Analogs of the anti-malaria drug mefloquine have broad-spectrum antifungal activity and are efficacious in a model of disseminated Candida auris infection.

Only three classes of antifungal drugs are currently in clinical use. Here, we report that derivatives of the malarial drug mefloquine have broad-spectrum antifungal activity including difficult-to-treat molds and endemic fungi. Pharmacokinetic and efficacy studies of NSC-4377 indicate that it penetrates the central nervous system and is active against Candida auris in vivo. These data strongly support the further development of mefloquine analogs as a potentially new class of antifungal molecules.

Analogues of the anti-malaria drug mefloquine have broad spectrum antifungal activity and are efficacious in a model of disseminated Candida auris infection.

Only three classes of antifungal drugs are currently in clinical use. Here, we report that derivatives of the malarial drug mefloquine have broad spectrum antifungal activity including difficult to treat molds and endemic fungi. Pharmacokinetic and efficacy studies of NSC-4377 indicate it penetrates the central nervous system and is active against Candida auris in vivo. These data strongly support the further development of mefloquine analogs as a potentially new class of antifungal molecules.

Similarities and distinctions in the activation of the Candida glabrata Pdr1 regulatory pathway by azole and non-azole drugs.

Incidences of fluconazole (FLC) resistance among Candida glabrata clinical isolates is a growing issue in clinics. The pleiotropic drug response (PDR) network in C . glabrata confers azole resistance and is defined primarily by the Zn 2 Cys 6 zinc cluster-containing transcription factor Pdr1 and target genes such as CDR1 , that encodes an ATP-binding cassette transporter protein thought to act as a FLC efflux pump. Mutations in the PDR1 gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in Candida spp. We have used a firefly luciferase reporter gene driven by the CDR1 promoter to demonstrate two distinct patterns of CDR1 promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid CDR1 induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of CDR1 induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of CDR1 overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity. Importance: Candida glabrata has emerged as the second-leading cause of candidiasis due in part to its ability to acquire high level resistance to azole drugs, a major class of antifungal, that acts to block the biosynthesis of the fungal sterol ergosterol. The presence of azole drugs causes the induction of a variety of genes involved in controlling susceptibility to this drug class including drug transporters and ergosterol biosynthetic genes such as ERG11 . We found that the presence of azole drugs leads to an induction of genes encoding drug transporters and ERG11 , while exposure of C. glabrata cells to antifungals of the phenothiazine class of drugs caused a much faster and larger induction of drug transporters but not ERG11 . Coupled with further genetic analyses of the effects of azole and phenothiazine drugs, our data indicate that these compounds are sensed and responded to differentially in the yeast cell.

Discovery of CO2 tolerance genes associated with virulence in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a ubiquitous soil fungus and airborne pathogen that causes over 180,000 deaths each year. Cryptococcus must adapt to host CO2 levels to cause disease, but the genetic basis for this adaptation is unknown. We utilized quantitative trait loci mapping with 374 progeny from a cross between a CO2-tolerant clinical isolate and a CO2-sensitive environmental isolate to identify genetic regions regulating CO2 tolerance. To identify specific quantitative trait genes, we applied fine mapping through bulk segregant analysis of near-isogenic progeny with distinct tolerance levels to CO2. We found that virulence among near-isogenic strains in a murine model of cryptococcosis correlated with CO2 tolerance. Moreover, we discovered that sensitive strains may adapt in vivo to become more CO2 tolerant and more virulent. These findings highlight the underappreciated role of CO2 tolerance and its importance in the ability of an opportunistic environmental pathogen to cause disease.

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

Synthesis and Antifungal Activity of Stereoisomers of Mefloquine Analogs.

Cryptococcal neoformans and Candida albicans are among the most prevalent causes of life-threatening fungal infections globally. The high mortality associated with these infections despite current antifungal therapy highlights the need for new drugs. In our previous work, we demonstrated that an analogue of the clinically used antimalarial mefloquine, (8-chloro-2-(4-chlorophenyl)quinolin-4-yl)(piperidin-2-yl)methanol (4377), has both antifungal activity and the ability to penetrate the central nervous system. Herein we describe the synthesis and antifungal assay of all four stereoisomers of 4377. All four stereoisomers retain potent antifungal activity with the erythro enantiomers having MIC values of 1 and 4 µg/mL against C. neoformans and C. albicans, respectively, and threo enantiomers, MIC values of 2 and 8 µg/mL, respectively. These results indicate that the stereochemistry of the piperidine methanol group is not critical for the antifungal properties of 4377 and gives guidance to future medicinal chemistry optimization efforts.

Temporal dynamics of Candida albicans morphogenesis and gene expression reveals distinctions between in vitro and in vivo filamentation.

Candida albicans is a common human fungal pathogen that is also a commensal of the oral cavity and gastrointestinal tract. C. albicans pathogenesis is linked to its transition from budding yeast to filamentous morphologies including hyphae and pseudohyphae. The centrality of this virulence trait to C. albicans pathobiology has resulted in extensive characterization of a wide range of factors associated with filamentation with a strong focus on transcriptional regulation. The vast majority of these experiments have used in vitro conditions to induce the yeast-to-filament transition. Taking advantage of in vivo approaches to quantitatively characterize both morphology and gene expression during filamentation during mammalian infection, we have investigated the dynamics of these two aspects of filamentation in vivo and compared them to in vitro filament induction with "host-like" tissue culture media supplemented with serum at mammalian body temperature. Although filamentation shares many common features in the two conditions, we have found two significant differences. First, alternative carbon metabolism genes are expressed early during in vitro filamentation and late in vivo, suggesting significant differences in glucose availability. Second, C. albicans begins a hyphae-to-yeast transition after 4-h incubation while we find little evidence of hyphae-to-yeast transition in vivo up to 24 h post-infection. We show that the low rate of in vivo hyphae-to-yeast transition is likely due to the very low expression of PES1, a key driver of lateral yeast in vitro and that heterologous expression of PES1 is sufficient to trigger lateral yeast formation in vivo.IMPORTANCECandida albicans filamentation is correlated with virulence and is an intensively studied aspect of C. albicans biology. The vast majority of studies on C. albicans filamentation are based on in vitro induction of hyphae and pseudohyphae. Here we used an in vivo filamentation assay and in vivo expression profiling to compare the tempo of morphogenesis and gene expression between in vitro and in vivo filamentation. Although the hyphal gene expression profile is induced rapidly in both conditions, it remains stably expressed over a 12-h time course in vivo while it peaks after 4 h in vitro and is reduced. This reduced hyphal gene expression in vitro correlates with reduced hyphae and increased hyphae-to-yeast transition. By contrast, there is little evidence of hyphae-to-yeast transition in vivo.

The role of the C. albicans transcriptional repressor NRG1 during filamentation and disseminated candidiasis is strain dependent.

Candida albicans is one of the most common causes of superficial and invasive fungal diseases in humans. Its ability to cause disease is closely linked to its ability to undergo a morphological transition from budding yeast to filamentous forms (hyphae and pseudohyphae). The extent to which C. albicans strains isolated from patients undergo filamentation varies significantly. In addition, the filamentation phenotypes of mutants involving transcription factors that positively regulate hyphal morphogenesis can also vary from strain to strain. Here, we characterized the virulence, in vitro and in vivo filamentation, and in vitro and in vivo hypha-associated gene expression profiles for four poorly filamenting C. albicans isolates and their corresponding deletion mutants of the repressor of filamentation NRG1. The two most virulent strains, 57055 and 78048, show robust in vivo filamentation but are predominately yeast phase under in vitro hypha induction; the two low-virulence strains (94015 and 78042) do not undergo filamentation well under either condition. In vitro, deletion of NRG1 increases hyphae formation in the SC5314 derivative SN250, but only pseudohyphae are formed in the clinical isolates. Deletion of NRG1 modestly increased the virulence of 78042, which was accompanied by increased expression of hypha-associated genes without an increase in filamentation. Strikingly, deletion of NRG1 in 78048 reduced filamentation in vivo, expression of candidalysin (ECE1), and virulence without dramatically altering establishment of infection. Thus, the function of the conserved repressor NRG1 in C. albicans shows strain-based heterogeneity during infection.IMPORTANCEClinical isolates of the human fungal pathogen Candida albicans show significant variation in their ability to undergo in vitro filamentation and in the function of well-characterized transcriptional regulators of filamentation. Here, we show that Nrg1, a key repressor of filamentation and filament specific gene expression in standard reference strains, has strain-dependent functions, particularly during infection. Most strikingly, loss of NRG1 function can reduce filamentation, hypha-specific gene expression such as the toxin candidalysin, and virulence in some strains. Our data emphasize that the functions of seemingly fundamental and well-conserved transcriptional regulators such as Nrg1 are contextual with respect to both environment and genetic backgrounds.

The anti-cancer efficacy of a novel phenothiazine derivative is independent of dopamine and serotonin receptor inhibition.

Introduction: An attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of primary brain tumors and brain metastases. Phenothiazines (PTZs) have demonstrated anti-cancer properties through a variety of mechanisms. However, it remains unclear whether these effects are entirely separate from their activity as dopamine and serotonin receptor (DR/5-HTR) antagonists. Methods: In this study, we evaluated the anti-cancer efficacy of a novel PTZ analog, CWHM-974, that was shown to be 100-1000-fold less potent against DR/5-HTR than its analog fluphenazine (FLU). Results: CWHM-974 was more potent than FLU against a panel of cancer cell lines, thus clearly demonstrating that its anti-cancer effects were independent of DR/5-HTR signaling. Our results further suggested that calmodulin (CaM) binding may be necessary, but not sufficient, to explain the anti-cancer effects of CWHM-974. While both FLU and CWHM-974 induced apoptosis, they induced distinct effects on the cell cycle (G0/G1 and mitotic arrest respectively) suggesting that they may have differential effects on CaM-binding proteins involved in cell cycle regulation. Discussion: Altogether, our findings indicated that the anti-cancer efficacy of the CWHM-974 is separable from DR/5-HTR antagonism. Thus, reducing the toxicity associated with phenothiazines related to DR/5-HTR antagonism may improve the potential to repurpose this class of drugs to treat brain tumors and/or brain metastasis.

Comparative dynamics of gene expression during in vitro and in vivo Candida albicans filamentation.

Candida albicans is one of them most common causes of fungal disease in humans and is a commensal member of the human microbiome. The ability of C. albicans to cause disease is tightly correlated with its ability to undergo a morphological transition from budding yeast to a filamentous form (hyphae and pseudohyphae). This morphological transition is accompanied by the induction of a set of well characterized hyphae-associated genes and transcriptional regulators. To date, the vast majority of data regarding this process has been based on in vitro studies of filamentation using a range of inducing conditions. Recently, we developed an in vivo imaging approach that allows the direct characterization of morphological transition during mammalian infection. Here, we couple this imaging assay with in vivo expression profiling to characterize the time course of in vivo filamentation and the accompanying changes in gene expression. We also compare in vivo observations to in vitro filamentation using a medium (RPMI 1640 tissue culture medium with 10% bovine calf serum) widely used to mimic host conditions. From these data, we make the following conclusions regarding in vivo and in vitro filamentation. First, the transcriptional programs regulating filamentation are rapidly induced in vitro and in vivo. Second, the tempo of filamentation in vivo is prolonged relative to in vitro filamentation and the period of high expression of genes associated with that process is also prolonged. Third, hyphae are adapting to changing infection environments after filamentation has reached steady-state.

Cryptococcus neoformans adapts to the host environment through TOR-mediated remodeling of phospholipid asymmetry.

Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients. Host CO2 concentrations are 100-fold higher than the external environment and strains unable to grow at host CO2 concentrations are not pathogenic. Using a genetic screening and transcriptional profiling approach, we report that the TOR pathway is critical for C. neoformans adaptation to host CO2 partly through Ypk1-dependent remodeling of phosphatidylserine asymmetry at the plasma membrane. We also describe a C. neoformans ABC/PDR transporter (PDR9) that is highly expressed in CO2-sensitive environmental strains, suppresses CO2-induced phosphatidylserine/phospholipid remodeling, and increases susceptibility to host concentrations of CO2. Interestingly, regulation of plasma membrane lipid asymmetry by the TOR-Ypk1 axis is distinct in C. neoformans compared to S. cerevisiae. Finally, host CO2 concentrations suppress the C. neoformans pathways that respond to host temperature (Mpk1) and pH (Rim101), indicating that host adaptation requires a stringent balance among distinct stress responses.

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms.

Multidrug resistance (MDR) transporters such as ATP-Binding Cassette (ABC) and Major Facilitator Superfamily proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased MDR transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of Candida drug resistance (CDR) transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 is one of the few examples of a molecule in which relatively small structural modifications significantly reduced susceptibility to multidrug transporter-mediated resistance.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism.

IMPORTANCE: Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates biofilm formation and oral commensalism.

Candida albicans is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of C. albicans . The C. albicans reference strain SC5314 is a ROB1 heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform. An analysis of 224 sequenced C. albicans genomes indicates that SC5314 is the only ROB1 heterozygote documented to date and that the dominant allele contains a proline at position 946. Remarkably, the ROB1 alleles are functionally distinct and the rare ROB1 946S allele supports increased filamentation in vitro and increased biofilm formation in vitro and in vivo, suggesting it is a phenotypic gain-of-function allele. SC5314 is amongst the most highly filamentous and invasive strains characterized to date. Introduction of the ROB1 946S allele into a poorly filamenting clinical isolate increases filamentation and conversion of an SC5314 laboratory strain to a ROB1 946S homozygote increases in vitro filamentation and biofilm formation. In a mouse model of oropharyngeal infection, the predominant ROB1 946P allele establishes a commensal state while the ROB1 946S phenocopies the parent strain and invades into the mucosae. These observations provide an explanation for the distinct phenotypes of SC5314 and highlight the role of heterozygosity as a driver of C. albicans phenotypic heterogeneity. Importance: Candida albicans is a commensal fungus that colonizes human oral cavity and gastrointestinal tracts but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogenous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These finding explain, in part, the outlier phenotype of the reference strain and highlight the role of heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

The Mystery of Candida albicans Hyphal Morphogenesis in the Macrophage Phagolysosome.

C. albicans transitions between budding yeast and filamentous hyphal forms in a process that is tightly associated with its virulence. This transition also occurs after the fungus has been phagocytosed by macrophages. A number of somewhat discordant models have been proposed for the environmental characteristics of the phagolysosome that induce this transition. H. B. Wilson and M. C. Lorenz (Infect Immun 91:e00087-23, 2023, https://doi.org/10.1128/iai.00087-23) revisited these models and found that none of them explained morphogenesis in the macrophage.

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms.

Multidrug resistance (MDR) transporters such as ATP Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased multidrug resistance transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of CDR transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata , despite inducing CDR1 expression to high levels. Overall, CWHM-974 represents a unique example of a medicinal chemistry-based conversion of chemical scaffold from MDR-sensitive to MDR-resistant and, hence, active against fungi that have developed resistance to clinically used antifungals such as the azoles.

Bacterial structural genomics target enabled by a recently discovered potent fungal acetyl-CoA synthetase inhibitor.

The compound ethyl-adenosyl monophosphate ester (ethyl-AMP) has been shown to effectively inhibit acetyl-CoA synthetase (ACS) enzymes and to facilitate the crystallization of fungal ACS enzymes in various contexts. In this study, the addition of ethyl-AMP to a bacterial ACS from Legionella pneumophila resulted in the determination of a co-crystal structure of this previously elusive structural genomics target. The dual functionality of ethyl-AMP in both inhibiting ACS enzymes and promoting crystallization establishes its significance as a valuable resource for advancing structural investigations of this class of proteins.

Novel Keto-Alkyl-Pyridinium Antifungal Molecules Active in Models of In Vivo Candida albicans Vascular Catheter Infection and Ex Vivo Candida auris Skin Colonization.

New antifungal therapies are needed for both systemic, invasive infections in addition to superficial infections of mucosal and skin surfaces as well as biofilms associated with medical devices. The resistance of biofilm and biofilm-like growth phases of fungi contributes to the poor efficacy of systemic therapies to nonsystemic infections. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2 to 16 µg/mL) against medically important yeasts and molds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus. Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce Candida albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial preclinical data suggest that molecules of this class may warrant further study and development for nonsystemic applications.

A Dual-Readout High-Throughput Screening Assay for Small Molecules Active Against Aspergillus Fumigatus.

Human fungal infections caused by molds have been on the rise in recent years. These infections have high mortality rates compared to other fungal infections, and yet treatment options are limited due to resistance to clinical antifungals and lack of broad-spectrum activity against molds. Technical challenges associated with molds have limited large-throughput screening efforts for anti-mold compounds: therefore, we adapted an assay for use with A. fumigatus to help fill the gap in robust screening platforms for these organisms. This assay measures the release of the cytosolic enzyme adenylate kinase (AK) as a measure of fungal cell lysis and can also detect inhibition of germination as a reduction in the secretion of AK during vegetative growth. The ability to detect both lysis and inhibition of germination facilitates the identification of a wide range of compounds with different mechanisms of action, creating a strong screening platform for the identification of novel, anti-mold compounds.

Candida albicans Oropharyngeal Infection Is an Exception to Iron-Based Nutritional Immunity.

Candida albicans is a commensal of the human gastrointestinal tract and a common cause of human fungal disease, including mucosal infections, such as oropharyngeal candidiasis and disseminated infections of the bloodstream and deep organs. We directly compared the in vivo transcriptional profile of C. albicans during oral infection and disseminated infection of the kidney to identify niche specific features. Overall, 97 genes were differentially expressed between the 2 infection sites. Virulence-associated genes, such as hyphae-specific transcripts, were expressed similarly in the 2 sites. Genes expressed during growth in a poor carbon source (ACS1 and PCK1) were upregulated in oral tissue relative to kidney. Most strikingly, C. albicans in oral tissue shows the transcriptional hallmarks of an iron replete state while in the kidney it is in the expected iron starved state. Interestingly, C. albicans expresses genes associated with a low zinc environment in both niches. Consistent with these expression data, strains lacking transcription factors that regulate iron responsive genes (SEF1, HAP5) have no effect on virulence in a mouse model of oral candidiasis. During microbial infection, the host sequesters iron, zinc, and other metal nutrients to suppress growth of the pathogen in a process called nutritional immunity. Our results indicate that C. albicans is subject to iron and zinc nutritional immunity during disseminated infection but not to iron nutritional immunity during oral infection. IMPORTANCE Nutritional immunity is a response by which infected host tissue sequesters nutrients, such as iron, to prevent the microbe from efficiently replicating. Microbial pathogens subjected to iron nutritional immunity express specific genes to compensate for low iron availability. By comparing the gene expression profiles of the common human fungal pathogen Candida albicans in 2 infection sites, we found that C. albicans infecting the kidney has the transcriptional profile of iron starvation. By contrast, the C. albicans expression profile during oropharyngeal infection indicates the fungus is not iron starved. Two transcription factors that activate the transcriptional response to iron starvation are not required for C. albicans virulence during oral infection but are required for disseminated infection of the kidney. Thus, our results indicate that C. albicans is subject to nutritional iron immunity during disseminated infection but not during oropharyngeal infection, and highlight niche specific differences in the host-Candida albicans interaction.