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Multiple cellular activities, including protein and lipid synthesis, ribosome biogenesis, and metabolic processes, are regulated by the target of rapamycin (TOR) pathway. Recent research suggests that the TOR might play an important role in various physiological functions of pathogenic fungi, such as nutrient sensing, stress response, and cell cycle progression. Given their robust immunosuppressant and antitumor activities, TOR inhibitors are widely used in clinical settings. In the present study, a microdilution checkerboard-based approach was employed to assess the interactions between the oral mammalian target of rapamycin (mTOR) inhibitor everolimus (EVL) and antifungal agents in the treatment of Aspergillus species derived from 35 clinical isolates in vitro. The results revealed that EVL exhibited promising inhibitory synergy with itraconazole (ITC), posaconazole (POS), and amphotericin B (AMB) for 85.7%, 74.2%, and 71.4%, respectively. In contrast, EVL exhibited minimal synergistic inhibitory activity (14.3%) when applied in combination with voriconazole (VRC). Antagonistic interactions were not observed. In vivo experiments conducted in Galleria mellonella revealed that EVL in combination with antifungal agents improved the larva survival rates in the ITC, VRC, POS, and AMB groups by 18.3%, 13.3%, 26.7%, and 13.3%, respectively. These data suggest that the combination treatment with antifungal agents and antifungal agents holds promise as a means of alleviating clinical aspergillosis.

Ribosomes are the basis for protein production, whose biogenesis is essential for cells to drive growth and proliferation. Ribosome biogenesis is highly regulated in accordance with cellular energy status and stress signals. In eukaryotic cells, response to stress signals and the production of newly-synthesized ribosomes require elements to be transcribed by the three RNA polymerases (RNA pols). Thus, cells need the tight coordination of RNA pols to adjust adequate components production for ribosome biogenesis which depends on environmental cues. This complex coordination probably occurs through a signaling pathway that links nutrient availability with transcription. Several pieces of evidence strongly support that the Target of Rapamycin (TOR) pathway, conserved among eukaryotes, influences the transcription of RNA pols through different mechanisms to ensure proper ribosome components production. This review summarizes the connection between TOR and regulatory elements for the transcription of each RNA pol in the budding yeast Saccharomyces cerevisiae. It also focuses on how TOR regulates transcription depending on external cues. Finally, it discusses the simultaneous coordination of the three RNA pols through common factors regulated by TOR and summarizes the most important similarities and differences between S. cerevisiae and mammals.

ABSTRACT In this study, 18 predicted membrane-localized ABC transporters of Candida glabrata were deleted individually to create a minilibrary of knockouts (KO). The transporter KOs were analyzed for their susceptibility toward antimycotic drugs. Although CgYOR1 has previously been reported to be upregulated in various azole-resistant clinical isolates of C. glabrata, deletion of this gene did not change the susceptibility to any of the tested azoles. Additionally, Cgyor1Δ showed no change in susceptibility toward oligomycin, which is otherwise a well-known substrate of Yor1 in other yeasts. The role of CgYor1 in azole susceptibility only became evident when the major transporter CgCDR1 gene was deleted. However, under nitrogen-depleted conditions, Cgyor1Δ demonstrated an azole-susceptible phenotype, independent of CgCdr1. Notably, Cgyor1Δ cells also showed increased susceptibility to target of rapamycin (TOR) and calcineurin inhibitors. Moreover, increased phytoceramide levels in Cgyor1Δ and the deletions of regulators downstream of TOR and the calcineurin signaling cascade (Cgypk1Δ, Cgypk2Δ, Cgckb1Δ, and Cgckb2Δ) in the Cgyor1Δ background and their associated fluconazole (FLC) susceptibility phenotypes confirmed their involvement. Collectively, our findings show that TOR and calcineurin signaling govern CgYor1-mediated azole susceptibility in C. glabrata. IMPORTANCE The increasing incidence of Candida glabrata infections in the last 40 years is a serious concern worldwide. These infections are usually associated with intrinsic azole resistance and increasing echinocandin resistance. Efflux pumps, especially ABC transporter upregulation, are one of the prominent mechanisms of azole resistance; however, only a few of them are characterized. In this study, we analyzed the mechanisms of azole resistance due to a multidrug resistance-associated protein (MRP) subfamily ABC transporter, CgYor1. We demonstrate for the first time that CgYor1 does not transport oligomycin but is involved in azole resistance. Under normal growing conditions its function is masked by major transporter CgCdr1; however, under nitrogen-depleted conditions, it displays its azole resistance function independently. Moreover, we propose that the azole susceptibility due to removal of CgYor1 is not due to its transport function but involves modulation of TOR and calcineurin cascades.

ABSTRACT Melanin is an antioxidant polyphenol pigment required for the pathogenicity of many fungal pathogens, but comprehensive regulatory mechanisms remain unidentified. In this study, we systematically analyzed melanin-regulating signaling pathways in Cryptococcus neoformans and identified four melanin-regulating core transcription factors (TFs), Bzp4, Usv101, Mbs1, and Hob1, required for induction of the laccase gene (LAC1). Bzp4, Usv101, and Mbs1 independently regulate LAC1 induction, whereas Hob1 controls Bzp4 and Usv101 expression. Both Bzp4 and Usv101 are localized in the cytoplasm under nutrient-rich conditions (i.e., in the presence of yeast extract-peptone-dextrose [YPD] medium) but translocate into the nucleus upon nutrient starvation (i.e., in the presence of yeast nitrogen base [YNB] medium without glucose), and Mbs1 is constitutively localized in the nucleus. Notably, the cAMP pathway is not involved in regulation of the four TFs, but the high-osmolarity glycerol response (HOG) pathway negatively regulates induction of BZP4 and LAC1. Next, we searched for potential kinases upstream of the core TFs and identified nine core kinases; their deletion led to defective melanin production and LAC1 induction. Deletion of GSK3 or KIC1 abolished induction of LAC1 and BZP4 and perturbed nuclear translocation of Bzp4. Notably, Gsk3 also regulated expression of HOB1, USV101, and MBS1, indicating that it is a critical melanin-regulating kinase. Finally, an RNA sequencing-based transcriptome analysis of the wild-type strain and of bzp4Δ, usv101Δ, hob1Δ, and mbs1Δ strains under nutrient-rich and nutrient-starved conditions revealed that the melanin-regulating core TFs govern redundant and distinct classes of genes involved in a variety of biological processes. IMPORTANCE Melanins are dark green, brown, or black pigments that serve as antioxidant, reactive oxygen species (ROS) scavengers that protect fungal pathogens from radiation and host immune responses. Cryptococcus neoformans, the major etiological agent of fungal meningoencephalitis, also utilizes melanin as a key virulence factor. In this basidiomycete pathogen, melanin production is regulated by the cAMP and high-osmolarity glycerol response (HOG) pathways, and yet its complex signaling networks remain poorly described. In this study, we uncovered novel melanin synthesis regulatory networks consisting of core transcription factors (TFs), including Bzp4, Usv101, Hob1, and Mbs1, and core kinases Gsk3 and Kic1. These networks were identified through coupling systematic analyses of the expression and epistatic relationships of TF and kinase mutant libraries in the presence of diverse melanin substrates with transcriptome profiling of the core TF mutants. Thus, this report provides comprehensive insight into the melanin-regulating pathways in C. neoformans and other fungal pathogens.

ABSTRACT Human infection with Cryptococcus causes up to a quarter of a million AIDS-related deaths annually and is the most common cause of nonviral meningitis in the United States. As an opportunistic fungal pathogen, Cryptococcus neoformans is distinguished by its ability to adapt to diverse host environments, including plants, amoebae, and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified high expression levels of an alternative carbon nutrient transporter gene, STL1, to be associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role for STL1 in mammalian pathogenesis and revealed that STL1 expression is regulated by a novel multigene regulatory mechanism involving the CAC2 subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the global regulator of virulence gene VAD1 was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein, Ecm15, in turn required for nuclear transport of the Cac2 protein. Derepression of STL1 by the CAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of the STL1 enhancer element. Derepression of STL1 resulted in enhanced survival and growth of the fungus in the presence of low-nutrient, alternative carbon sources, facilitating virulence in mice. This study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host. IMPORTANCE Cryptococcus is a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of nonviral meningitis in the United States. The fungus is carried in about 10% of the adult population and, after reactivation, causes disease in a wide variety of immunosuppressed individuals, including the HIV infected and patients receiving transplant conditioning, cancer therapy, or corticosteroid therapy for autoimmune diseases. The fungus is widely carried in the soil but can also cause infections in plants and mammals. However, the mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to inhabit these diverse environments. Further studies identified a nutrient transporter gene, STL1, to be upregulated under low nutrients and to be associated with early fungicidal activity, a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in adaptation to the human host may help to design better methods of control and treatment of widely dispersed fungal pathogens such as Cryptococcus.

ABSTRACT 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. IMPORTANCE Candida 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.

ABSTRACT The target of rapamycin complex 1 (TORC1) pathway is a highly conserved signaling pathway across eukaryotes that integrates nutrient and stress signals to regulate the cellular growth rate and the transition into and maintenance of dormancy. The majority of the pathway’s components, including the central TOR kinase, have been lost in the apicomplexan lineage, and it is unknown how these organisms detect and respond to nutrient starvation in its absence. Plasmodium falciparum encodes a putative ortholog of the RNA polymerase (Pol) III repressor Maf1, which has been demonstrated to modulate Pol III transcription in a TOR-dependent manner in a number of organisms. Here, we investigate the role of P. falciparum Maf1 (PfMaf1) in regulating RNA Pol III expression under conditions of nutrient starvation and other stresses. Using a transposon insertion mutant with an altered Maf1 expression profile, we demonstrated that proper Maf1 expression is necessary for survival of the dormancy-like state induced by prolonged amino acid starvation and is needed for full recovery from other stresses that slow or stall the parasite cell cycle. This Maf1 mutant is defective in the downregulation of pre-tRNA synthesis under nutrient-limiting conditions, indicating that the function of Maf1 as a stress-responsive regulator of structural RNA transcription is conserved in P. falciparum. Recent work has demonstrated that parasites carrying artemisinin-resistant K13 alleles display an enhanced ability to recover from drug-induced growth retardation. We show that one such artemisinin-resistant line displays greater regulation of pre-tRNA expression and higher survival upon prolonged amino acid starvation, suggesting that overlapping, PfMaf1-associated pathways may regulate growth recovery from both artemisinin treatment and amino acid starvation. IMPORTANCE Eukaryote organisms sense changes in their environment and integrate this information through signaling pathways to activate response programs to ensure survival. The TOR pathway is a well-studied signaling pathway found throughout eukaryotes that is known to integrate a variety of signals to regulate organismal growth in response to starvation and other stresses. The human malaria parasite Plasmodium falciparum appears to have lost the TOR pathway over the course of evolution, and it is unclear how the parasite modulates its growth in response to starvation and drug treatment. Here, we show that Maf1, a protein regulated by TOR in other eukaryotes, plays an important role in maintaining the parasite’s viability in the face of starvation and other forms of stress. This suggests that PfMaf1 is a component of a yet-to-be-described nutrient and stress response pathway.

Chlamydospore formation is a characteristic of many fungal species, among them the closely related human-pathogenic dimorphic yeasts Candida albicans and C. dubliniensis. Whereas function and regulation of filamentation are well-studied in these species, the basis of chlamydospore formation is mostly unknown. Here, we investigate the contribution of environmental and genetic factors and identified central proteins involved in species-specific regulation of chlamydosporulation.We show that specific nutrient levels strongly impact chlamydospore initiation, with starvation favoring sporulation and elevated levels of saccharides or peptone inhibiting it. Thresholds for these nutritional effects differ between C. albicans and C. dubliniensis, which explain species-specific chlamydospore formation on certain diagnostic media. A C. albicans nrg1Δ mutant phenocopied C. dubliniensis, putting Nrg1 regulation at the basis of species-specific chlamydospore formation under various conditions.By screening a series of potential chlamydospore regulators, we identified the TOR and cAMP-pathways as crucial for sporulation. As rapamycin treatment blocked chlamydosporulation, a low basal Tor1 activity seems to be essential. In addition, TOR effector pathways play an important role, and loss of the NCR (nitrogen catabolite repression) gene regulators Gat1 and Gln3 reduced chlamydospore formation. A severe reduction was seen for a C. albicans gcn4Δ deletion strain, implicating a link between regulation of amino acid biosynthesis and chlamydospore development. On the other hand, deletion of the GTPase gene RAS1 and the adenylyl cyclase gene CYR1 caused a defect in chlamydospore formation that was mostly rescued by cAMP supplementation. Thus, cAMP-signaling is a second major pathway to control chlamydospore production. Finally, we confirmed light exposure to have a repressive effect on chlamydosporulation. However, permanent illumination only reduced, but not abolished chlamydospore production of C. albicans whereas C. dubliniensis sporulation was unaffected.In summary, we describe novel environmental factors which determine chlamydosporulation and propose a first model for the regulatory network of chlamydospore formation by different Candida species.