Identification of Irpex and Rhodotorula on surveillance bronchoscopy in a pediatric lung transplant recipient: A case report and review of literature of these atypical fungal organisms.

BACKGROUND: Invasive fungal disease (IFD) is a frequent complication in pediatric lung transplant recipients, occurring in up to 12% of patients in the first year. Risk factors for infection include impaired lung defenses and intense immunosuppressive regimens. While most IFD occurs from Aspergillus, other fungal conidia are continuously inhaled, and infections with fungi on a spectrum of human pathogenicity can occur. CASE REPORT: We report a case of a 17-year-old lung transplant recipient in whom Irpex lacteus and Rhodotorula species were identified during surveillance bronchoscopy. She was asymptomatic and deemed to be colonized by Irpex lacteus and Rhodotorula species following transplant. 2 years after transplantation, she developed a fever, respiratory symptoms, abnormal lung imaging, and histological evidence of acute and chronic bronchitis on transbronchial biopsy. After developing symptoms concerning for a pulmonary infection and graft dysfunction, she was treated for a presumed IFD. Unfortunately, further diagnostic testing could not be performed at this time given her tenuous clinical status. Despite the initiation of antifungal therapy, her graft function continued to decline resulting in a second lung transplantation. CONCLUSIONS: This case raises the concern for IFD in lung transplant recipients from Irpex species. Further investigation is needed to understand the pathogenicity of this organism, reduce the incidence and mortality of IFD in lung transplant recipients, and refine the approach to diagnosis and manage the colonization and isolation of rare, atypical fungal pathogens in immunocompromised hosts.

Candida albicans' inorganic phosphate transport and evolutionary adaptation to phosphate scarcity.

Phosphorus is essential in all cells' structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. In this process, initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity were eventually lost as populations presumably gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations' fitness recovery in Pi scarcity, while that of others like membrane stress responses diverged from these fitness phenotypes. C. albicans therefore has diverse options to reconfigure Pi management during prolonged scarcity. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth and virulence.

In Support of Universal Admission Testing for SARS-CoV-2 During Significant Community Transmission.

Many hospitals have stopped or are considering stopping universal admission testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We discuss reasons why admission testing should still be part of a layered system to prevent hospital-acquired SARS-CoV-2 infections during times of significant community transmission. These include the morbidity of SARS-CoV-2 in vulnerable patients, the predominant contribution of presymptomatic and asymptomatic people to transmission, the high rate of transmission between patients in shared rooms, and data suggesting surveillance testing is associated with fewer nosocomial infections. Preferences of diverse patient populations, particularly the hardest-hit communities, should be surveyed and used to inform prevention measures. Hospitals' ethical responsibility to protect patients from serious infections should predominate over concerns about costs, labor, and inconvenience. We call for more rigorous data on the incidence and morbidity of nosocomial SARS-CoV-2 infections and more research to help determine when to start, stop, and restart universal admission testing and other prevention measures.

Glycerophosphocholine provision rescues Candida albicans growth and signaling phenotypes associated with phosphate limitation.

IMPORTANCE: Candida albicans is the most commonly isolated species from patients suffering from invasive fungal disease. C. albicans is most commonly a commensal organism colonizing a variety of niches in the human host. The fungus must compete for resources with the host flora to acquire essential nutrients such as phosphate. Phosphate acquisition and homeostasis have been shown to play a key role in C. albicans virulence, with several genes involved in these processes being required for normal virulence and several being upregulated during infection. In addition to inorganic phosphate (Pi), C. albicans can utilize the lipid-derived metabolite glycerophosphocholine (GPC) as a phosphate source. As GPC is available within the human host, we examined the role of GPC in phosphate homeostasis in C. albicans. We find that GPC can substitute for Pi by many though not all criteria and is likely a relevant physiological phosphate source for C. albicans.

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats.

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

Effect of clinician information sessions on diagnostic testing for Chagas disease.

BACKGROUND: Chagas disease is a potentially life-threatening neglected disease of poverty that is endemic in continental Latin America. Caused by Trypanosoma cruzi (T. cruzi), it is one of six parasitic diseases in the United States targeted by the Centers for Disease Control as a public health problem in need of action. An estimated 300,000 people are infected with T. cruzi in the United States (US). Although its morbidity, mortality and economic burden are high, awareness of Chagas disease is lacking among many healthcare providers in the US. The purpose of this analysis is to determine if the number of diagnostic tests performed at a community health center serving an at-risk population for Chagas disease increased after information sessions. A secondary aim was to determine if there was a difference by provider type, i.e., nurse practitioner vs. physician, or by specialty in the number of patients screened. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a retrospective data analysis of the number of Chagas serology tests performed at a community health center before and after information sessions for clinicians. A time series analysis was conducted focusing on the Adult and Family Medicine Departments at East Boston Neighborhood Health Center (EBNHC). Across all departments there were 1,957 T. cruzi tests performed before the sessions vs. 2,623 after the sessions. Interrupted time series analysis across departments indicated that testing volume was stable over time prior to the sessions (pre-period slope = +4.1 per month; p = 0.12), followed by an immediate shift after the session (+51.6; p = 0.03), while testing volume remained stable over time after the session (post-period slope = -6.0 per month; p = 0.11). CONCLUSION/SIGNIFICANCE: In this study, Chagas testing increased after information sessions. Clinicians who began testing their patients for Chagas disease after learning of the importance of this intervention added an extra, potentially time-consuming task to their already busy workdays without external incentives or recognition.

Inflammasome-mediated GSDMD activation facilitates escape of Candida albicans from macrophages.

Candida albicans is the most common cause of fungal sepsis. Inhibition of inflammasome activity confers resistance to polymicrobial and LPS-induced sepsis; however, inflammasome signaling appears to protect against C. albicans infection, so inflammasome inhibitors are not clinically useful for candidiasis. Here we show disruption of GSDMD, a known inflammasome target and key pyroptotic cell death mediator, paradoxically alleviates candidiasis, improving outcomes and survival of Candida-infected mice. Mechanistically, C. albicans hijacked the canonical inflammasome-GSDMD axis-mediated pyroptosis to promote their escape from macrophages, deploying hyphae and candidalysin, a pore-forming toxin expressed by hyphae. GSDMD inhibition alleviated candidiasis by preventing C. albicans escape from macrophages while maintaining inflammasome-dependent but GSDMD-independent IL-1ß production for anti-fungal host defenses. This study demonstrates key functions for GSDMD in Candida's escape from host immunity in vitro and in vivo and suggests that GSDMD may be a potential therapeutic target in C. albicans-induced sepsis.

Perceived barriers to Chagas disease screening among a diverse group of prenatal care providers.

BACKGROUND: Chagas disease is a vector borne infection of poverty endemic to Latin America which affects an estimated 40,000 women of child-bearing age in the United States (US). In the US Chagas disease is concentrated among individuals who have lived in endemic areas. Prenatal diagnosis and treatment are needed to prevent congenital transmission. The objective of this study was to assess perceived barriers to Chagas disease screening among prenatal care providers in Obstetrics/Gynecology and Family Medicine Departments of a tertiary care safety-net hospital caring for a significant at-risk population. METHODOLOGY/PRINCIPAL FINDINGS: An anonymous survey was distributed to 178 Obstetrics/Gynecology and Family Medicine practitioners. Of the 66 respondents, 39% thought Chagas screening was very important, and 48% somewhat important as a public health initiative. One third judged screening patients during clinic visits as very important. Most respondents (64%) reported being familiar with Chagas disease. However, only 32% knew how to order a test and only 22% reported knowing what to do if a test was positive. CONCLUSIONS/SIGNIFICANCE: These findings will be incorporated into measures to facilitate full implementation of Chagas screening, and can inform initiatives at other centers who wish to address this deeply neglected infection among their patient families. Greater integration of information on Chagas disease screening and treatment in medical and nursing education curricula can contribute to addressing this disease with the focus that its potentially fatal sequelae merit.

Trends in Pediatric Candidemia: Epidemiology, Anti-Fungal Susceptibility, and Patient Characteristics in a Children's Hospital.

Candida bloodstream infections (CBSIs) have decreased among pediatric populations in the United States, but remain an important cause of morbidity and mortality. Species distributions and susceptibility patterns of CBSI isolates diverge widely between children and adults. The awareness of these patterns can inform clinical decision-making for empiric or pre-emptive therapy of children at risk for candidemia. CBSIs occurring from 2006-2016 among patients in a large children's hospital were analyzed for age specific trends in incidence rate, risk factors for breakthrough-CBSI, and death, as well as underlying conditions. Candida species distributions and susceptibility patterns were evaluated in addition to the anti-fungal agent use. The overall incidence rate of CBSI among this complex patient population was 1.97/1000 patient-days. About half of CBSI episodes occurred in immunocompetent children and 14% in neonatal intensive care unit (NICU) patients. Anti-fungal resistance was minimal: 96.7% of isolates were fluconazole, 99% were micafungin, and all were amphotericin susceptible. Liposomal amphotericin was the most commonly prescribed anti-fungal agent included for NICU patients. Overall, CBSI-associated mortality was 13.7%; there were no deaths associated with CBSI among NICU patients after 2011. Pediatric CBSI characteristics differ substantially from those in adults. The improved management of underlying diseases and antimicrobial stewardship may further decrease morbidity and mortality from CBSI, while continuing to maintain low resistance rates among Candida isolates.

Phosphate in Virulence of Candida albicans and Candida glabrata.

Candida species are the most commonly isolated invasive human fungal pathogens. A role for phosphate acquisition in their growth, resistance against host immune cells, and tolerance of important antifungal medications is becoming apparent. Phosphorus is an essential element in vital components of the cell, including chromosomes and ribosomes. Producing the energy currency of the cell, ATP, requires abundant inorganic phosphate. A comparison of the network of regulators and effectors that controls phosphate acquisition and intracellular distribution, the PHO regulon, between the model yeast Saccharomyces cerevisiae, a plant saprobe, its evolutionarily close relative C. glabrata, and the more distantly related C. albicans, highlights the need to coordinate phosphate homeostasis with adenylate biosynthesis for ATP production. It also suggests that fungi that cope with phosphate starvation as they invade host tissues, may link phosphate acquisition to stress responses as an efficient mechanism of anticipatory regulation. Recent work indicates that connections among the PHO regulon, Target of Rapamycin Complex 1 signaling, oxidative stress management, and cell wall construction are based both in direct signaling links, and in the provision of phosphate for sufficient metabolic intermediates that are substrates in these processes. Fundamental differences in fungal and human phosphate homeostasis may offer novel drug targets.

Phosphoric Metabolites Link Phosphate Import and Polysaccharide Biosynthesis for Candida albicans Cell Wall Maintenance.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans' tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.IMPORTANCECandida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

Candida albicans Dispersed Cells Are Developmentally Distinct from Biofilm and Planktonic Cells.

Candida albicans surface-attached biofilms such as those formed on intravenous catheters with direct access to the bloodstream often serve as a nidus for continuous release of cells capable of initiating new infectious foci. We previously reported that cells dispersed from a biofilm are yeast cells that originate from the top-most hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm dispersal yeast cells displayed enhanced virulence-associated characteristics and drug resistance. However, little is known about their molecular properties. To address that issue, in this study we aimed to define the molecular characteristics of these biofilm dispersal cells. We found that the inducer of dispersal, PES1, genetically interacts with the repressor of filamentation, NRG1, in a manner consistent with the definition of dispersed cells as yeast cells. Further, using a flow biofilm model, we performed comprehensive comparative RNA sequencing on freshly dispersed cells in order to identify unique transcriptomic characteristics. Gene expression analysis demonstrated that dispersed cells largely inherit a biofilm-like mRNA profile. Strikingly, however, dispersed cells seemed transcriptionally reprogrammed to acquire nutrients such as zinc and amino acids and to metabolize alternative carbon sources, while their biofilm-associated parent cells did not induce the same high-affinity transporters or express gluconeogenetic genes, despite exposure to the same nutritional signals. Collectively, the findings from this study characterize cell dispersal as an intrinsic step of biofilm development which generates propagules more adept at colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before the cells experience the associated external signals.IMPORTANCECandida albicans surface-attached biofilms serve as a reservoir of cells to perpetuate and expand an infection; cells released from biofilms on catheters have direct access to the bloodstream. Biofilm dispersal yeast cells exhibit enhanced adhesion, invasion, and biofilm formation compared to their planktonic counterparts. Here, we show using transcriptome sequencing (RNA-seq) that dispersed yeast cells are developmentally distinct from the cells in their parent biofilms as well as from planktonic yeast cells. Dispersal cells possess an anticipatory expression pattern that primes them to infect new sites in the host, to survive in nutrient-starved niches, and to invade new sites. These studies identified dispersal cells as a unique proliferative cell type of the biofilm and showed that they could serve as targets for antibiofilm drug development in the future.

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence.

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.

The Human Gut Microbial Metabolome Modulates Fungal Growth via the TOR Signaling Pathway.

Candida albicans is well known as a major human fungal pathogen, but it is also a permanent resident of healthy gastrointestinal tracts. Recent studies have shown that the human gut microbial metabolome represents an interesting source of bioactive molecules with a significant degree of chemical diversity. Some of these bioactive molecules may have useful antivirulence activities. For instance, intestinal bacterial species belonging to the Lachnospiraceae family were found to secrete molecules that attenuate Salmonella pathogenicity and repress the expression of virulence genes. Here, we have investigated whether the microbial gut metabolome (GM) contains molecules that might promote the commensal lifestyle and/or inhibit the expression of virulence of C. albicans in the intestine. We found that metabolites from human feces inhibited the growth of C. albicans and other opportunistic yeasts. A genetic screen in C. albicans suggested that TOR is the molecular target of the antifungal molecule(s) of the GM. In addition, we found that the GM metabolites inhibit both C. albicans hyphal growth and the invasion of human enterocytes. The antigrowth and antivirulence activities were partially recapitulated by secretions from Roseburia spp. and Bacteroides ovatus strains, respectively. This study demonstrates that the antimicrobial activity of the GM can be extended to a eukaryotic pathogen, C. albicans, illuminating the antagonistic interkingdom interactions between a fungus and intestinal commensal bacteria. IMPORTANCECandida albicans is a natural component of the human microbiota but also an opportunistic pathogen that causes life-threatening infections. The human gastrointestinal tract is the main reservoir of C. albicans, from where systemic infections originate as a consequence of the disruption of the intestinal mucosal barrier. Recent studies provided convincing evidence that overgrowth of C. albicans and other related species in the gut is predominantly associated with chronic intestinal inflammatory bowel diseases. Here, we showed, for the first time, the antagonistic interkingdom interactions between C. albicans and common intestinal commensal bacteria. From a therapeutic perspective, administering a defined bacterial community, such as the one described here with anti-Candida activity, could provide potential therapeutic protection against gastrointestinal inflammatory diseases.

The Candida albicans TOR-Activating GTPases Gtr1 and Rhb1 Coregulate Starvation Responses and Biofilm Formation.

Target of rapamycin complex 1 (TORC1) is an essential regulator of metabolism in eukaryotic cells and in the fungal pathogen Candida albicans regulates morphogenesis and nitrogen acquisition. Gtr1 encodes a highly conserved GTPase that in Saccharomyces cerevisiae regulates nitrogen sensing and TORC1 activation. Here, we characterize the role of C. albicans GTR1 in TORC1 activation and compare it with the previously characterized GTPase Rhb1. A homozygous gtr1/gtr1 mutant exhibited impaired TORC1-mediated phosphorylation of ribosomal protein S6 and increased susceptibility to rapamycin. Overexpression of GTR1 impaired nitrogen starvation-induced filamentous growth, MEP2 expression, and growth in bovine serum albumin as the sole nitrogen source. Both GTR1 and RHB1 were shown to regulate genes involved in ribosome biogenesis, amino acid biosynthesis, and expression of biofilm growth-induced genes. The rhb1/rhb1 mutant exhibited a different pattern of expression of Sko1-regulated genes and increased susceptibility to Congo red and calcofluor white. The homozygous gtr1/gtr1 mutant exhibited enhanced flocculation phenotypes and, similar to the rhb1/rhb1 mutant, exhibited enhanced biofilm formation on plastic surfaces. In summary, Gtr1 and Rhb1 link nutrient sensing and biofilm formation and this connectivity may have evolved to enhance the competitiveness of C. albicans in niches where there is intense competition with other microbes for space and nutrients. IMPORTANCECandida albicans is the major fungal pathogen of humans and is responsible for a wide range of infections, including life-threatening systemic infections in susceptible hosts. Target of rapamycin complex 1 (TORC1) is an essential regulator of metabolism in this fungus, and components of this complex are under increased investigation as targets for new antifungal drugs. The present study characterized the role of GTR1, encoding a putative GTPase, in TORC1 activation. This study shows that GTR1 encodes a protein required for activation of TORC1 activity in response to amino acids and regulation of nitrogen starvation responses. GTR1 mutants show increased cell-cell adhesion and biofilm formation and increased expression of genes involved in these processes. This study demonstrates that starvation responses and biofilm formation are coregulated by GTR1 and suggests that these responses are linked to compete with the microbiome for space and nutrients.

Skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems.

Nuclear factor of activated T cells (NFAT) is activated in innate immune cells downstream of pattern recognition receptors, but little is known about NFAT's functions in innate immunity compared with adaptive immunity. We show that early activation of NFAT balances the two major phases of the innate response to Candida albicans skin infections: the protective containment (abscess) and the elimination (expulsion) phases. During the early containment phase, transforming growth factor-ß (TGF-ß) induces the deposit of collagen around newly recruited polymorphonuclear cells to prevent microbial spreading. During the elimination phase, interferon-γ (IFN-γ) blocks differentiation of fibroblasts into myofibroblasts by antagonizing TGF-ß signaling. IFN-γ also induces the formation of plasmin that, in turn, promotes abscess capsule digestion and skin ulceration for microbial discharge. NFAT controls innate IFN-γ production and microbial expulsion. This cross-talk between the innate immune and the fibrinolytic systems also occurs during infection with Staphylococcus aureus and is a protective response to minimize tissue damage and optimize pathogen elimination.

Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition.

The Target of Rapamycin (TOR) pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans Eukaryotic Target of Rapamycin complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking unknown components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than the WT. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1 but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and a constitutively active Gtr1Q67L mutant suppresses TORC1-related defects. In Saccharomyces cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence, connections from phosphate homeostasis (PHO) to TORC1 may differ between C. albicans and S. cerevisiae The converse direction of signaling from TORC1 to the PHO regulon previously observed in S. cerevisiae was genetically shown in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, a Food and Drug Administration-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes require significant amounts of phosphate. Our study shows that phosphate availability is monitored and also controlled by TORC1 and that TORC1 can be indirectly targeted by inhibiting Pho84.

Fungi that Infect Humans.

Fungi must meet four criteria to infect humans: growth at human body temperatures, circumvention or penetration of surface barriers, lysis and absorption of tissue, and resistance to immune defenses, including elevated body temperatures. Morphogenesis between small round, detachable cells and long, connected cells is the mechanism by which fungi solve problems of locomotion around or through host barriers. Secretion of lytic enzymes, and uptake systems for the released nutrients, are necessary if a fungus is to nutritionally utilize human tissue. Last, the potent human immune system evolved in the interaction with potential fungal pathogens, so few fungi meet all four conditions for a healthy human host. Paradoxically, the advances of modern medicine have made millions of people newly susceptible to fungal infections by disrupting immune defenses. This article explores how different members of four fungal phyla use different strategies to fulfill the four criteria to infect humans: the Entomophthorales, the Mucorales, the Ascomycota, and the Basidiomycota. Unique traits confer human pathogenic potential on various important members of these phyla: pathogenic Onygenales comprising thermal dimorphs such as Histoplasma and Coccidioides; the Cryptococcus spp. that infect immunocompromised as well as healthy humans; and important pathogens of immunocompromised patients-Candida, Pneumocystis, and Aspergillus spp. Also discussed are agents of neglected tropical diseases important in global health such as mycetoma and paracoccidiomycosis and common pathogens rarely implicated in serious illness such as dermatophytes. Commensalism is considered, as well as parasitism, in shaping genomes and physiological systems of hosts and fungi during evolution.

Beauvericin Potentiates Azole Activity via Inhibition of Multidrug Efflux, Blocks Candida albicans Morphogenesis, and Is Effluxed via Yor1 and Circuitry Controlled by Zcf29.

Invasive fungal infections are a leading cause of human mortality. Effective treatment is hindered by the rapid emergence of resistance to the limited number of antifungal drugs, demanding new strategies to treat life-threatening fungal infections. Here, we explore a powerful strategy to enhance antifungal efficacy against leading human fungal pathogens by using the natural product beauvericin. We found that beauvericin potentiates the activity of azole antifungals against azole-resistant Candida isolates via inhibition of multidrug efflux and that beauvericin itself is effluxed via Yor1. As observed in Saccharomyces cerevisiae, we determined that beauvericin inhibits TOR signaling in Candida albicans To further characterize beauvericin activity in C. albicans, we leveraged genome sequencing of beauvericin-resistant mutants. Resistance was conferred by mutations in transcription factor genes TAC1, a key regulator of multidrug efflux, and ZCF29, which was uncharacterized. Transcriptional profiling and chromatin immunoprecipitation coupled to microarray analyses revealed that Zcf29 binds to and regulates the expression of multidrug transporter genes. Beyond drug resistance, we also discovered that beauvericin blocks the C. albicans morphogenetic transition from yeast to filamentous growth in response to diverse cues. We found that beauvericin represses the expression of many filament-specific genes, including the transcription factor BRG1 Thus, we illuminate novel circuitry regulating multidrug efflux and establish that simultaneously targeting drug resistance and morphogenesis provides a promising strategy to combat life-threatening fungal infections.