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Iron is an essential nutrient required as a cofactor for many biological processes. As a fungal commensal-pathogen of humans, Candida albicans encounters a range of bioavailable iron levels in the human host and maintains homeostasis with a conserved regulatory circuit. How C. albicans senses and responds to iron availability is unknown. In model yeasts, regulation of the iron homeostasis circuit requires monothiol glutaredoxins (Grxs), but their functions beyond the regulatory circuit are unclear. Here, we show Grx3 is required for virulence and growth on low iron for C. albicans. To explore the global roles of Grx3, we applied a proteomic approach and performed in vivo cross-linked tandem affinity purification coupled with mass spectrometry. We identified a large number of Grx3 interacting proteins that function in diverse biological processes. This included Fra1 and Bol2/Fra2, which function with Grxs in intracellular iron trafficking in other organisms. Grx3 interacts with and regulates the activity of Sfu1 and Hap43, components of the C. albicans iron regulatory circuit. Unlike the regulatory circuit, which determines expression or repression of target genes in response to iron availability, Grx3 amplifies levels of gene expression or repression. Consistent with the proteomic data, the grx3 mutant is sensitive to heat shock, oxidative, nitrosative, and genotoxic stresses, and shows growth dependence on histidine, leucine, and tryptophan. We suggest Grx3 is a conserved global regulator of iron-dependent processes occurring within the cell., Author summary Mammalian pathogens occupy a diverse set of niches within the host organism. These niches vary in iron and oxygen availability. As a commensal and pathogen of humans, its ability to regulate iron uptake and utilization in response to bioavailable iron level is critical for its survival in different host environments encompassing a broad range of iron levels. This study aims to understand how C. albicans senses and responds to iron level to regulate multiple aspects of its biology. The cytosolic monothiol glutaredoxin Grx3 is a critical regulator of C. albicans iron homeostasis and virulence. Taking a proteomic approach, we identified a large list of Grx3 associated proteins of diverse functions, including iron-sulfur trafficking, iron homeostasis, metabolism redox homeostasis, protein translation, DNA maintenance and repair. In support of these protein associations, Grx3 is important for all these processes. Thus, Grx3 is a global regulator of iron homeostasis and other iron dependent cellular processes.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality., Author summary All organisms tailor genetic programs to the available nutrients, such as sources of carbon. Here we define two key regulators of the genetic programs for carbon source utilization in the fungal pathogen Candida albicans. The two regulators have many shared roles, yet are partially specialized to control carbon acquisition and metabolism, respectively. In addition, the regulators together control traits associated with pathogenicity, an indication that carbon regulation is integrated into the pathogenicity program. Finally, the regulators help to explain a long-standing riddle—that the central carbon regulator Snf1 is essential for C. albicans viability.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype., Author summary Opportunistic fungal pathogens commonly acquire extra copies of chromosomes that can provide a fitness benefit under acute stress such as exposure to antifungal agents but how these extra copies affect fungal life-style and interactions with their hosts is poorly understood. Here we show that in C. albicans the acquisition of specific whole chromosome trisomies during oropharyngeal infection in mice results in a commensal-like phenotype. Our data indicate that trisomies of chromosomes 5 and 6 alter several related virulence-associated traits that affect how the host recognizes and responds to C. albicans during oropharyngeal infection, thereby inducing this commensal-like phenotype. Whole genome sequencing revealed that trisomies were mostly maintained in subsequent oral infections and that de novo mutations that arose were not shared among strains. We hypothesize that both in vivo and in vitro phenotypes are likely the result of allelic imbalance of specific genes on the trisomic chromosomes, rather than due to whole chromosome trisomy.

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Localization of specific mRNAs is an important mechanism through which cells achieve polarity and direct asymmetric growth. Based on a framework established in Saccharomyces cerevisiae, we describe a She3-dependent RNA transport system in Candida albicans, a fungal pathogen of humans that grows as both budding (yeast) and filamentous (hyphal and pseudohyphal) forms. We identify a set of 40 mRNAs that are selectively transported to the buds of yeast-form cells and to the tips of hyphae, and we show that many of the genes encoded by these mRNAs contribute to hyphal development, as does the transport system itself. Although the basic system of mRNA transport is conserved between S. cerevisiae and C. albicans, we find that the cargo mRNAs have diverged considerably, implying that specific mRNAs can easily move in and out of transport control over evolutionary timescales. The differences in mRNA cargos likely reflect the distinct selective pressures acting on the two species., Author Summary Generation of cellular polarity – asymmetry in shape, protein distribution, and/or sub-cellular function – is an essential feature of most eukaryotic cells and underlies such diverse processes as differentiation, mating, nutrient acquisition, and growth. Localization of specific mRNAs is one mechanism through which cells achieve polarity. We describe an RNA transport system in Candida albicans, a fungal pathogen of humans, that grows in both single cell (budding yeast) and filamentous (hyphal and pseudohyphal) forms. Hyphae are chains of elongated cells that remain attached after cell division and exhibit highly polarized growth at their tips. We show that the C. albicans She3-dependent RNA transport system binds to 40 mRNAs and transports these mRNAs to yeast buds and to the tips of hyphae. Both the transport system itself and many of the genes encoded by transported mRNAs are required for normal growth and function of hyphae. Although the basic transport mechanism appears conserved with that of the model yeast, Saccharomyces cerevisiae, the cargo mRNAs are largely distinct. The apparently rapid evolution of the transported mRNAs probably reflects distinct selective pressures acting on the two organisms.