A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata

Abstract

Iron is an essential nutrient for all living organisms and human pathogens employ a battery of factors to scavenge iron from the high-affinity iron-binding host proteins. In the present study, we have elucidated, via a candidate gene approach, major iron acquisition and homoeostatic mechanisms operational in an opportunistic human fungal pathogen Candida glabrata. Phenotypic, biochemical and molecular analysis of a set of 13 C. glabrata strains, deleted for proteins potentially implicated in iron metabolism, revealed that the high-affinity reductive iron uptake system is required for utilization of alternate carbon sources and for growth under both in vitro iron-limiting and in vivo conditions. Furthermore, we show for the first time that the cysteine-rich CFEM (common in fungal extracellular membranes) domain-containing cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3, are essential for maintenance of intracellular iron content, adherence to epithelial cells and virulence. Consistent with their roles in iron homoeostasis, mitochondrial aconitase activity was lower and higher in mutants disrupted for high-affinity iron transport, and haemolysin respectively. Additionally, we present evidence that the mitochondrial frataxin, CgYfh1, is pivotal to iron metabolism. Besides yielding insights into major in vitro and in vivo iron acquisition strategies, our findings establish high-affinity iron uptake mechanisms as critical virulence determinants in C. glabrata.

The Phosphoinositide 3-Kinase Regulates Retrograde Trafficking of the Iron Permease CgFtr1 and Iron Homeostasis in Candida glabrata

“…2A, 13 iron homeostasis genes including genes encoding proteins involved in high-affinity iron uptake (CgFet3, a multicopper oxidase), lowaffinity ion transport (CgFet4, a low-affinity ion transporter), and siderophore-iron acquisition (CgSit1, a siderophore-iron transporter) were differentially regulated in the Cgvps34⌬ mutant. Of note, the high-affinity iron transport system in C. glabrata is activated and repressed under iron-depleted and iron-repleted conditions, respectively (10,19). Overall, our RNA-Seq analysis revealed that genes involved in iron transport were down-regulated, whereas genes implicated in iron utilization/iron-sulfur (Fe-S) cluster binding were up-regulated in the Cgvps34⌬ mutant ( Fig.…”

“…low-affinity system composed of CgFet4, and the siderophore uptake system comprised of CgSit1 transporter, are required for iron acquisition (10). As shown in Fig.…”

“…Although C. glabrata neither synthesizes nor secretes high-affinity ironchelating compounds, siderophores, it does possess the sole siderophore transporter, CgSit1, which is essential for utilization of ferrichrome as an iron source under iron-deficient conditions, and for iron-dependent survival in macrophages (10,18). Lastly, two components, identified so far, of the hostspecific iron acquisition system are the putative hemolysin (CgMam3) and the cell wall structural protein (CgCcw14), which are required for survival in systemic infections in mice (10).…”

“…Besides acting as a cofactor forseveralmetalloproteinsinvolvedinfundamentalcellularprocesses of oxygen transport, respiration, energy metabolism, and DNA synthesis and repair, iron modulates the transcriptional activation of virulence genes of pathogenic fungi (16,17). To survive and proliferate in varied iron host niches including the gastrointestinal tract, blood, oral cavity, and genitourinary tract, C. glabrata has developed at least four distinct types of iron acquisition systems (10).…”

“…The high-affinity iron uptake system in C. glabrata is composed of an iron transporter (CgFtr1), a cell surface multicopper oxidase (CgFet3), a P-type Cu-ATPase (CgCcc2), and many members of the ferric reductase (CgFre) family, and is required for proliferation under in vitro iron-limited conditions as well as in murine disseminated candidiasis infection model (10). Conversely, the low-affinity iron transport system, typified by the nonspecific ion transporter, CgFet4, is dispensable for growth in the iron-poor environment (10).…”