Glutathione biosynthesis in the yeast pathogens Candida glabrata and Candida albicans: essential in C. glabrata, and essential for virulence in C. albicans

Abstract

Redox pathways play a key role in pathogenesis. Glutathione, a central molecule in redox homeostasis in yeasts, is an essential metabolite, but its requirements can be met either from endogenous biosynthesis or from the extracellular milieu. In this report we have examined the importance of glutathione biosynthesis in two major human opportunistic fungal pathogens, Candida albicans and Candida glabrata. As the genome sequence of C. glabrata had suggested the absence of glutathione transporters, we initially investigated exogenous glutathione utilization in C. glabrata by disruption of the MET15 gene, involved in methionine biosynthesis. We observed an organic sulphur auxotrophy in a C. glabrata met15Δ strain; however, unlike its Saccharomyces cerevisiae counterpart, the C. glabrata met15Δ strain was unable to grow on exogenous glutathione. This inability to grow on exogenous glutathione was demonstrated to be due to the lack of a functional glutathione transporter, despite the presence of a functional glutathione degradation machinery (the Dug pathway). In the absence of the ability to obtain glutathione from the extracellular medium, we examined and could demonstrate that γ-glutamyl cysteine synthase, the first enzyme of glutathione biosynthesis, was essential in C. glabrata. Further, although γ-glutamyl cysteine synthase has been reported to be non-essential in C. albicans, we report here for what is believed to be the first time that the enzyme is required for survival in human macrophages in vitro, as well as for virulence in a murine model of disseminated candidiasis. The essentiality of γ-glutamyl cysteine synthase in C. glabrata, and its essentiality for virulence in C. albicans, make the enzyme a strong candidate for antifungal development.

CgCYN1, a Plasma Membrane Cystine-specific Transporter of Candida glabrata with Orthologues Prevalent among Pathogenic Yeast and Fungi

“…3E). Cystathionine is also present at low levels in blood plasma (3). Not surprisingly, we observed that cystathionine could also be used efficiently as a sulfur source in this yeast.…”

“…Cellular Localization of HA-tagged CgCYN1-For the localization studies Cgmet15⌬CYN1⌬ was transformed with the 3 The abbreviations used are: ARC, American Radiolabeled Chemicals; PGK, phosphoglycerate kinase; TEF, translation elongation factor. HA-tagged CgCYN1, and the transformants were used for the indirect immunofluorescence as described earlier (16).…”

“…Source in C. glabrata-C. glabrata met15⌬ strains, like S. cerevisiae met15⌬ strains, are organic sulfur auxotrophs and have been previously shown to utilize both cysteine and methionine as a sulfur source suggesting the existence of both forward and reverse transsulfuration pathways in C. glabrata (3). We used this C. glabrata met15⌬ strain to test for the growth on cystine.…”

“…Sulfur is one of the essential requirements being a part of two important amino acids cysteine and methionine, as well as several other essential sulfur-containing compounds including glutathione and coenzyme A. Yeast pathogens must fulfill this requirement of sulfur by retrieving them from their human host. C. glabrata, like many yeast, can use both inorganic and organic forms of sulfur and have been demonstrated to have both forward and reverse transsulfuration pathways for the conversion of cysteine to methionine and also from methionine to cysteine (3). Although the sulfur assimilatory pathways were found to be very similar to that of S. cerevisiae, one important difference was found in the utilization of glutathione where C. glabrata was found to lack the ability to transport glutathione because of the absence of a high affinity glutathione transporter thus relying solely on the endogenous biosynthesis of glutathione for its survival (3).…”

“…C. glabrata, like many yeast, can use both inorganic and organic forms of sulfur and have been demonstrated to have both forward and reverse transsulfuration pathways for the conversion of cysteine to methionine and also from methionine to cysteine (3). Although the sulfur assimilatory pathways were found to be very similar to that of S. cerevisiae, one important difference was found in the utilization of glutathione where C. glabrata was found to lack the ability to transport glutathione because of the absence of a high affinity glutathione transporter thus relying solely on the endogenous biosynthesis of glutathione for its survival (3). Interestingly, when one looks at the presence of sulfur compounds in the human blood plasma, glutathione is found in negligible amounts, whereas the most predominant organic sulfur form found in blood plasma is cystine (the oxidized form of cysteine) (4).…”