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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 ?M) 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 ?M) 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.

SUBMITTER: Berges M

PROVIDER: S-EPMC6311696 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Project description: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 (760 g/l) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron transporters were found induced by Fur regulation during low iron (11 g/l) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were found significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some initial intermediates (phenylpyruvate, oxo-isocaproate, 3-hydroxy-butanoyl-CoA, crotonyl-CoA) were found accumulated, while some end product like isocaproate and butanoate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were found 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 found induced while the formation of a V-type, mostly proton-pumping, ATP-consuming 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. An intensive remodeling of the cell wall was observed most likely to increase antibiotic resistance. Polyamine biosynthesis (spe) was found induced leading to an accumulation of spermine, spermidine and putrescine. The fur mutant lost most of its flagella. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, significant decrease in metabolic iron utilization and protection during the complex transition.

Project description: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 (760 g/l) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron transporters were found induced by Fur regulation during low iron (11 g/l) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were found significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some initial intermediates (phenylpyruvate, oxo-isocaproate, 3-hydroxy-butanoyl-CoA, crotonyl-CoA) were found accumulated, while some end product like isocaproate and butanoate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were found 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 found induced while the formation of a V-type, mostly proton-pumping, ATP-consuming 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. An intensive remodeling of the cell wall was observed most likely to increase antibiotic resistance. Polyamine biosynthesis (spe) was found induced leading to an accumulation of spermine, spermidine and putrescine. The fur mutant lost most of its flagella. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, significant decrease in metabolic iron utilization and protection during the complex transition.

Project description:Clostridiodes difficile (C. difficile) was ranked an "urgent threat" by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the "hypervirulent" strain, R20291, make it difficult to assign a "one size fits all" mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

Project description:The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits.IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.

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Iron Regulation in Clostridioides difficile.

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