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.

An RNA-centric global view ofClostridioides difficilereveals broad activity of Hfq in a clinically important Gram-positive bacterium

“…One such example is the Fur-regulated sRNA FsrA in B. subtilis, that regulates mRNA targets involved in iron transport and metabolism (Gaballa et al, 2008;Pi and Helmann, 2017) in a very similar way to RyhB in Enterobacteriaceae (Masse and Gottesman, 2002;Masse et al, 2005). The genome of C. difficile 630 lacks any sequence conservation to FsrA but the transcriptomic response of a C. difficile 630 Fur mutant involves the down-regulation of similar genes and pathways as in B. subtilis (Berges et al, 2018). Therefore, future analyses of transcriptional responses to iron starvation and other defined stress conditions might reveal novel sRNAs in C. difficile that have functionally related members in other bacteria.…”

TheClostridioides difficilespecies problem: global phylogenomic analysis uncovers three ancient, toxigenic, genomospecies

“…The C-III metal transport gene cluster encoded a chelator of heavy metal ions and a multi-component transport system with specificity for iron, nickel and glutathione. The conserved spermidine operon found in all C. difficile clades is thought to play an important role in various stress responses including during iron limitation 51 . The additional, divergent spermidine transporters found in C-III were similar to regions in closely related genera Romboutsia and Paeniclostridium (data not shown).…”

The battle for iron in enteric infections

“…Clostridium difficile also significantly changes the composition of its cell wall in response to iron deficiency, presumably to protect itself from other microorganisms, antibiotics or host immune responses. 91 Moreover, iron depletion revealed significant up-regulation of C. difficile genes associated with virulence, including polyamine and histidine biosynthesis and uptake, as well as several flagella-associated genes, which represent well-known factors involved in adherence. 92 The diverse changes in gene expression mediated by Fur underpin how sensing and responding to iron availability is synonymous with sensing the diverse environments to which a pathogen is exposed as it moves through the host.…”

“…From these, the operon feo1 was consistently shown to be highly induced under iron-depleted conditions. [90][91][92] In addition to Feo systems, other Fur-regulated putative iron transporters might also import free Fe 2+ from the external milieu, such as proteins similar to P-type cation transporters, and the lowaffinity zinc transporter ZupT. 90,91 Direct and siderophore mediated uptake of Tf-and Lf-bound Fe 3+…”

“…134 The genome of C. difficile encodes several ferritin-, ferric-, and siderophore-bound iron uptake systems, which are almost exclusively Fur-regulated and highly responsive to iron fluctuations. 91,135 Previously, it was shown that the catecholate's precursor spermidine biosynthetic-and transport-coding genes (speAHEB and potABCD, respectively), as well as the catecholate siderophore import system YclNOPQ, are Fur-regulated and highly induced in C. difficile grown under iron-limiting conditions. 91,136 Moreover, iron deprivation also induced expression of putative ferric hydroxamate uptake and sulphonate ABC transporter systems, which might facilitate iron uptake during iron-poor conditions.…”