and complemented strains. (A) Cultures were sporulated on SMC agar for 24 h, harvested in PBS, and fluorescently labeled with a membrane stain (FM4-64) and DNA stain (Hoechst 33258). Both the ΔCD0106 and ΔCD3494 mutants appeared to produce normal phase-bright spores. The ΔCD0125 mutant failed to produce visible mature spores, and sporulation appeared to halt at forespore formation (white arrowheads). The ΔCD3567 mutant also did not produce mature spores, and only small, phase-dark spores were observed (black arrowheads). Complementation of the ΔCD0125 and ΔCD3567 mutants restored normal spore production. (B and C) Higher magnification micrographs of ΔCD0125 and ΔCD3567 mutants showing severe sporulation defects.CC BY · PubMed Central
FIG 4 Germination efficiency of CD0106 and CD3494 isogenic deletion mutants. Spores of the parental strain 630Δerm (WT), ΔCD0106, ΔCD3494, and complemented strains were produced on solid media and purified. (A and B) Purified spores were resuspended in BHIS supplemented with the germinant taurocholate (0.5%), and initiation of germination was monitored by measuring the drop in OD600 due to Ca2+-DPA release from the core. Data are expressed as the ratio of OD600 at each time point to t0. The ΔCD0106 mutant did not appear to germinate during the course of the experiment, and complementation restored germination to wild-type levels. The ΔCD3494 mutant appeared to germinate with a small delay compared to the parental strain but otherwise appeared to germinate normally. (C) Germinating suspensions were sampled, fixed in formaldehyde, and visualized by phase-contrast microscopy. In agreement with panel A, the ΔCD0106 mutant did not germinate in response to taurocholate, whereas the ΔCD3494 mutant appeared to germinate normally. Time (in minutes) is shown above the photos.
FIG 4 Germination efficiency of CD0106 and CD3494 isogenic deletion mutants. Spores of the parental strain 630Δerm (WT), ΔCD0106, ΔCD3494, and complemented strains were produced on solid media and purified. (A and B) Purified spores were resuspended in BHIS supplemented with the germinant taurocholate (0.5%), and initiation of germination was monitored by measuring the drop in OD600 due to Ca2+-DPA release from the core. Data are expressed as the ratio of OD600 at each time point to t0. The ΔCD0106 mutant did not appear to germinate during the course of the experiment, and complementation restored germination to wild-type levels. The ΔCD3494 mutant appeared to germinate with a small delay compared to the parental strain but otherwise appeared to germinate normally. (C) Germinating suspensions were sampled, fixed in formaldehyde, and visualized by phase-contrast microscopy. In agreement with panel A, the ΔCD0106 mutant did not germinate in response to taurocholate, whereas the ΔCD3494 mutant appeared to germinate normally. Time (in minutes) is shown above the photos.CC BY · PubMed Central
FIG 5 Comparison of genes required for sporulation with published regulons for Spo0A, σE, σF, σG, and σK. The 798 genes identified in our screen were cross-referenced with the previously identified regulons of Spo0A (41) and σE, σF, σG, and σK (12). (A) Each gene is shown as a single point and colored according to the regulon; those genes not identified in the 5 published regulons are shown as black points. Dashed red lines indicate a log2FC of 2 and −2. (B) Log2FC in the spore transposon library of every gene previously identified as belonging to the σE, σF, σG, and σK regulons colored by functional class, as defined in reference 12. The box-and-whisker plots show the interquartile range (IQR), with the median marked (black line). The whiskers indicate the highest/lowest point within 1.5× IQR of the upper/lower quartile.
FIG 5 Comparison of genes required for sporulation with published regulons for Spo0A, σE, σF, σG, and σK. The 798 genes identified in our screen were cross-referenced with the previously identified regulons of Spo0A (41) and σE, σF, σG, and σK (12). (A) Each gene is shown as a single point and colored according to the regulon; those genes not identified in the 5 published regulons are shown as black points. Dashed red lines indicate a log2FC of 2 and −2. (B) Log2FC in the spore transposon library of every gene previously identified as belonging to the σE, σF, σG, and σK regulons colored by functional class, as defined in reference 12. The box-and-whisker plots show the interquartile range (IQR), with the median marked (black line). The whiskers indicate the highest/lowest point within 1.5× IQR of the upper/lower quartile.CC BY · PubMed Central

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Metadata

Authors
Dembek, M., Barquist, L., Boinett, C. J., Cain, A. K., Mayho, M., Lawley, T. D., Fairweather, N. F., & Fagan, R. P.
Affiliations
  • Imperial College London (GB)
  • University of Würzburg (DE)
  • Wellcome Sanger Institute (GB)
  • University of Sheffield (GB)
Corresponding author
Fairweather, Neil F.
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2015-05
Volume
6
Issue
2
Pages
e02383-e02383
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.02383-14
PMC
PMC4358009
OpenAlex
W1989733081
Cited by
185
References
53
Citations by year
2026: 14 · 2025: 12 · 2024: 18 · 2023: 19 · 2022: 14 · 2021: 19 · 2020: 17 · 2019: 20 · 2018: 15 · 2017: 18 · 2016: 14 · 2015: 5
FWCI
13.51
Type
journal article
Language
en
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Online Publication Service of Würzburg University (Würzburg University) (submittedVersion, cc-by)
  • Newcastle University ePrints (Newcastle Univesity)
Funders
  • Alexander von Humboldt-Stiftung
  • Wellcome Trust (098051, 089875/Z/09/A)
  • Medical Research Council (098051, G0800170)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Clostridium difficile and Clostridium perfringens research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Health Sciences › Medicine › Infectious Diseases › Antimicrobial Resistance in Staphylococcus
Keywords
Clostridium difficile, gene essentiality, bacterial sporulation, transposon mutagenesis, TraDIS, transposon mutant library, antibiotic-associated diarrhea, sporulation, therapeutic target, bacterial virulence
MeSH terms
DNA Transposable Elements, Genes, Bacterial, Genetic Testing, Humans, Spores, Bacterial, Mutagenesis, Insertional, Clostridioides difficile, Genes, Essential, High-Throughput Screening Assays

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