Article Figures & Data
Figures
- FIG 1
C. difficile alters gene transcription in response to CP treatment and infection. (A) Combined RNA-seq and Nanostring results showing global changes in C. difficile gene expression in response to CP in medium and significantly different changes in gene expression during infection compared to that in medium, respectively. (i and ii) Sense and antisense genes, respectively, in C. difficile strain R20291. (iii and iv) Each bar represents a gene significantly upregulated or downregulated, respectively, in medium containing calprotectin compared to that in medium alone. Each level of color gradation represents a 2.5-fold change. Genes with expression > or <7.5-fold change are represented with bars that hit the maximum limit. (v) Green bars show phage genes present in RNA-seq. (vi and vii) Each bar represents genes from Nanostring panel with significantly upregulated or downregulated expression, respectively, during infection relative to expression in medium. (B) Genes identified in the RNA-seq analysis with significantly different expression grouped into predicted functional categories. (C) Log2 fold change of genes from the Nanostring panel significantly upregulated or downregulated during infection compared to expression in medium. #, genes similarly upregulated or downregulated in RNA-seq and Nanostring panel; ^, gene with expression found to be significantly upregulated according to RNA-seq in response to calprotectin but significantly downregulated according to Nanostring during infection.
- FIG 2
Calprotectin-mediated Zn limitation leads to increased expression of proline fermentation genes. (A) Relative prdA expression normalized to rpoB via qPCR in TY medium with or without 30 mM l-proline, 42 μM or 150 μM ZnCl2, 42 μM MnCl2, calprotectin site I mutant (CP ΔSI), or site I and site II (CP ΔSI/SII) mutant. (B) Relative prdA expression normalized to rpoB via qPCR in TY medium with or without 30 mM l-proline, 75 μM TPEN, or 75 μM ZnCl2. *, P < 0.05 one-way ANOVA followed by Tukey’s multiple-comparison test; ns, not significant.
- FIG 3
Proline fermentation contributes to C. difficile growth in vitro. (A) Genes involved in proline fermentation. Dark triangles indicate location of intron insertion using ClosTron mutagenesis. Arrow indicates the location of the selenocysteine codon in the gene of the main catalytic subunit for the proline reductase, prdB. (B) Growth of WT, prdB::CT, and prdR::CT strains in TY medium containing 30 mM l-proline as measured by OD600. (C) 5-Aminovalerate concentrations in spent media of WT C. difficile, prdB::CT, and prdR::CT strains. Error bars indicate standard deviations. nd, not detected; *, P < 0.05 between WT and prdB::CT at indicated time point; **, P < 0.05 between WT and prdR::CT at indicated time point by two-way ANOVA and Dunnett’s multiple comparisons.
- FIG 4
Proline fermentation contributes to C. difficile growth during primary and relapse infection. (A) Model of antibiotic-induced CDI and relapse. (B and C) C. difficile abundances in the feces of mice coinfected with WT C. difficile and the prdB::CT or prdR::CT mutant, respectively. Each dot represents CFU from an individual mouse. Shaded boxes represent the times when mice were provided vancomycin to induce relapse. Bars represent geometric means. (D and E) The competitive indices (CIs) (input/output ratios) of WT to mutant C. difficile in mice included in data shown in panels B and C. Each dot represents the CI for an individual mouse. Bars represent the geometric means. (F) 5-Aminovalerate concentrations in the feces of naive mice, mice provided cefoperazone treatment only, or mice provided cefoperazone treatment and subsequently infected with either WT C. difficile or the prdB::CT mutant. *, P < 0.05 by unpaired t test.
- FIG 5
Se and Zn limitation decrease C. difficile proline fermentation. (A) 5-Aminovalerate concentrations in spent medium of C. difficile grown in the presence of 75 μM TPEN. TY was supplemented with l-proline (30 mM final concentration). Each dot represents a single replicate. Bacteria were grown to early exponential phase (OD600 of ∼0.2) (B) The intracellular NAD+/NADH ratios of WT or prdB::CT C. difficile grown in TY medium with or without 75 μM TPEN or 75 μM ZnCl2. (C) Intracellular selenium abundance in C. difficile grown in TY medium with or without 75 μM TPEN or 10 μM Na2SeO3 determined through ICP-MS. (D) 5-Aminovalerate concentrations in spent medium from C. difficile grown in TY medium with or without 75 μM TPEN, 10 μM Na2SeO3, or 75 μM ZnCl2 at the indicated time points. Error bars indicate standard deviations. (E) PrdB peptide (TAVIVQR) abundance relative to abundance of an SlpA S-layer peptide (LYNLVNTQLDK). The PrdB peptide is downstream of the selenocysteine residue. Data represent 2 to 3 replicates. Error bars indicate standard deviations. *, P < 0.05 by unpaired t test.
- FIG 6
Calprotectin restricts C. difficile proline fermentation during infection. (A) Abundance of Zn and Se in the feces of WT C57BL/6 or S100A9−/− mice infected with WT C. difficile. Box plots represent the median, maximum, and minimum values; N = 8 to 10 mice per time point per genotype. (B) C. difficile abundance in coinfected mice at day 2 postinfection. Each dot represents CFU from a single mouse. *, P < 0.05, ln-transformed data. (C) The competitive indices of WT C57BL/6 and S100A9−/− mice coinfected with WT and prdB::CT C. difficile included in the data shown in panel B. *, P < 0.05 by unpaired t test; error bars represent the standard errors of the means.
Tables
- TABLE 1
Bacterial strains and plasmids used in this study
- TABLE 2
Oligonucleotide sequences used in this study
↵a Boldface font indicates region in primer with sequence similarity to C. difficile.
TABLE S1
C. difficile genes with significantly different expression when treated with calprotectin. Results from RNA sequencing comparing C. difficile gene expression of bacteria treated with 0.35 mg/ml CP compared to that in medium only. Download Table S1, XLSX file, 0.1 MB.
Copyright © 2019 Lopez et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
Target genes and sequences from Nanostring panel. Download Table S2, XLSX file, 0.1 MB.
Copyright © 2019 Lopez et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S1
Iron chelation does not increase prdA transcription. Relative prdA expression normalized to that of rpoB via qPCR in TY medium with or without 30 mM l-proline or 50 μM 2,2-dipyridyl. *, P < 0.05 by unpaired t test. Download FIG S1, TIF file, 0.7 MB.
Copyright © 2019 Lopez et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
TPEN treatment and Se supplementation do not change PrdR abundance. PrdR peptide (NIGILPVLR) abundance relative to abundance of an SlpA S-layer peptide (LYNLVNTQLDK). Data presented on similar scale as shown in Fig. 5E. Data represent 2 to 3 replicates. Download FIG S2, TIF file, 0.6 MB.
Copyright © 2019 Lopez et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
