Role of Glutathione Metabolism of Treponema denticola in Bacterial Growth and Virulence Expression

Abstract

Hydrogen sulfide (H 2 S) is a major metabolic end product detected in deep periodontal pockets that is produced by resident periodontopathic microbiota associated with the progression of periodontitis. Treponema denticola, a member of the subgingival biofilm at disease sites, produces cystalysin, an enzyme that catabolizes cysteine, releasing H 2 S. The metabolic pathway leading to H 2 S formation in periodontal pockets has not been determined. We used a variety of thiol compounds as substrates for T. denticola to produce H 2 S. Our results indicate that glutathione, a readily available thiol source in periodontal pockets, is a suitable substrate for H 2 S production by this microorganism. In addition to H 2 S, glutamate, glycine, ammonia, and pyruvate were metabolic end products of metabolism of glutathione. Cysteinyl glycine (Cys-Gly) was also catabolized by the bacteria, yielding glycine, H 2 S, ammonia, and pyruvate. However, purified cystalysin could not catalyze glutathione and Cys-Gly degradation in vitro. Moreover, the enzymatic activity(ies) in T. denticola responsible for glutathione breakdown was inactivated by trypsin or proteinase K, by heating (56°C) and freezing (؊20°C), by sonication, and by exposure to N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK). These treatments had no effect on degradation of cysteine by the purified enzyme. In this study we delineated an enzymatic pathway for glutathione metabolism in the oral spirochete T. denticola; our results suggest that glutathione metabolism plays a role in bacterial nutrition and potential virulence expression.Treponema denticola is a predominant cultivable spirochete found in the gingival crevice and has been implicated in the development of the subgingival ecology of periodontal pockets (18,33,41,43). A number of studies have shown that there is a relationship between the emergence of oral treponemes and the transition from health to periodontitis (21,30,39,47). It has also been proposed that T. denticola belongs to Socransky's Red complex, which may be related to biofilm virulence (21). While it has been shown in vitro that T. denticola produces multiple potential virulence factors (16,17,20,25,26,35,44), the exact role or activity of these factors in the in vivo environment remains to be determined. One of these factors, the production of volatile sulfur compounds, including hydrogen sulfide (H 2 S), could contribute to pathogenic changes in the host tissues (16). High levels of H 2 S (up to 2 mM) have been detected in infected periodontal pockets, while low levels have been detected in clinically healthy sites (22,34,36,42). In vitro, H 2 S has been shown to be cytotoxic for a variety of host cells, including gingival fibroblasts and epithelial cells (4, 10, 40, 48).Several reports have described H 2 S formation from metabolism of human serum proteins, cysteine, and glutathione by oral bacteria (6,37,38). Despite these observations, the metabolic pathways leading to H 2 S production from glutathione by oral bacteria have not been delineated. P...

Virulence Factors of the Oral Spirochete Treponema denticola

“…T. denticola is the only known oral bacterium to contain the three-step pathway required to produce H 2 S, from the abundant host tripeptide, glutathione (Chu et al, 2008). This pathway contains cysteinylglycinase, γ-glutamyltransferase, and the novel enzyme, cystalysin (Makinen and Makinen, 1997;Chu et al, 2002). H 2 S production via cystalysin has been shown to be the mechanism by which T. denticola disrupts erythrocyte membranes, thereby explaining its original description as a hemolysin (Chu et al, 1995).…”

Gene Regulation, Two Component Regulatory Systems, and Adaptive Responses in Treponema Denticola

“…Important to both metabolism and virulence, T. denticola strains encode an array of 18-25 proteases. Two proteases that have been experimentally characterized in some detail are dentilisin (Chi et al 2003;Ellen et al 2000;Ishihara et al 1998;McDowell et al 2009;Miller et al 2016;Yamazaki et al 2006) and cystalysin (Chu et al 2002(Chu et al , 2008Krupka et al 2000;Zhang et al 2010). Both play important roles in T. denticola pathogenesis.…”

Role for Recombinant γ-Glutamyltransferase from Treponema denticola in Glutathione Metabolism

“…A recent study examined glutathione metabolism in T. denticola (9). Based on these data, a pathway of three steps was proposed.…”

“…The last is then cleaved into glycine and L-cysteine. Finally, Lcysteine is degraded, releasing pyruvate, ammonia, and H 2 S. Key enzymes involved in glutathione metabolism may include ␥-glutamyltransferase (GGT), cysteinylglycinase, and L-cysteine desulfhydrase (cystalysin) (9,11,20). In previous studies, the cystalysin from T. denticola has been cloned (8,11).…”

“…Thiol sources for H 2 S production in vivo can be multiple, including serum proteins, L-cysteine, and glutathione (8,9,11,30). Initial research focused mainly on L-cysteine (8,11).…”