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.

Figure 1. Metabolic map of prdR regulated genes.

Conversion of glucose to pyruvate (glycolysis) and oxidation of amino acids to carboxylic acids (Stickland metabolism) generate NADH. Proline reductase (1), glycine reductase (2) and the succinate-acetyl CoA pathway to butyrate (3, 4) generate NAD⁺. Solid lines indicate single enzyme steps, whereas dotted lines indicate multienzyme steps in the pathways. Specific pathways and genes that encode the relevant enzymes are denoted as follows: (1) D-proline reductase (CD3236–3244, prd); (2) glycine reductase (CD2348-CD2358, grd); (3) succinate reduction (CD2344–CD2338); (4) conversion of acetyl CoA to butyryl CoA (CD1054–CD1059); (5) ethanolamine utilization (CD1906-CD1925, eut); and (6) alcohol dehydrogenase (CD2966, adhE). In the prdR mutant, the prd genes were underexpressed, but the other indicated pathways were all overexpressed at least 5-fold compared to the wild-type.

Figure 2. Effects of a rex mutation on expression of NAD⁺-regeneration genes.

Expression of the prdA (A), CD2344 (B), bcd2 (C), and grdE (D) genes was measured by qRT-PCR in wild- type, rex mutant and complemented rex mutant strains. Cells were grown in TY medium with or without supplementation with 30 mM L-proline to mid-exponential phase as described under Experimental procedures. The means and standard error of the means of at least three biological replicates are shown.

Figure 3. Gel mobility shift assays of Rex binding to the upstream regions of potential target genes.

DNA samples corresponding to upstream regions of potential Rex target genes were created by PCR using radioactive oligonucleotides listed in Table S4. For each potential target, the radioactive DNA was incubated with varying concentrations of purified, His-tagged Rex protein with or without the addition of NAD⁺. The samples were then subjected to electrophoresis in non-denaturing polyacrylamide gels and analyzed by autoradiography.

Figure 4. Impact of NADH/NAD⁺ ratios on binding of Rex to target sites for the adhE. grdE and CD2344 genes.

As described in Figure 3, radioactive DNAs carrying the adhE, grdE and CD2344 Rex-binding sites were incubated with Rex protein in the absence or presence of 10 mM NAD⁺ and at varying concentrations of NADH.

Figure 5. Impact of varying concentrations of NAD⁺ on binding of Rex to target sites for the adhE, grdE, bcd2 and CD2344 genes.

As described in Figure 3, radioactive DNAs carrying the adhE, grdE, bcd2 and CD2344 Rex-binding sites were incubated with Rex protein in the presence of varying concentrations of NAD⁺ with and without 1 mM NADH.

Figure 6. DNase I footprinting of the Rex binding site upstream of the CD2344 coding region.

(A) Binding of Rex to the CD2344 regulatory region as detected by a footprinting assay. Radiolabelled DNA fragments were incubated with increasing amounts of purified Rex with 10 mM NAD⁺ (left panel), or with a mixture of 10 mM NAD⁺ and 1 mM NADH (middle panel) or no effectors (right panel). Rex concentrations used (nM) are indicated above each lane. The black bars delineate the regions of protection corresponding to Rex binding sites (BS-1, BS-2 and BS-3). (B) Sequence of the CD2344 regulatory region. Two sites of transcription initiation as determined by 5’-RACE are indicated in bold below the broken arrow. The CodY-protected area (Dineen et al., 2010) is in bold. Potential −10 and −35 regions of the promoter are indicated. The sequences protected by Rex in DNase I footprinting experiments located using a G/A ladder (see Experimental procedures) are underlined and predicted binding sites are italicized.

Figure 7. Impact of deletion of the apparent Rex binding site on Rex binding in vitro.

Radioactive DNA samples carrying the apparent wild-type Rex-binding site upstream of the bcd2 coding region or a deletion in the apparent site were tested for binding by purified Rex protein in vitro.

Figure 8. Alignment of Rex binding motifs.

(A) To search for a consensus Rex binding site, the protected regions of the CD2344, grd and bcd2 genes were aligned and compared to the consensus proposed by Ravcheev et al. (Ravcheev et al., 2012). The location of the apparent binding motif is presented with respect to the start codon of the gene in question. (B) A motif logo based on the six C. difficile Rex-binding sites was generated using the MEME algorithm (http://meme.nbcr.net/meme/).

Figure 9. Impact of Rex and PrdR on virulence.

(A) Effects of a rex mutation on toxin gene expression. Expression of the tcdA gene was measured by qRT-PCR in strains JIR8094 (WT), LB-CD24 (rex mutant) and LB-CD8 (prdR mutant). Cells were grown in CDMM for 24 h. The amount of tcdA mRNA was normalized to that of the internal control transcript rpoA. The means and standard error of the means of at least three biological replicates are shown. (B) Virulence of rex and prdR mutants in the hamster model of CDI. Kaplan-Meier survival curves of clindamycin-treated Syrian hamsters inoculated with 1,000 spores of C. difficile JIR8094 (wild-type, black line) or the rex mutant (dotted line) or the prdR mutant (dashed line). Uninfected control animals were treated with clindamycin, but did not receive any C. difficile. Animals showing signs of C. difficile infection (wet tail, poor fur coat, lethargy) were euthanized.

Figure 10. Model for regulation of NAD⁺-regenerating pathways.

Proline reductase (PR) is proposed to be the preferred pathway for conversion of NADH to NAD⁺. When proline is present in an amount sufficient to activate PrdR, PR is expressed and produces enough NAD⁺ to activate Rex as a repressor of alternative NAD⁺-regenerating pathways. When proline is consumed, Rex loses DNA-binding activity and the alternative pathways are derepressed.