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Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report thatput1-/-andput2-/- strains, carrying defectiveProlineUTilization genes, display remarkable proline sensitivity withput2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate P5C, which inhibits mitochondrial respiration. Theput1-/- andput2-/-mutations attenuate virulence inDrosophilaand murine candidemia models. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages ofC. albicanscells colonizing a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not ofput2-/-cells. Multiple members of theCandidaspecies complex, includingC. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1Δ cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1Δ mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence., Author summary Candida albicans is an opportunistic fungal pathogen that exists as a benign member of the human microbiome. Immunosuppression, or microbial dysbiosis, can predispose an individual to infection, enabling this fungus to evade innate immune cells and initiate a spectrum of pathologies, including superficial mucocutaneous or even life-threatening invasive infections. Infectious growth is attributed to an array of virulence characteristics, a major one being the ability to switch morphologies from round yeast-like to elongated hyphal cells. Here we report that mitochondrial proline catabolism is required to induce hyphal growth of C. albicans cells in phagosomes of engulfing macrophages, which is key to evade killing by macrophages. The finding that proline catabolism, also required for the utilization of arginine and ornithine, is required to sustain the energy demands of hyphal growth underscores the central role of mitochondria in fungal virulence. In contrast to existing dogma, we show that in C. albicans, mitochondrial function is subject to glucose repression, amino acid-induced signals are strictly dependent on Ras1 and the SPS-sensor is the primary sensor of extracellular amino acids. The results provide a clear example of how C. albicans cells sense and respond to host nutrients to ensure proper nutrient uptake and survival.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer ofSaccharomyces cerevisiaeand a crucial element in the phosphate homeostatic system of this model yeast. We found that loss ofCandida albicansPho84 attenuated virulence inDrosophilaand murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS):pho84-/-cells were no more susceptible than wild typeC. albicansto neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized.pho84-/-mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 inpho84-/-cells, whileSOD3overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression ofSOD3expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulatingSOD3transcription was observed inPHO84wild type cells. Sod3 levels were not the only factor driving oxidative stress effects onpho84-/-cells, though, because overexpressingSOD3did not ameliorate these cells’ hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function ofS. cerevisiaePho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess inpho84-/-null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizingC. albicansto oxidative stress.

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells’ oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells’ hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress., Author summary Candida albicans is the species most often isolated from patients with invasive fungal disease, and is also a common colonizer of healthy people. It is well equipped to compete for nutrients with bacteria co-inhabiting human gastrointestinal mucous membranes, since it possesses multiple transporters to internalize important nutrients like sugars, nitrogen sources, and phosphate. During infection, the fungus needs to withstand human defense cells that attack it with noxious chemicals, among which reactive oxygen species (ROS) are critical. We found that a high-affinity phosphate transporter, Pho84, is required for C. albicans’ ability to successfully invade animal hosts and to eliminate ROS. Levels of a fungal enzyme that breaks down ROS, Sod3, were decreased in cells lacking Pho84. A connection between this phosphate transporter and the ROS-detoxifying enzyme was identified in the Target of Rapamycin (TOR) pathway, to which Pho84 is known to provide activating signals when phosphate is abundant. Small molecules that block Pho84 activity impair the ability of C. albicans to detoxify ROS. Since humans manage phosphate differently than fungi and have no Pho84 homolog, a drug that inhibits Pho84 could disable the defense of the fungus against the host.

The C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) influences many steps in the synthesis of an mRNA and helps control the final destiny of the mature transcript. ADAR2 edits RNA by converting adenosine to inosine within double-stranded or structured RNA. Site-selective A-to-I editing often occurs at sites near exon/intron borders, where it depends on intronic sequences for substrate recognition. It is therefore essential that editing precedes splicing. We have investigated whether there is coordination between ADAR2 editing and splicing of the GluR-B pre-mRNA. We show that the CTD is required for efficient editing at the R/G site one base upstream of a 5′-splice site. The results suggest that the CTD enhances editing at the R/G site by preventing premature splicing that would remove the intronic recognition sites for ADAR2. Editing at the GluR-B Q/R site, 24 bases upstream of the intron 11 5′-splice site, stimulates splicing at this intron. Furthermore, unlike previously studied introns, the CTD actually inhibits excision of intron 11, which includes the complementary recognition sequences for the Q/R editing site. In summary, these results show that the CTD and ADAR2 function together to enforce the order of events that allows editing to precede splicing, and they furthermore suggest a new role for the CTD as a coordinator of two interdependent pre-mRNA processing events.

The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality-of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N -acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO 2 and reduced temperatures (30°C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.

The fungal pathogen Candida albicans is a common cause of opportunistic infections in humans. We report that wild-type Drosophila melanogaster (OrR) flies are susceptible to virulent C. albicans infections and have established experimental conditions that enable OrR flies to serve as model hosts for studying C. albicans virulence. After injection into the thorax, wild-type C. albicans cells disseminate and invade tissues throughout the fly, leading to lethality. Similar to results obtained monitoring systemic infections in mice, well-characterized cph1Δ efg1Δ and csh3Δ fungal mutants exhibit attenuated virulence in flies. Using the OrR fly host model, we assessed the virulence of C. albicans strains individually lacking functional components of the SPS sensing pathway. In response to extracellular amino acids, the plasma membrane localized SPS-sensor (Ssy1, Ptr3, and Ssy5) activates two transcription factors (Stp1 and Stp2) to differentially control two distinct modes of nitrogen acquisition (host protein catabolism and amino acid uptake, respectively). Our results indicate that a functional SPS-sensor and Stp1 controlled genes required for host protein catabolism and utilization, including the major secreted aspartyl protease SAP2, are required to establish virulent infections. By contrast, Stp2, which activates genes required for amino acid uptake, is dispensable for virulence. These results indicate that nutrient availability within infected hosts directly influences C. albicans virulence.