Iron Regulation in Clostridioides difficile

Abstract

The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (15 μM) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron and siderophore transporter genes were induced by Fur during low iron (0.2 μM) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some intermediates (phenylpyruvate, 2-oxo-isocaproate, 3-hydroxy-butyryl-CoA, crotonyl-CoA) accumulated, while end products like isocaproate and butyrate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB) of the F₀F₁-type was induced while the formation of a ATP-consuming, proton-pumping V-type ATPase (atpDBAFCEKI) was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. The fur mutant showed an increased sensitivity to vancomycin and polymyxin B. An intensive remodeling of the cell wall was observed, Polyamine biosynthesis (spe) was induced leading to an accumulation of spermine, spermidine, and putrescine. The fur mutant lost most of its flagella and motility. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.

Keywords: Fur; cell wall; iron regulation; iron transport; metabolism; polyamine.

FIGURE 1

Growth of wild type and fur mutant growth at low and high iron concentration. Growth curves of Clostridioides difficile wild type and the corresponding fur mutant in CDM medium with 15 μM iron sulfate (high iron, black for wild type and green symbols for the fur mutant) or 0.2 μM (low iron, red for wild type and blue symbols for the fur mutant) are shown. Growth was monitored every two in at least five independent cultivations. Arrows indicate time points of sampling for the systems biology (Omics) approaches. Standard deviations are indicated.

FIGURE 2

(A) Principle component analysis (PCA) of transcriptome data from previously published datasets and this study concerning Fur regulated adaptation to low/no iron conditions and sequence logo for the deduced Fur binding site. Data are shown as triplicates recorded after growth in iron rich medium [red, wild type – this study, yellow, same – from (Ho and Ellermeier, 2015), green, same from (Hastie et al., 2018), blue – fur mutant, this study, brown – from (Ho and Ellermeier, 2015)]. (B) Position weight matrices deduced Fur binding site sequence logo is shown on the bottom (see also Supplementary Table S7).

FIGURE 3

Overview of the overall adaptation strategies of C. difficile to low iron conditions. Transcriptome (RNA-Seq), proteome and metabolome data were integrated into a general adaptation strategy model. Shown are enzymes (bold) and metabolites (non-bold), generally upregulated pathways are shown in black, while downregulated pathways are labeled in gray. The changes in abundance of the corresponding mRNAs, proteins and metabolites between low iron and high iron and/or a comparison between the fur mutant and the wild type strain are indicated in the following code: Transcriptome (RNA–Seq) data are shown as squares, proteome data as circles, metabolome data as triangles (cytoplasmic metabolome, peak up, exo-metabolome, peak down). Green indicates higher abundance and red indicates a reduction of the cellular abundances of the corresponding molecules. Filled symbols indicate the same effect in both conditions (high versus low iron and wild type versus fur mutant), open symbols indicate the effect in only one condition, blue symbols indicate contrary effects. Cut off values were a log₂ fold change of 2 for transcriptome and proteome and a fold change of 1.5 for metabolic data. Iron-dependent reactions are labeled by brown circles and letters (Fd, ferredoxin; FeS, iron sulfur clusters, Fe²⁺). Fe-ABC, YclNOPQ; OH, hydroxy-group; CoA, coenzyme A; Me, methyl group. For details, see Table 1, and the Supplementary Tables.

FIGURE 4

Scanning/transmission electron microscopic images and motility assays of C. difficile wild type and the corresponding fur mutant. (A) CDMM agar filled glass tubes containing 0.2 μM (-Fe) and 15 μM (+Fe) iron sulfate were inoculated with wild type (wt) and the corresponding fur mutant strain and incubated anaerobically for 24 h. Scanning electron microscopy picture of C. difficile (B, right panel) and the fur (mutant B, left panel) grown in CDMM containing 0.2 μM (-Fe) and 15 μM (+Fe) iron sulfate are shown. Negative staining (C) also depicts less flagellation of the fur mutant and no detectable other appendage-like structures on the surface of C. difficile like pili or fimbriae. Bars represent 2 μm in (B), top 2 images and 1 μm in all other images.

FIGURE 5

Growth of wild type and fur mutant growth at high iron concentration in the presence of vancomycin and polymyxin B. Growth curves of C. difficile wild type and the corresponding fur mutant in CDM medium with 15 μM iron sulfate, without (A) and with the addition of 0.3 mg vancomycin/l (B) and 150 mg polymyxin B/l (C) are shown. Black symbols are used for the wild type and green symbols for the fur mutant. Growth was monitored every 2 h in at least five independent cultivations. Standard deviations are indicate.