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Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017-2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

Candida auris has become a serious threat to public health. The mechanisms of how this fungal pathogen adapts to the mammalian host are poorly understood. Here we report the rapid evolution of an adaptive C. auris multicellular aggregative morphology in the murine host during systemic infection. C. auris aggregative cells accumulate in the brain and exhibit obvious advantages over the single-celled yeast-form cells during systemic infection. Genetic mutations, specifically de novo point mutations in genes associated with cell division or budding processes, underlie the rapid evolution of this aggregative phenotype. Most mutated C. auris genes are associated with the regulation of cell wall integrity, cytokinesis, cytoskeletal properties, and cellular polarization. Moreover, the multicellular aggregates are notably more recalcitrant to the host antimicrobial peptides LL-37 and PACAP relative to the single-celled yeast-form cells. Overall, to survive in the host, C. auris can rapidly evolve a multicellular aggregative morphology via genetic mutations.

The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in healthcare settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five Food and Drug Administration (FDA)-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris. To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only twofold to fivefold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1) and in the drug transporter CDR1. These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more susceptible to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. IMPORTANCE The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated 8-hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris. Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest twofold to fivefold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500-fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated 8-hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris. Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.

Sexual reproduction is prevalent in eukaryotic organisms and plays a critical role in the evolution of new traits and in the generation of genetic diversity. Environmental factors often have a direct impact on the occurrence and frequency of sexual reproduction in fungi. The regulatory effects of atmospheric relative humidity (RH) on sexual reproduction and pathogenesis in plant fungal pathogens and in soil fungi have been extensively investigated. However, the knowledge of how RH regulates the lifecycles of human fungal pathogens is limited. In this study, we report that low atmospheric RH promotes the development of mating projections and same-sex (homothallic) mating in the human fungal pathogen Candida albicans. Low RH causes water loss in C. albicans cells, which results in osmotic stress and the generation of intracellular reactive oxygen species (ROS) and trehalose. The water transporting aquaporin Aqy1, and the G-protein coupled receptor Gpr1 function as cell surface sensors of changes in atmospheric humidity. Perturbation of the trehalose metabolic pathway by inactivating trehalose synthase or trehalase promotes same-sex mating in C. albicans by increasing osmotic or ROS stresses, respectively. Intracellular trehalose and ROS signal the Hog1-osmotic and Hsf1-Hsp90 signaling pathways to regulate the mating response. We, therefore, propose that the cell surface sensors Aqy1 and Gpr1, intracellular trehalose and ROS, and the Hog1-osmotic and Hsf1-Hsp90 signaling pathways function coordinately to regulate sexual mating in response to low atmospheric RH conditions in C. albicans.

Candida vulturna belongs to the Candida haemulonii species complex and is phylogenetically related to C. auris. We report a C. vulturna outbreak among persons in Shanxi Province, China, during 2019-2022. Isolates were resistant to multiple antifungal drugs and exhibited enhanced adhesion and biofilm formation properties.

Candida parapsilosis is one of the most commen causes of life-threatening candidaemia, particularly in premature neonates, individuals with cancer of the haematopoietic system, and recipients of organ transplants. Historically, drug-susceptible strains have been linked to clonal outbreaks. However, worldwide studies started since 2018 have reported severe outbreaks among adults caused by fluconazole-resistant strains. Outbreaks caused by fluconazole-resistant strains are associated with high mortality rates and can persist despite strict infection control strategies. The emergence of resistance threatens the efficacy of azoles, which is the most widely used class of antifungals and the only available oral treatment option for candidaemia. The fact that most patients infected with fluconazole-resistant strains are azole-naive underscores the high potential adaptability of fluconazole-resistant strains to diverse hosts, environmental niches, and reservoirs. Another concern is the multidrug-resistant and echinocandin-tolerant C parapsilosis isolates, which emerged in 2020. Raising awareness, establishing effective clinical interventions, and understanding the biology and pathogenesis of fluconazole-resistant C parapsilosis are urgently needed to improve treatment strategies and outcomes.

Catheter-associated urinary tract infections (CAUTIs) account for 40% of hospital-acquired infections (HAIs). As 20 to 50% of hospitalized patients receive catheters, CAUTIs are one of the most common HAIs, resulting in increased morbidity, mortality, and health care costs. Candida albicans is the second most common CAUTI uropathogen, yet relative to its bacterial counterparts, little is known about how fungal CAUTIs are established. Here, we show that the catheterized bladder environment induces Efg1- and fibrinogen (Fg)-dependent biofilm formation that results in CAUTI. In addition, we identify the adhesin Als1 as the critical fungal factor for C. albicans Fg-urine biofilm formation. Furthermore, we show that in the catheterized bladder, a dynamic and open system, both filamentation and attachment are required, but each by themselves are not sufficient for infection. Our study unveils the mechanisms required for fungal CAUTI establishment, which may aid in the development of future therapies to prevent these infections.

Candida auris is an emerging multidrug-resistant fungal pathogen and a new global threat to human health. A unique morphological feature of this fungus is its multicellular aggregating phenotype, which has been thought to be associated with defects in cell division. In this study, we report a new aggregating form of two clinical C. auris isolates with increased biofilm forming capacity due to enhanced adherence of adjacent cells and surfaces. Unlike the previously reported aggregating morphology, this new aggregating multicellular form of C. auris can become unicellular after treatment with proteinase K or trypsin. Genomic analysis demonstrated that amplification of the subtelomeric adhesin gene ALS4 is the reason behind the strain's enhanced adherence and biofilm forming capacities. Many clinical isolates of C. auris have variable copy numbers of ALS4, suggesting that this subtelomeric region exhibits instability. Global transcriptional profiling and quantitative real-time PCR assays indicated that genomic amplification of ALS4 results in a dramatic increase in overall levels of transcription. Compared to the previously characterized nonaggregative/yeast-form and aggregative-form strains of C. auris, this new Als4-mediated aggregative-form strain of C. auris displays several unique characteristics in terms of its biofilm formation, surface colonization, and virulence.

Candida albicans and Streptococcus mutans are known to synergistically interact with each other in the oral cavity. For example, glucosyltransferase B (GtfB), secreted by S. mutans, can bind to the C. albicans cell surface, promoting dual-species biofilm formation. However, the fungal factors mediating interactions with S. mutans are unknown. The C. albicans adhesins Als1, Als3, and Hwp1 are key players in C. albicans single-species biofilm formation, but their roles, if any, in interacting with S. mutans have not been assessed. Here, we investigated the roles of the C. albicans cell wall adhesins Als1, Als3, and Hwp1 on forming dual-species biofilms with S. mutans. We assessed the abilities of the C. albicans wild-type als1Δ/Δ, als3Δ/Δ, als1Δ/Δ/als3Δ/Δ, and hwp1Δ/Δ strains to form dual-species biofilms with S. mutans by measuring optical density, metabolic activity, cell enumeration, biomass, thickness, and architecture of the biofilms. We observed that the C. albicans wild-type strain formed enhanced dual-species biofilms in the presence of S. mutans in these different biofilm assays, confirming that C. albicans and S. mutans synergistically interact in the context of biofilms. Our results reveal that C. albicans Als1 and Hwp1 are major players in interacting with S. mutans, since dual-species biofilm formation was not enhanced when the als1Δ/Δ or hwp1Δ/Δ strains were cultured with S. mutans in dual-species biofilms. Als3, however, does not seem to play a clear role in interacting with S. mutans in dual-species biofilm formation. Overall, our data suggest that the C. albicans adhesins Als1 and Hwp1 function to modulate interactions with S. mutans and could be potential targets for future therapeutics.

Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast-to-hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. We isolated and cataloged >100 mucin O-glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, we identified three structures (core 1, core 1 + fucose and core 2 + galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, this work identifies mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.

The recently emerged fungal pathogen Candida auris often displays resistance to one or more antifungal drugs. Its infections have been identified in at least 40 countries on six continents to date. Here we report a case of C. auris candidemia in a patient in Xiamen, a city in south China. We also review currently reported cases of C. auris infection in China and compare the genetic and biological features of C. auris strains isolated from this country. Our phylogenetic analysis indicates that there are at least two C. auris genetic clades present in China (the South African clade and the south Asian clade) that display opposite mating type loci (one is MTL a and the other is MTLα). We also found that there are several distinct features among the clinical isolates studied, including the expression of virulence factors, antifungal susceptibilities, and cellular morphologies, and that these features could be associated with the mating-type of the isolate. For example, C. auris MTL a isolates generally secreted higher levels of secreted aspartyl proteases (Saps) at ambient environmental temperatures. Taken together, this study demonstrates that C. auris clinical isolates from China exhibit diversity in both biological and genetic features.

Candida auris is a multidrug-resistant human fungal pathogen that has recently emerged worldwide. It can cause life-threatening disseminated infections in humans, with mortality rates upwards of 50%. The molecular mechanisms underlying its multidrug resistance and pathogenic properties are largely unknown. Few methods exist for genome editing in C. auris, all of which rely on selectable markers that limit the number of modifications that can be made. Here, we present a markerless CRISPR/Cas9-mediated genome editing system in C. auris. Using this system, we successfully deleted genes of interest and subsequently reconstituted them at their native loci in isolates across all five C. auris clades. This system also enabled us to introduce precision genome edits to create translational fusions and single point mutations. Using Cas5 as a test case for this system, we discovered a conserved role for Cas5 in the caspofungin response between Candida albicans and C. auris. Overall, the development of a system for precise and facile genome editing in C. auris that can allow edits to be made in a high-throughput manner is a major step forward in improving our understanding of this important human fungal pathogen. IMPORTANCE Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Few tools are available for genome editing in C. auris. Here, we present a markerless genome editing system for C. auris that relies on CRISPR/Cas9 technology and works to modify the genomes of all known C. auris clades. Using this system, we discovered a conserved role for Cas5 in the caspofungin response between C. albicans and C. auris. Overall, the development of a system for facile genome editing in C. auris is a major step forward in improving our understanding of this important human fungal pathogen.

A striking feature of pathogenic Candida species is morphological plasticity that facilitates environmental adaptation and host infection. Candida auris is an emerging multidrug-resistant fungal pathogen first described in Japan in 2009. In this study, we demonstrate that clinical isolates of C. auris have multiple colony and cellular morphologies including the yeast, filamentous, aggregated, and elongated forms. This phenotypic diversity has been observed in eight clinical isolates of C. auris representing four major genetic clades, suggesting that it could be a general characteristic. We further demonstrate that different cell types of C. auris exhibit distinct antifungal resistance and virulence properties in a Galleria mellonella infection model. Our findings imply that morphological diversity is an important biological feature of C. auris and could be a contributor to its emergence and rapid prevalence worldwide.Lay summaryCandida auris is an emerging multidrug-resistant fungal pathogen. Morphological analyses indicate that filamentation is a general feature of clinical isolates of C. auris. This ability is associated with antifungal resistance and virulence.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER-resident protein, inositol-requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1-mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence-related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin-induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1-mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

Fungal infections are increasing in prevalence worldwide. The paucity of available antifungal drug classes, combined with the increased occurrence of multidrug resistance in fungi, has led to new clinical challenges in the treatment of fungal infections. Candida auris is a recently emerged multidrug resistant human fungal pathogen that has become a worldwide public health threat. C. auris clinical isolates are often resistant to one or more antifungal drug classes, and thus, there is a high unmet medical need for the development of new therapeutic strategies effective against C. auris. Additionally, C. auris possesses several virulence traits, including the ability to form biofilms, further contributing to its drug resistance, and complicating the treatment of C. auris infections. Here we assessed red, green, and blue visible lights alone and in combination with photosensitizing compounds for their efficacies against C. auris biofilms. We found that (1) blue light inhibited and disrupted C. auris biofilms on its own and that the addition of photosensitizing compounds improved its antibiofilm potential; (2) red light inhibited and disrupted C. auris biofilms, but only in combination with photosensitizing compounds; and (3) green light inhibited C. auris biofilms in combination with photosensitizing compounds, but had no effects on disrupting C. auris biofilms. Taken together, our findings suggest that photodynamic therapy could be an effective non-drug therapeutic strategy against multidrug resistant C. auris biofilm infections.

Candida albicans is one of the most common fungal pathogens of humans. Prior work introduced the planarian Schmidtea mediterranea as a new model system to study the host response to fungal infection at the organismal level. In the current study, we analyzed host-pathogen changes that occurred in situ during early infection with C. albicans. We found that the transcription factor Bcr1 and its downstream adhesin Als3 are required for C. albicans to adhere to and colonize the planarian epithelial surface, and that adherence of C. albicans triggers a multi-system host response that is mediated by the Dectin signaling pathway. This infection response is characterized by two peaks of stem cell divisions and transcriptional changes in differentiated tissues including the nervous and the excretory systems. This response bears some resemblance to a wound-like response to physical injury; however, it takes place without visible tissue damage and it engages a distinct set of progenitor cells. Overall, we identified two C. albicans proteins that mediate epithelial infection of planarians and a comprehensive host response facilitated by diverse tissues to effectively clear the infection.

Candida albicans, an opportunistic fungal pathogen, is a significant cause of human infections, particularly in immunocompromised individuals. Phenotypic plasticity between two morphological phenotypes, yeast and hyphae, is a key mechanism by which C. albicans can thrive in many microenvironments and cause disease in the host. Understanding the decision points and key driver genes controlling this important transition and how these genes respond to different environmental signals is critical to understanding how C. albicans causes infections in the host. Here we build and analyze a Boolean dynamical model of the C. albicans yeast to hyphal transition, integrating multiple environmental factors and regulatory mechanisms. We validate the model by a systematic comparison to prior experiments, which led to agreement in 17 out of 22 cases. The discrepancies motivate alternative hypotheses that are testable by follow-up experiments. Analysis of this model revealed two time-constrained windows of opportunity that must be met for the complete transition from the yeast to hyphal phenotype, as well as control strategies that can robustly prevent this transition. We experimentally validate two of these control predictions in C. albicans strains lacking the transcription factor UME6 and the histone deacetylase HDA1, respectively. This model will serve as a strong base from which to develop a systems biology understanding of C. albicans morphogenesis.

Fungal infections are increasing in prevalence worldwide, especially in immunocompromised individuals. Given the emergence of drug-resistant fungi and the fact that there are only three major classes of antifungal drugs available to treat invasive fungal infections, there is a need to develop alternative therapeutic strategies effective against fungal infections. Candida albicans is a commensal of the human microbiota that is also one of the most common fungal pathogens isolated from clinical settings. C. albicans possesses several virulence traits that contribute to its pathogenicity, including the ability to form drug-resistant biofilms, which can make C. albicans infections particularly challenging to treat. Here, we explored red, green, and blue visible lights alone and in combination with common photosensitizing compounds for their efficacies at inhibiting and disrupting C. albicans biofilms. We found that blue light inhibited biofilm formation and disrupted mature biofilms on its own and that the addition of photosensitizing compounds improved its antibiofilm potential. Red and green lights, however, inhibited biofilm formation only in combination with photosensitizing compounds but had no effects on disrupting mature biofilms. Taken together, these results suggest that photodynamic therapy may be an effective non-drug treatment for fungal biofilm infections that is worthy of further exploration.

The human fungal pathogen Candida albicans can form biofilms on biotic and abiotic surfaces, which are inherently resistant to antifungal drugs. We screened the Chembridge Small Molecule Diversity library containing 30,000 "drug-like" small molecules and identified 45 compounds that inhibited biofilm formation. These 45 compounds were then tested for their abilities to disrupt mature biofilms and for combinatorial interactions with fluconazole, amphotericin B, and caspofungin, the three antifungal drugs most commonly prescribed to treat Candida infections. In the end, we identified one compound that moderately disrupted biofilm formation on its own and four compounds that moderately inhibited biofilm formation and/or moderately disrupted mature biofilms only in combination with either caspofungin or fluconazole. No combinatorial interactions were observed between the compounds and amphotericin B. As members of a diversity library, the identified compounds contain "drug-like" chemical backbones, thus even seemingly "weak hits" could represent promising chemical starting points for the development and the optimization of new classes of therapeutics designed to target Candida biofilms.

Candida albicans is an opportunistic fungal pathogen of humans known for its ability to cause a wide range of infections. One major virulence factor of C. albicans is its ability to form hyphae that can invade host tissues and cause disseminated infections. Here, we introduce a method based on atomic force microscopy to investigate C. albicans hyphae in situ on silicone elastomer substrates, focusing on the effects of temperature and antifungal drugs. Hyphal growth rates differ significantly for measurements performed at different physiologically relevant temperatures. Furthermore, it is found that fluconazole is more effective than caspofungin in suppressing hyphal growth. We also investigate the effects of antifungal drugs on the mechanical properties of hyphal cells. An increase in Young's modulus and a decrease in adhesion force are observed in hyphal cells subjected to caspofungin treatment. Young's moduli are not significantly affected following treatment with fluconazole; the adhesion force, however, increases. Overall, our results provide a direct means of observing the effects of environmental factors and antifungal drugs on C. albicans hyphal growth and mechanics with high spatial resolution.IMPORTANCECandida albicans is one of the most common pathogens of humans. One important virulence factor of C. albicans is its ability to form elongated hyphae that can invade host tissues and cause disseminated infections. Here, we show the effect of different physiologically relevant temperatures and common antifungal drugs on the growth and mechanical properties of C. albicans hyphae using atomic force microscopy. We demonstrate that minor temperature fluctuations within the normal range can have profound effects on hyphal cell growth and that different antifungal drugs impact hyphal cell stiffness and adhesion in different ways.

The human fungal pathogen Candida albicans maintains pathogenic and commensal states primarily through cell wall functions. The echinocandin antifungal drug caspofungin inhibits cell wall synthesis and is widely used in treating disseminated candidiasis. Signaling pathways are critical in coordinating the adaptive response to cell wall damage (CWD). C. albicans executes a robust transcriptional program following caspofungin-induced CWD. A comprehensive analysis of signaling pathways at the transcriptional level facilitates the identification of prospective genes for functional characterization and propels the development of novel antifungal interventions. This review article focuses on the molecular functions and signaling crosstalk of the C. albicans transcription factors Sko1, Rlm1, and Cas5 in caspofungin-induced CWD signaling.

ABSTRACTCandida albicans is one of the most common fungal pathogens of humans. Prior work introduced the planarian Schmidtea mediterranea as a new model system to study the host response to fungal infection at the organismal level. In the current study, we analyzed host-pathogen changes that occurred in situ during early infection with C. albicans. We found that the transcription factor Bcr1 and its downstream adhesin Als3 are required for C. albicans to adhere to and colonize the planarian epithelial surface, and that adherence of C. albicans triggers a multi-system host response that is mediated by the Dectin signaling pathway. This infection response is characterized by two peaks of stem cell divisions and transcriptional changes in differentiated tissues including the nervous and the excretory systems. This response bears some resemblance to a wound-like response to physical injury; however, it takes place without visible tissue damage and it engages a distinct set of progenitor cells. Overall, we identified two C. albicans proteins that mediate epithelial infection of planarians and a comprehensive host response facilitated by diverse tissues to effectively clear the infection.

Mucus is an important host innate defense factor that lines most epithelial cell layers of the body and provides crucial physical and biological protection against pathogenic microorganisms. Mucins are the main glycoproteins of mucus that are responsible for interacting with microorganisms and are critical for the antimicrobial properties of mucus. The mechanisms by which microorganisms interact with mucins are poorly understood, especially in terms of fungi, and these interactions are continually evolving. Work in bacterial pathogens has shown that mucins inhibit bacterial virulence traits, including quorum sensing, toxin secretion and biofilm formation. Among the fungal clade, the common opportunistic human fungal pathogen and commensal Candida albicans engages in constant battle with the host innate immune system. This battle creates strong selective pressures for C. albicans to evolve in response to the host. Recent work in C. albicans found that mucins inhibit specific virulence traits, such as surface adherence, filamentation, biofilm formation and the production of secreted proteases. Here we review the current knowledge of microbial interactions with mucins, with a special emphasis on the interactions between C. albicans and mucins.

Antimicrobial peptides and proteins play critical roles in the host defense against invading pathogens. We recently discovered that recombinantly expressed human and mouse serum amyloid A1 (rhSAA1 and rmSAA1, respectively) proteins have potent antifungal activities against the major human fungal pathogen Candida albicans At high concentrations, rhSAA1 disrupts C. albicans membrane integrity and induces rapid fungal cell death. In the present study, we find that rhSAA1 promotes cell aggregation and targets the C. albicans cell wall adhesin Als3. Inactivation of ALS3 in C. albicans leads to a striking decrease in cell aggregation and cell death upon rhSAA1 treatment, suggesting that Als3 plays a critical role in SAA1 sensing. We further demonstrate that deletion of the transcriptional regulators controlling the expression of ALS3, such as AHR1, BCR1, and EFG1, in C. albicans results in similar effects to that of the als3/als3 mutant upon rhSAA1 treatment. Global gene expression profiling indicates that rhSAA1 has a discernible impact on the expression of cell wall- and metabolism-related genes, suggesting that rhSAA1 treatment could lead to a nutrient starvation effect on C. albicans cells.

Candida albicans is a major human fungal pathogen, capable of switching among a range of morphological types, such as the yeast form, including white and opaque cell types and the GUT (gastrointestinally induced transition) cell type, the filamentous form, including hyphal and pseudohyphal cell types, and chlamydospores. This ability is associated with its commensal and pathogenic life styles. In response to pheromone, C. albicans cells are able to form long mating projections resembling filaments. This filamentous morphology is required for efficient sexual mating. In the current study, we report the genetic regulatory mechanisms controlling the development of mating projections in C. albicans. Ectopic expression of MTLα1 in "a" cells induces the secretion of α-pheromone and promotes the development of mating projections. Using this inducible system, we reveal that members of the pheromone-sensing pathway (including the pheromone receptor), the Ste11-Hst7-Cek1/2 mediated MAPK signalling cascade, and the RAM pathway are essential for the development of mating projections. However, the cAMP/PKA signalling pathway and a number of key regulators of filamentous growth such as Hgc1, Efg1, Flo8, Tec1, Ume6, and Rfg1 are not required for mating projection formation. Therefore, despite the phenotypic similarities between filaments and mating projections in C. albicans, distinct mechanisms are involved in the regulation of these two morphologies.

Candida albicans is a commensal member of the human microbiota that colonizes multiple niches in the body including the skin, oral cavity, and gastrointestinal and genitourinary tracts of healthy individuals. It is also the most common human fungal pathogen isolated from patients in clinical settings. C. albicans can cause a number of superficial and invasive infections, especially in immunocompromised individuals. The ability of C. albicans to succeed as both a commensal and a pathogen, and to thrive in a wide range of environmental niches within the host, requires sophisticated transcriptional regulatory programs that can integrate and respond to host specific environmental signals. Identifying and characterizing the transcriptional regulatory networks that control important developmental processes in C. albicans will shed new light on the strategies used by C. albicans to colonize and infect its host. Here, we discuss the transcriptional regulatory circuits controlling three major developmental processes in C. albicans: biofilm formation, the white-opaque phenotypic switch, and the commensal-pathogen transition. Each of these three circuits are tightly knit and, through our analyses, we show that they are integrated together by extensive regulatory crosstalk between the core regulators that comprise each circuit.

The human fungal pathogen Candida albicans is constantly exposed to environmental challenges impacting the cell wall. Signaling pathways coordinate stress adaptation and are essential for commensalism and virulence. The transcription factors Sko1, Cas5, and Rlm1 control the response to cell wall stress caused by the antifungal drug caspofungin. Here, we expand the Sko1 and Rlm1 transcriptional circuit and demonstrate that Rlm1 activates Sko1 cell wall stress signaling. Caspofungin-induced transcription of SKO1 and several Sko1-dependent cell wall integrity genes are attenuated in an rlm1Δ/Δ mutant strain when compared to the treated wild-type strain but not in a cas5Δ/Δ mutant strain. Genome-wide chromatin immunoprecipitation (ChIP-seq) results revealed numerous Sko1 and Rlm1 directly bound target genes in the presence of caspofungin that were undetected in previous gene expression studies. Notable targets include genes involved in cell wall integrity, osmolarity, and cellular aggregation, as well as several uncharacterized genes. Interestingly, we found that Rlm1 does not bind to the upstream intergenic region of SKO1 in the presence of caspofungin, indicating that Rlm1 indirectly controls caspofungin-induced SKO1 transcription. In addition, we discovered that caspofungin-induced SKO1 transcription occurs through self-activation. Based on our ChIP-seq data, we also discovered an Rlm1 consensus motif unique to C. albicans. For Sko1, we found a consensus motif similar to the known Sko1 motif for Saccharomyces cerevisiae. Growth assays showed that SKO1 overexpression suppressed caspofungin hypersensitivity in an rlm1Δ/Δ mutant strain. In addition, overexpression of the glycerol phosphatase, RHR2, suppressed caspofungin hypersensitivity specifically in a sko1Δ/Δ mutant strain. Our findings link the Sko1 and Rlm1 signaling pathways, identify new biological roles for Sko1 and Rlm1, and highlight the complex dynamics underlying cell wall signaling.

First described in 2009 in Japan, the emerging multidrug-resistant fungal pathogen Candida auris is becoming a worldwide public health threat that has been attracting considerable attention due to its rapid and widespread emergence over the past decade. The reasons behind the recent emergence of this fungus remain a mystery to date. Genetic analyses indicate that this fungal pathogen emerged simultaneously in several different continents, where 5 genetically distinct clades of C. auris were isolated from distinct geographical locations. Although C. auris belongs to the CTG clade (its constituent species translate the CTG codon as serine instead of leucine, as in the standard code), C. auris is a haploid fungal species that is more closely related to the haploid and often multidrug-resistant species Candida haemulonii and Candida lusitaniae and is distantly related to the diploid and clinically common fungal pathogens Candida albicans and Candida tropicalis. Infections and outbreaks caused by C. auris in hospitals settings have been rising over the past several years. Difficulty in its identification, multidrug resistance properties, evolution of virulence factors, associated high mortality rates in patients, and long-term survival on surfaces in the environment make C. auris particularly problematic in clinical settings. Here, we review progress made over the past decade on the biological and clinical aspects of C. auris. Future efforts should be directed toward understanding the mechanistic details of its biology, epidemiology, antifungal resistance, and pathogenesis with a goal of developing novel tools and methods for the prevention, diagnosis, and treatment of C. auris infections.

Biofilms formed by the human fungal pathogen Candida albicans are naturally resistant to many of the antifungal agents commonly used in the clinic. We screened a library containing 1600 clinically tested drug compounds to identify compounds that inhibit C. albicans biofilm formation. The compounds that emerged from the initial screen were validated in a secondary screen and then tested for (1) their abilities to disrupt mature biofilms and (2) for synergistic interactions with representatives of the three antifungal agents most commonly prescribed to treat Candida infections, fluconazole, amphotericin B, and caspofungin. Twenty compounds had antibiofilm activity in at least one of the secondary assays and several affected biofilms but, at the same concentration, had little or no effect on planktonic (suspension) growth of C. albicans. Two calcium channel blockers, a selective serotonin reuptake inhibitor, and an azole-based proton pump inhibitor were among the hits, suggesting that members of these three classes of drugs or their derivatives may be useful for treating C. albicans biofilm infections.

Biofilms, structured and densely packed communities of microbial cells attached to surfaces, are considered to be the natural growth state for a vast majority of microorganisms. The ability to form biofilms is an important virulence factor for most pathogens, including the opportunistic human fungal pathogen Candida albicans. C. albicans is one of the most prevalent fungal species of the human microbiota that asymptomatically colonizes healthy individuals. However, C. albicans can also cause severe and life-threatening infections when host conditions permit (e.g., through alterations in the host immune system, pH, and resident microbiota). Like many other pathogens, this ability to cause infections depends, in part, on the ability to form biofilms. Once formed, C. albicans biofilms are often resistant to antifungal agents and the host immune response, and can act as reservoirs to maintain persistent infections as well as to seed new infections in a host. The majority of C. albicans clinical isolates are heterozygous (a/α) at the mating type-like (MTL) locus, which defines Candida mating types, and are capable of forming robust biofilms when cultured in vitro. These "conventional" biofilms, formed by MTL-heterozygous (a/α) cells, have been the primary focus of C. albicans biofilm research to date. Recent work in the field, however, has uncovered novel mechanisms through which biofilms are generated by C. albicans cells that are homozygous or hemizygous (a/a, a/Δ, α/α, or α/Δ) at the MTL locus. In these studies, the addition of pheromones of the opposite mating type can induce the formation of specialized "sexual" biofilms, either through the addition of synthetic peptide pheromones to the culture, or in response to co-culturing of cells of the opposite mating types. Although sexual biofilms are generally less robust than conventional biofilms, they could serve as a protective niche to support genetic exchange between mating-competent cells, and thus may represent an adaptive mechanism to increase population diversity in dynamic environments. Although conventional and sexual biofilms appear functionally distinct, both types of biofilms are structurally similar, containing yeast, pseudohyphal, and hyphal cells surrounded by an extracellular matrix. Despite their structural similarities, conventional and sexual biofilms appear to be governed by distinct transcriptional networks and signaling pathways, suggesting that they may be adapted for, and responsive to, distinct environmental conditions. Here we review sexual biofilms and compare and contrast them to conventional biofilms of C. albicans.

The sensing and efficient utilization of environmental nutrients are critical for the survival of microorganisms in environments where nutrients are limited, such as within mammalian hosts. Candida albicans is a common member of the human microbiota as well as an opportunistic fungal pathogen. The amide derivative sugar N-acetlyglucosamine (GlcNAc) is an important signaling molecule for C. albicans that could be a major nutrient source for this fungus in host settings. In this article, we review progress made over the past two decades on GlcNAc utilization, sensing, and functions in C. albicans and its related fungal species. GlcNAc sensing and catabolic pathways have been intensively studied in C. albicans. The C. albicans protein Ngt1 represents the first identified GlcNAc-specific transporter in eukaryotic organisms. In C. albicans, GlcNAc not only induces morphological transitions including the yeast to hyphal transition and the white to opaque phenotypic switch, but it also promotes fungal cell death. The Ras-cAMP/PKA signaling pathway plays critical roles in regulating these processes. Given the importance of GlcNAc sensing and utilization in C. albicans, targeting GlcNAc associated pathways and key pathway components could be promising in the development of new antifungal strategies.

Biofilms formed by the fungal pathogen Candida albicans are resistant to many of the antifungal agents commonly used in the clinic. Previous reports suggest that protease inhibitors, specifically inhibitors of aspartyl proteases, could be effective antibiofilm agents. We screened three protease inhibitor libraries, containing a total of 80 compounds for the abilities to prevent C. albicans biofilm formation and to disrupt mature biofilms. The compounds were screened individually and in the presence of subinhibitory concentrations of the most commonly prescribed antifungal agents for Candida infections: fluconazole, amphotericin B, or caspofungin. Although few of the compounds affected biofilms on their own, seven aspartyl protease inhibitors inhibited biofilm formation when combined with amphotericin B or caspofungin. Furthermore, nine aspartyl protease inhibitors disrupted mature biofilms when combined with caspofungin. These results suggest that the combination of standard antifungal agents together with specific protease inhibitors may be useful in the prevention and treatment of C. albicans biofilm infections.

Mammalian serum amyloid A (SAA) is a major acute phase protein that shows a massive increase in plasma concentration during inflammation. In the present study, we demonstrate that the expression of mouse SAA1 in serum was increased when infected with Candida albicans, a major human fungal pathogen, in a systemic infection model. We then set out to investigate the antifungal activity of SAA proteins against C. albicans Recombinant human and mouse SAA1 (rhSAA1 and rmSAA1) were expressed and purified in Escherichia coli Both rhSAA1 and rmSAA1 exhibited a potent antifungal activity against C. albicans We further demonstrate that rhSAA1 binds to the cell surface of C. albicans, disrupts cell membrane integrity, and induces rapid fungal cell death in C. albicans Our finding expands the known functions of SAA1 and provides new insight into host-Candida interactions during fungal infection.

Venom is a known source of novel antimicrobial natural products. The substantial, increasing number of these discoveries have unintentionally culminated in the misconception that venom and venom-producing glands are largely sterile environments. Culture-dependent and -independent studies on the microbial communities in venom microenvironments reveal the presence of archaea, algae, bacteria, fungi, protozoa, and viruses. Venom-centric microbiome studies are relatively sparse to date with the adaptive advantages that venom-associated microbes might offer to their hosts, or that hosts might provide to venom-associated microbes, remaining largely unknown. We highlight the potential for the discovery of venom microbiomes within the adaptive landscape of venom systems. The considerable number of convergently evolved venomous animals, juxtaposed with the comparatively few known studies to identify microbial communities in venom, provides new possibilities for both biodiversity and therapeutic discoveries. We present an evidence-based argument for integrating microbiology as part of venomics (i.e., venom-microbiomics) and introduce iVAMP, the Initiative for Venom Associated Microbes and Parasites (https://ivamp-consortium.github.io/), as a growing collaborative consortium. We express commitment to the diversity, inclusion and scientific collaboration among researchers interested in this emerging subdiscipline through expansion of the iVAMP consortium.

Candida albicans is one of the most common fungal pathogens of humans. Currently, there are limitations in the evaluation of C. albicans infection in existing animal models, especially in terms of understanding the influence of specific infectious stages of the fungal pathogen on the host. We show that C. albicans infects, grows and invades tissues in the planarian flatworm Schmidtea mediterranea, and that the planarian responds to infection by activating components of the host innate immune system to clear and repair host tissues. We study different stages of C. albicans infection and demonstrate that planarian stem cells increase division in response to fungal infection, a process that is likely evolutionarily conserved in metazoans. Our results implicate MORN2 and TAK1/p38 signaling pathways as possible mediators of the host innate immune response to fungal infection. We propose the use of planarians as a model system to investigate host-pathogen interactions during fungal infections.

Candida albicans is a normal member of the human microbiota that asymptomatically colonizes healthy individuals, however it is also an opportunistic pathogen that can cause severe infections, especially in immunocompromised individuals. The medical impact of C. albicans depends, in part, on its ability to form biofilms, communities of adhered cells encased in an extracellular matrix. Biofilms can form on both biotic and abiotic surfaces, such as tissues and implanted medical devices. Once formed, biofilms are highly resistant to antifungal agents and the host immune system, and can act as a protected reservoir to seed disseminated infections. Here, we present several in vitro biofilm protocols, including protocols that are optimized for high-throughput screening of mutant libraries and antifungal compounds. We also present protocols to examine specific stages of biofilm development and protocols to evaluate interspecies biofilms that C. albicans forms with interacting microbial partners. © 2018 by John Wiley & Sons, Inc.

Candida albicans is among the most prevalent fungal species of the human microbiota and asymptomatically colonizes healthy individuals. However, it is also an opportunistic pathogen that can cause severe, and often fatal, bloodstream infections. The medical impact of C. albicans typically depends on its ability to form biofilms, which are closely packed communities of cells that attach to surfaces, such as tissues and implanted medical devices. In this Review, we provide an overview of the processes involved in the formation of C. albicans biofilms and discuss the core transcriptional network that regulates biofilm development. We also consider some of the advantages that biofilms provide to C. albicans in comparison with planktonic growth and explore polymicrobial biofilms that are formed by C. albicans and certain bacterial species.

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

Fungal pathogens such as Candida albicans exhibit several survival mechanisms to evade attack by antifungals and colonise host tissues. Rta3, a member of the Rta1-like family of lipid-translocating exporters has a 7-transmembrane domain topology, similar to the G-protein-coupled receptors and is unique to the fungal kingdom. Our findings point towards a role for the plasma membrane localised Rta3 in providing tolerance to miltefosine, an analogue of alkylphosphocholine, by maintaining mitochondrial energetics. Concurrent with miltefosine susceptibility, the rta3Δ/Δ strain displays increased inward translocation (flip) of fluorophore-labelled phosphatidylcholine (PC) across the plasma membrane attributed to enhanced PC-specific flippase activity. We also assign a novel role to Rta3 in the Bcr1-regulated pathway for in vivo biofilm development. Transcriptome analysis reveals that Rta3 regulates expression of Bcr1 target genes involved in cell surface properties, adhesion, and hyphal growth. We show that rta3Δ/Δ mutant is biofilm-defective in a rat venous catheter model of infection and that BCR1 overexpression rescues this defect, indicating that Bcr1 functions downstream of Rta3 to mediate biofilm formation in C. albicans. The identification of this novel Rta3-dependent regulatory network that governs biofilm formation and PC asymmetry across the plasma membrane will provide important insights into C. albicans pathogenesis.

Candida albicans and Streptococcus oralis are ubiquitous oral commensal organisms. Under host-permissive conditions these organisms can form hypervirulent mucosal biofilms. C. albicans biofilm formation is controlled by 6 master transcriptional regulators: Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1. The objective of this work was to test whether any of these regulators play a role in cross-kingdom interactions between C. albicans and S. oralis in oral mucosal biofilms, and identify downstream target gene(s) that promote these interactions. Organotypic mucosal constructs and a mouse model of oropharyngeal infection were used to analyze mucosal biofilm growth and fungal gene expression. By screening 6 C. albicans transcription regulator reporter strains we discovered that EFG1 was strongly activated by interaction with S. oralis in late biofilm growth stages. EFG1 gene expression was increased in polymicrobial biofilms on abiotic surfaces, mucosal constructs and tongue tissues of mice infected with both organisms. EFG1 was required for robust Candida-streptococcal biofilm growth in organotypic constructs and mouse oral tissues. S. oralis stimulated C. albicans ALS1 gene expression in an EFG1-dependent manner, and Als1 was identified as a downstream effector of the Efg1 pathway which promoted C. albicans-S. oralis coaggregation interactions in mixed biofilms. We conclude that S. oralis induces an increase in EFG1 expression in C. albicans in late biofilm stages. This in turn increases expression of ALS1, which promotes coaggregation interactions and mucosal biofilm growth. Our work provides novel insights on C. albicans genes which play a role in cross-kingdom interactions with S. oralis in mucosal biofilms.

Morphological plasticity such as the yeast-to-hyphae transition is a key virulence factor of the human fungal pathogen Candida albicans. Hyphal formation is controlled by a multilayer regulatory network composed of environmental sensing, signaling, transcriptional modulators as well as chromatin modifications. Here, we demonstrate a novel role for the replication-independent HIR histone chaperone complex in fungal morphogenesis. HIR operates as a crucial modulator of hyphal development, since genetic ablation of the HIR complex subunit Hir1 decreases sensitivity to morphogenetic stimuli. Strikingly, HIR1-deficient cells display altered transcriptional amplitudes upon hyphal initiation, suggesting that Hir1 affects transcription by establishing transcriptional thresholds required for driving morphogenetic cell-fate decisions. Furthermore, ectopic expression of the transcription factor Ume6, which facilitates hyphal maintenance, rescues filamentation defects of hir1Δ/Δ cells, suggesting that Hir1 impacts the early phase of hyphal initiation. Hence, chromatin chaperone-mediated fine-tuning of transcription is crucial for driving morphogenetic conversions in the fungal pathogen C. albicans.

Candida albicans is the most common cause of life-threatening fungal infections in humans, especially in immunocompromised individuals. Crucial to its success as an opportunistic pathogen is the considerable dynamism of its genome, which readily undergoes genetic changes generating new phenotypes and shaping the evolution of new strains. Candida africana is an intriguing C. albicans biovariant strain that exhibits remarkable genetic and phenotypic differences when compared with standard C. albicans isolates. Candida africana is well-known for its low degree of virulence compared with C. albicans and for its inability to produce chlamydospores that C. albicans, characteristically, produces under certain environmental conditions. Chlamydospores are large, spherical structures, whose biological function is still unknown. For this reason, we have sequenced, assembled, and annotated the whole transcriptomes obtained from an efficient C. albicans chlamydospore-producing clinical strain (GE1), compared with the natural chlamydospore-negative C. africana clinical strain (CBS 11016). The transcriptomes of both C. albicans (GE1) and C. africana (CBS 11016) clinical strains, grown under chlamydospore-inducing conditions, were sequenced and assembled into 7,442 (GE1 strain) and 8,370 (CBS 11016 strain) high quality transcripts, respectively. The release of the first assembly of the C. africana transcriptome will allow future comparative studies to better understand the biology and evolution of this important human fungal pathogen.

Candida albicans biofilms have a significant medical impact due to their rapid growth on implanted medical devices, their resistance to antifungal drugs, and their ability to seed disseminated infections. Biofilm assays performed in vitro allow for rapid, high-throughput screening of gene deletion libraries or antifungal compounds and typically serve as precursors to in vivo studies. Here, we compile and discuss the protocols for several recently published C. albicansin vitro biofilm assays. We also describe improved versions of these protocols as well as novel in vitro assays. Finally, we consider some of the advantages and disadvantages of these different types of assays.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Morphological transitions and metabolic regulation are critical for the human fungal pathogen Candida albicans to adapt to the changing host environment. In this study, we generated a library of central metabolic pathway mutants in the tricarboxylic acid (TCA) cycle, and investigated the functional consequences of these gene deletions on C. albicans biology. Inactivation of the TCA cycle impairs the ability of C. albicans to utilize non-fermentable carbon sources and dramatically attenuates cell growth rates under several culture conditions. By integrating the Ras1-cAMP signaling pathway and the heat shock factor-type transcription regulator Sfl2, we found that the TCA cycle plays fundamental roles in the regulation of CO2 sensing and hyphal development. The TCA cycle and cAMP signaling pathways coordinately regulate hyphal growth through the molecular linkers ATP and CO2. Inactivation of the TCA cycle leads to lowered intracellular ATP and cAMP levels and thus affects the activation of the Ras1-regulated cAMP signaling pathway. In turn, the Ras1-cAMP signaling pathway controls the TCA cycle through both Efg1- and Sfl2-mediated transcriptional regulation in response to elevated CO2 levels. The protein kinase A (PKA) catalytic subunit Tpk1, but not Tpk2, may play a major role in this regulation. Sfl2 specifically binds to several TCA cycle and hypha-associated genes under high CO2 conditions. Global transcriptional profiling experiments indicate that Sfl2 is indeed required for the gene expression changes occurring in response to these elevated CO2 levels. Our study reveals the regulatory role of the TCA cycle in CO2 sensing and hyphal development and establishes a novel link between the TCA cycle and Ras1-cAMP signaling pathways.

UnlabelledCandida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation.ImportanceBiofilm formation by the opportunistic fungal pathogen C. albicans is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by C. albicans biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during C. albicans biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from C. albicans and also from additional pathogenic Candida species. Furthermore, SAP5 and SAP6 deletions confirm that both proteases are required for proper biofilm development in vitro and in vivo We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of Candida biofilm-associated infections.

Microorganisms rarely exist as single species in natural environments. The opportunistic fungal pathogen Candida albicans and lactic acid bacteria (LAB) are common members of the microbiota of several human niches such as the mouth, gut and vagina. Lactic acid bacteria are known to suppress filamentation, a key virulence feature of C. albicans, through the production of lactic acid and other metabolites. Here we report that C. albicans cells switch between two heritable cell types, white and opaque, to undergo filamentation to adapt to diversified environments. We show that acidic pH conditions caused by LAB and low temperatures support opaque cell filamentation, while neutral pH conditions and high temperatures promote white cell filamentation. The cAMP signalling pathway and the Rfg1 transcription factor play major roles in regulating the responses to these conditions. This cell type-specific response of C. albicans to different environmental conditions reflects its elaborate regulatory control of phenotypic plasticity.

A major virulence attribute of Candida albicans is its ability to form biofilms, densely packed communities of cells adhered to a surface. These biofilms are intrinsically resistant to conventional antifungal therapeutics, the host immune system, and other environmental factors, making biofilm-associated infections a significant clinical challenge. Here, we review current knowledge on the development, regulation, and molecular mechanisms of C. albicans biofilms.

UnlabelledThe human commensal and opportunistic pathogen Candida albicans can switch between two distinct, heritable cell types, named "white" and "opaque," which differ in morphology, mating abilities, and metabolic preferences and in their interactions with the host immune system. Previous studies revealed a highly interconnected group of transcriptional regulators that control switching between the two cell types. Here, we identify Ssn6, the C. albicans functional homolog of the Saccharomyces cerevisiae transcriptional corepressor Cyc8, as a new regulator of white-opaque switching. In A: or α mating type strains, deletion of SSN6 results in mass switching from the white to the opaque cell type. Transcriptional profiling of ssn6 deletion mutant strains reveals that Ssn6 represses part of the opaque cell transcriptional program in white cells and the majority of the white cell transcriptional program in opaque cells. Genome-wide chromatin immunoprecipitation experiments demonstrate that Ssn6 is tightly integrated into the opaque cell regulatory circuit and that the positions to which it is bound across the genome strongly overlap those bound by Wor1 and Wor2, previously identified regulators of white-opaque switching. This work reveals the next layer in the white-opaque transcriptional circuitry by integrating a transcriptional regulator that does not bind DNA directly but instead associates with specific combinations of DNA-bound transcriptional regulators.ImportanceThe most common fungal pathogen of humans, C. albicans, undergoes several distinct morphological transitions during interactions with its host. One such transition, between cell types named "white" and "opaque," is regulated in an epigenetic manner, in the sense that changes in gene expression are heritably maintained without any modification of the primary genomic DNA sequence. Prior studies revealed a highly interconnected network of sequence-specific DNA-binding proteins that control this switch. We report the identification of Ssn6, which defines an additional layer of transcriptional regulation that is critical for this heritable switch. Ssn6 is necessary to maintain the white cell type and to properly express the opaque cell transcriptional program. Ssn6 does not bind DNA directly but rather associates with specific combinations of DNA-bound transcriptional regulators to control the switch. This work is significant because it reveals a new level of regulation of an important epigenetic switch in the predominant fungal pathogen of humans.

UnlabelledSingle-celled organisms have different strategies to sense and utilize nutrients in their ever-changing environments. The opportunistic fungal pathogen Candida albicans is a common member of the human microbiota, especially that of the gastrointestinal (GI) tract. An important question concerns how C. albicans gained a competitive advantage over other microbes to become a successful commensal and opportunistic pathogen. Here, we report that C. albicans uses N-acetylglucosamine (GlcNAc), an abundant carbon source present in the GI tract, as a signal for nutrient availability. When placed in water, C. albicans cells normally enter the G0 phase and remain viable for weeks. However, they quickly lose viability when cultured in water containing only GlcNAc. We term this phenomenon GlcNAc-induced cell death (GICD). GlcNAc triggers the upregulation of ribosomal biogenesis genes, alterations of mitochondrial metabolism, and the accumulation of reactive oxygen species (ROS), followed by rapid cell death via both apoptotic and necrotic mechanisms. Multiple pathways, including the conserved cyclic AMP (cAMP) signaling and GlcNAc catabolic pathways, are involved in GICD. GlcNAc acts as a signaling molecule to regulate multiple cellular programs in a coordinated manner and therefore maximizes the efficiency of nutrient use. This adaptive behavior allows C. albicans' more efficient colonization of the gut.ImportanceThe ability to rapidly and appropriately respond to nutrients in the environment is crucial to free-living microorganisms. To maximize the use of available nutrients, microorganisms often use a limiting nutritional component as a signal to coordinate multiple biological processes. The human fungal pathogen Candida albicans uses N-acetylglucosamine (GlcNAc) as a signal for the availability of external nutrient resources. GlcNAc induces rapid cell death in C. albicans due to the constitutive activation of oxidative metabolism and accumulation of reactive oxygen species (ROS), and multiple pathways are involved in its regulation. This study sheds light on the mechanisms of niche specialization of pathogenic fungi and raises the possibility that this cell death pathway could be an unexplored therapeutic target.