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Role of the global regulator Rex in control of NAD+ -regeneration in Clostridioides (Clostridium) difficile.

ABSTRACT: For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the glycine reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile.

SUBMITTER: Bouillaut L

PROVIDER: S-EPMC6561804 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Role of the global regulator Rex in control of NAD<sup>+</sup> -regeneration in Clostridioides (Clostridium) difficile.

Bouillaut Laurent L Dubois Thomas T Francis Michael B MB Daou Nadine N Monot Marc M Sorg Joseph A JA Sonenshein Abraham L AL Dupuy Bruno B

Molecular microbiology 20190402 6

For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline reductase (P ...[more]

PMID: 30882947

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Project description:Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent pathways encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinic acid or the late intermediate cobinamide (Cbi) and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in the btuFCD loci of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile.IMPORTANCE The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, although it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo but produces them when given cobamide precursors. A better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.

Project description:UnlabelledClostridium difficile is an anaerobic, Gram-positive, spore-forming opportunistic pathogen and is the most common cause of hospital-acquired infectious diarrhea. Although iron acquisition in the host is a key to survival of bacterial pathogens, high levels of intracellular iron can increase oxidative damage. Therefore, expression of iron acquisition mechanisms is tightly controlled by transcriptional regulators. We identified a C. difficile homologue of the master bacterial iron regulator Fur. Using targetron mutagenesis, we generated a fur insertion mutant of C. difficile. To identify the genes regulated by Fur in C. difficile, we used microarray analysis to compare transcriptional differences between the fur mutant and the wild type when grown in high-iron medium. The fur mutant had increased expression of greater than 70 transcriptional units. Using quantitative reverse transcriptase PCR (qRT-PCR), we analyzed several of the Fur-regulated genes identified by the microarray and verified that they are both iron and Fur regulated in C. difficile. Among those Fur- and iron-repressed genes were C. difficile genes encoding 7 putative cation transport systems of different classes. We found that Fur was able to bind the DNA upstream of three Fur-repressed genes in electrophoretic mobility shift assays. We also demonstrate that expression of Fur-regulated putative iron acquisition systems was increased during C. difficile infection using the hamster model. Our data suggest that C. difficile expresses multiple iron transport mechanisms in response iron depletion in vitro and in vivo.ImportanceClostridium difficile is the most common cause of hospital-acquired infectious diarrhea and has been recently classified as an "urgent" antibiotic resistance threat by the CDC. To survive and cause disease, most bacterial pathogens must acquire the essential enzymatic cofactor iron. While import of adequate iron is essential for most bacterial growth, excess intracellular iron can lead to extensive oxidative damage. Thus, bacteria must regulate iron import to maintain iron homeostasis. We demonstrate here that C. difficile regulates expression of several putative iron acquisition systems using the transcriptional regulator Fur. These import mechanisms are induced under iron-limiting conditions in vitro and during C. difficile infection of the host. This suggests that during a C. difficile infection, iron availability is limited in vivo.

Dataset Information

Role of the global regulator Rex in control of NAD+ -regeneration in Clostridioides (Clostridium) difficile.

Publications

Role of the global regulator Rex in control of NAD<sup>+</sup> -regeneration in Clostridioides (Clostridium) difficile.

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