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.
Highlights
Clostridioides difficile is a sporeforming, Gram-positive, anaerobic, toxins-producing pathogen leading to often hospital-acquired infections worldwide (Burke and Lamont, 2014)
The growth behavior of the wild type and the constructed fur mutant in logarithmic growth phase was almost identical when tested in the complex Brain-Heart-Infusion (BHI) medium independent of the addition of iron (Supplementary Figure S2)
Similar observations have been made for the fur mutant grown in complex TY medium before (Ho and Ellermeier, 2015)
Summary
Clostridioides difficile (formerly Clostridium difficile) is a sporeforming, Gram-positive, anaerobic, toxins-producing pathogen leading to often hospital-acquired infections worldwide (Burke and Lamont, 2014). Ferric reductases are excreted (Schroder et al, 2003) In pathogenic bacteria these iron-uptake mechanisms acquire iron directly from host proteins, including the iron-binding glycoproteins transferrin in serum and extracellular fluid, lactoferrin in mucosal secretions, and heme-containing proteins such as hemoglobin, haptoglobin, and hemopexin (Symeonidis, 2012). Bacteria have evolved various mechanisms to control iron homeostasis They carefully adjust their iron uptake and utilization strategies at the transcriptional level (Troxell and Hassan, 2013; Porcheron and Dozois, 2015). The ferric uptake regulator (Fur) protein is a transcriptional repressor of genes in iron uptake and utilization (Troxell and Hassan, 2013; Fillat, 2014; Porcheron and Dozois, 2015). Under iron starvation conditions, the Fur protein is inactive, which allows for the expression of Fur-regulated genes
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