Abstract
The phosphoinositide 3-kinase (PI3K), which phosphorylates phosphatidylinositol and produces PI3P, has been implicated in protein trafficking, intracellular survival, and virulence in the pathogenic yeast Candida glabrata. Here, we demonstrate PI3-kinase (CgVps34) to be essential for maintenance of cellular iron homeostasis. We examine how CgVps34 regulates the fundamental process of iron acquisition, and underscore its function in vesicular trafficking as a central determinant. RNA sequencing analysis revealed iron homeostasis genes to be differentially expressed upon CgVps34 disruption. Consistently, the Cgvps34Δ mutant displayed growth attenuation in low- and high-iron media, increased intracellular iron content, elevated mitochondrial aconitase activity, impaired biofilm formation, and extenuated mouse organ colonization potential. Furthermore, we demonstrate for the first time that C. glabrata cells respond to iron limitation by expressing the iron permease CgFtr1 primarily on the cell membrane, and to iron excess via internalization of the plasma membrane-localized CgFtr1 to the vacuole. Our data show that CgVps34 is essential for the latter process. We also report that macrophage-internalized C. glabrata cells express CgFtr1 on the cell membrane indicative of an iron-restricted macrophage internal milieu, and Cgvps34Δ cells display better survival in iron-enriched medium-cultured macrophages. Overall, our data reveal the centrality of PI3K signaling in iron metabolism and host colonization.
Highlights
The phosphoinositide 3-kinase (PI3K), which phosphorylates phosphatidylinositol and produces phosphatidylinositol 3-phosphate (PI3P), has been implicated in protein trafficking, intracellular survival, and virulence in the pathogenic yeast Candida glabrata
The Cgvps34⌬ Mutant Displays Elevated Intracellular ATP Levels—We have previously shown that deletion of C. glabrata phosphoinositide 3-kinase (CgVps34) results in cell death in human macrophages and highly attenuated virulence in mice (12)
We report an essential role for the phosphoinositide 3-kinase CgVps34 in the maintenance of iron homeostasis
Summary
The Cgvps34⌬ Mutant Displays Elevated Intracellular ATP Levels—We have previously shown that deletion of C. glabrata phosphoinositide 3-kinase (CgVps34) results in cell death in human macrophages and highly attenuated virulence in mice (12). Localization of CgFtr1-GFP in the Cgvps34⌬ mutant did not change in response to ironrepleted conditions, and remained primarily confined to the plasma membrane in the vast majority (95%) of cells (Fig. 10A) These data indicate that Cgvps34⌬ cells are deficient in the retrograde transport of CgFtr1-GFP from the plasma membrane in the high-iron environment, which could partly account for elevated susceptibility of the Cgvps34⌬ mutant to the surplus iron. Whereas survival rate of the Cgvps34⌬ mutant remained unaffected in iron-restricted macrophages, a 5-fold higher survival was observed in iron-supplemented macrophages compared with the RPMI medium-grown macrophages (Fig. 11D) These data suggest that higher extracellular iron levels promote the survival of Cgvps34⌬ cells in macrophages, which could be attributed, in part, to better growth of the Cgvps34⌬ mutant in iron-rich macrophages. These data indicate that attenuated virulence of the Cgvps34⌬ mutant in the systemic murine candidiasis model is probably a combined effect of lower colonization of host cells, and increased clearance by the host immune system
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