Selenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy metabolism. Following this initial characterization, we demonstrate that this pathway is linked to production of toxins by this organism. Finally, we show that interruption of selenium metabolism is a viable pathway for development of antimicrobials against this, and other selenoprotein dependent pathogens. We investigated whether Stickland reactions (paired amino acid fermentation) might be at the heart of C. difficile's bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating 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 and D-proline reductase. These selenoenzymes were expressed upon 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. D-proline reductase utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. The enzyme was found to be independent of divalent cations, and zinc was a potent inhibitor. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of D-proline reductase may differ significantly from similar enzymes from non-pathogenic species. C. difficile pathogenesis is due to the production of toxins, A and B, members of the large clostridial cytotoxin family. Previous studies have shown that toxin production by this organism is influenced by the composition of the growth medium. We examined the impact of Stickland acceptor amino acids (Stickland acceptors; glycine, proline and hydroxyproline) on growth kinetics and yield, protein synthesis, toxin production and gene expression. Although addition of Stickland acceptors moderately increases growth yield and total protein synthesis, there does not appear to be a clear impact on entry into stationary phase. Glycine dramatically increases the amount of toxin released into the growth medium. Conversely, the addition of hydroxyproline suppresses toxin production. We examine possible mechanisms of regulation and demonstrate that CodY, a regulator of toxin gene transcription does not appear to mediate this effect. Given the importance of selenium dependent Stickland reactions to C. difficile growth and toxin production we aimed to examine the efficacy of blocking such pathways as a means of antimicrobial development. Selenide is the only known substrate for selenophosphate synthetase, the first enzyme involved in the specific incorporation of selenium into selenoproteins. We have identified a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. The antimicrobial potential of blocking selenium metabolism is further demonstrated in the dental pathogen Treponema denticola. We show that auranofin blocks the growth this organism which also participates in Stickland fermentation. In addition, we provide evidence that the antimicrobial action of stannous salts against T. denticola is also mediated through inhibition of the metabolism of selenium. These studies clearly show that, at least in a subset of microbes that use selenium for the synthesis of selenoproteins, the need for this metalloid can be a useful target for future antimicrobial development.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-4822 |
| Date | 01 January 2009 |
| Creators | Rosario, Sarah |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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