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.

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity

“…12 Furthermore, it was shown that Hyp can induce the expression of an enzyme responsible for Pro reduction in C. difficile . 13 In contrast to the characterized pathway for Pro reduction (Fig. 2A), the enzymes and downstream metabolites involved in Hyp fermentation had not been identified until our work.…”

“…The presence of this pathway in the order Clostridiales was notable given that anaerobic metabolism of Hyp was first observed in Clostridiales. 13 These organisms carry out Stickland fermentation wherein pairs of amino acids are used as electron donors and acceptors in energy metabolism. 12 It was known as early as 1934 that Pro and Hyp can be used as electron acceptors in such fermentations.…”

“…Indeed, HypD is present in species previously demonstrated to use Hyp and Pro as electron acceptors ( C. difficile, Clostridium sporogenes ). 12 , 13 Notably, ∼65% of the Clostridiales species that possess the prd operon, which is essential for Pro reduction, also encode HypD. 13 , 14 In these species, the conserved proC gene (encoding P5CR) adjacent to hypD provides a strong indication that Hyp is metabolized to Pro and used as an electron acceptor.…”

“…12 , 13 Notably, ∼65% of the Clostridiales species that possess the prd operon, which is essential for Pro reduction, also encode HypD. 13 , 14 In these species, the conserved proC gene (encoding P5CR) adjacent to hypD provides a strong indication that Hyp is metabolized to Pro and used as an electron acceptor. In Stickland fermentation, Pro is metabolized via a 2-step pathway to 5-aminovalerate, which often accumulates as an end product (Fig.…”

“…For example, C. difficile can utilize both Hyp and Pro as electron acceptors. 13 Although the regulation of toxin production in C. difficile is not well understood, fermentation of Pro and other amino acids are thought to indirectly influence this process by modulating NADH/NAD + ratios in the cell. 52 Since metabolism of Hyp to Pro generates NAD + , this transformation may also affect virulence in C. difficile .…”