Analysis of proline reduction in the nosocomial pathogen Clostridium difficile

Abstract

Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and d-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme d-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only d-proline but not l-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.

FIG. 1.

Overview of Stickland reactions. Based on previous studies with C. sticklandii, E. acidaminophilum, and C. sporogenes (1, 7, 42, 43, 46), a schematic overview is presented on the coupled oxidation and reduction of pairs of amino acids (Stickland reactions). In addition to oxidation of amino acids, Stickland reactions may also couple to the oxidation of purines and sugars, based on early work by Barker (2). The thioredoxin (Trx) and thioredoxin reductase (TrxR) systems have been suggested by Andreesen et al. to be directly linked to glycine reductase based on the colocalization of genes encoding Trx and TrxR with components of the glycine reductase in several organisms (1). The means by which reducing potential couples to d-proline reductase to produce proton motive force is unknown, although proline-dependent production of ΔpH has been demonstrated in C. sporogenes (29, 30).

FIG. 2.

Growth of C. difficile requires selenium when cultured with Stickland pairs. Optical densities of cultures were measured at 22 h after inoculation (using a 1% inoculum), which was determined to be the peak density in growth curve studies (data not shown). Cultures were incubated at 37°C overnight in a 95% nitrogen-5% hydrogen atmosphere in a Coy anaerobic chamber. Selenium, when added, was in the form of selenite at a concentration of 1 μM. Torula yeast extract was used in the basal medium to reduce trace selenium normally present in TYPG medium (see Materials and Methods for details on TTYP medium composition). P, d-proline; G, glycine; A, alanine. The mean optical density from at least three individual cultures is plotted with the standard deviation plotted as error, and the experiment was reproduced at least three times.

FIG. 3.

Identification of putative genes encoding glycine reductase and d-proline reductase in C. difficile 630. The sequence data used for annotation were produced by the Pathogen Sequencing Group at the Wellcome Trust Sanger Centre and were obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/cd. A schematic representation of the genes encoded within two separate regions of the genomic DNA of Clostridium difficile 630, compared directly with similar operons in C. sticklandii, is shown. Numbers indicate the locations of the DNA coding regions within the linear genomic DNA sequence (Sanger Institute). (A) Alignment of the glycine reductase region from C. difficile 630 and C. sticklandii. (17). Primary putative assignment of genes in C. difficile was made by comparison with genes encoded by C. sticklandii (accession no. GI:11065682). The assignment of olgP (oligopeptidase) and proP (Xaa-Pro aminopeptidase) genes was made based on their homology to similar genes in Clostridium perfringens (accession no. GI:18145988) and Clostridium tetani (accession no. NP:782200), respectively. (B) Alignment of the proline reductase region from C. difficile 630 and C. sticklandii. Primary putative assignment of genes in C. difficile was made by comparison with genes encoded by C. sticklandii (accession no. GI:6899992).

FIG. 4.

Radiolabeling (⁷⁵Se) of C. difficile reveals increased proline reductase and glycine reductase upon the addition of glycine, d-proline, or l-4-hydroxyproline. (A) Cultures were grown in TYPG medium (see text) with ⁷⁵Se (10 μCi) for 24 h, harvested, lysed by sonication, and separated by 12% SDS-PAGE. The locations of molecular mass markers are indicated at the left (in kDa). Arrows indicate the selenoprotein present in cell extracts grown under the following conditions: lanes 1 and 5, TYPG alone (C); lanes 2 and 6, TYGP plus d-proline (10 mM) (P); lanes 3 and 7, TYGP plus glycine (10 mM) (G); lanes 4 and 8, TYGP plus d-proline and glycine (10 mM each) (PG). (B) Immunoblot to detect the presence of glycine reductase selenoprotein A (small subunit). After SDS-PAGE, the same extracts from panel A were transferred to a polyvinylidene difluoride membrane and probed with polyclonal antibodies raised against GR selenoprotein A from C. sticklandii (kindly provided by T. C. Stadtman, NHLBI, NIH). (C) l-Proline and l-4-hydroxyproline also induce the production of a selenoprotein in C. difficile (strain 9689). Lane 1, TYPG medium (no addition) (C); lane 2, TYPG plus glycine (10 mM) (G); lane 3, TYPG plus l-proline (10 mM) (P); lane 4, TYPG plus l-4-hydroxyproline (10 mM) (L-Hyp). The predicted selenoproteins are indicated by arrows based on the molecular weights of the genes annotated in Fig. 3.

FIG. 5.

Purification and substrate specificity of C. difficile d-proline reductase. (A) Fractions from sequential steps of purification of d-proline reductase are separated by SDS-PAGE (15%) after being stained with Coomassie blue. Lane 1, molecular mass marker (size in kDa indicated at left); lane 2, crude cell extract; lane 3, 60% ammonium sulfate fraction; lane 4, phenyl-Sepharose; lane 5, EAH Sepharose; lane 6, Sephacryl S-200. Five micrograms of protein was loaded in each lane. The identification of protein subunits was accomplished by Edman degradation (see text for details). PrdA* is a proteolytically cleaved product of the precursor subunit PrdA (21). (B) Substrate specificity of d-proline reductase. Proline racemase (PrdF; Fig. 3) was purified by affinity chromatography (see Materials and Methods) and used to elucidate the substrate specificity of d-proline reductase from C. difficile. Specific activity was measured by assaying for δ-aminovaleric acid production as previously described (41). Proline racemase (1 μg) was preincubated with proline substrate for 30 min (30°C) prior to the addition of d-proline reductase. Each proline substrate was present in the reaction at a concentration of 1 mM. ND indicates no detectable activity. Activity is expressed as nmol min⁻¹ mg⁻¹ protein.

FIG. 6.

Analysis of the requirement for divalent cations for d-proline reductase activity. The requirement for divalent cations for d-proline reductase activity was tested in the presence of several cations: (A) Mg²⁺, (B) Mn²⁺, (C) Ca²⁺, and (D) Zn²⁺. Specific activity is plotted versus cation concentration. The mean of these activities is plotted with the standard deviation shown as error. At least three independent enzyme assays are represented. Activity was determined by following the production of δ-aminovalerate as described in Materials and Methods.