Opportunistic Human Fungal Pathogen Research Articles

Insight into Virulence and Mechanisms of Amphotericin B Resistance in the Candida haemulonii Complex.

The Candida haemulonii complex includes emerging opportunistic human fungal pathogens with documented multidrug-resistance profiles. It comprises Candida haemulonii sensu stricto, Candida haemulonii var. vulnera, Candida duobushaemulonii, Candida pseudohaemulonii, and Candida vulturna. In recent years, rates of clinical isolation of strains from this complex have increased in multiple countries, including China, Malaysia, and Brazil. Biofilm formation, hydrolytic enzymes, surface interaction properties, phenotype switching and cell aggregation abilities, extracellular vesicles production, stress response, and immune evasion help these fungi to infect the host and exert pathological effects. Multidrug resistance profiles also enhance the threat they pose; they exhibit low susceptibility to echinocandins and azoles and an intrinsic resistance to amphotericin B (AMB), the first fungal-specific antibiotic. AMB is commonly employed in antifungal treatments, and it acts via several known mechanisms. Given the propensity of clinical Candida species to initiate bloodstream infections, clarifying how C. haemulonii resists AMB is of critical clinical importance. This review outlines our present understanding of the C. haemulonii complex's virulence factors, the mechanisms of action of AMB, and the mechanisms underlying AMB resistance.

Adiponectin pathway activation dampens inflammation and enhances alveolar macrophage fungal killing via LC3-associated phagocytosis.

Although innate immunity is critical for antifungal host defense against the human opportunistic fungal pathogen Aspergillus fumigatus, potentially damaging inflammation must be controlled. Adiponectin (APN) is an adipokine produced mainly in adipose tissue that exerts anti-inflammatory effects in adipose-distal tissues such as the lung. We observed 100% mortality and increased fungal burden and inflammation in neutropenic mice with invasive aspergillosis (IA) that lack APN or the APN receptors AdipoR1 or AdipoR2. Alveolar macrophages (AMs), early immune sentinels that detect and respond to lung infection, express both receptors, and APN-/- AMs exhibited an inflammatory/M1 phenotype that was associated with decreased fungal killing. Pharmacological stimulation of AMs with AdipoR agonist AdipoRon partially rescued deficient killing in APN-/- AMs that was dependent on both receptors. Finally, APN-enhanced fungal killing was associated with increased activation of the non-canonical LC3 pathway of autophagy. Thus, our study identifies a novel role for APN in LC3-mediated killing of A. fumigatus.

Nitrate assimilation compensates for cell wall biosynthesis in the absence of Aspergillus fumigatus phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen Aspergillus fumigatus, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δpgi mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein N-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δpgi mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δpgi mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in A. fumigatus.IMPORTANCEAspergillus fumigatus is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of A. fumigatus PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein N-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of A. fumigatus to address cell wall defects, which could hold promise for the treatment of infections.

Nanorobots to Treat Candida albicans Infection.

Candida albicans is an opportunistic fungal pathogen of humans. It causes a variety of infections ranging from superficial mucocutaneous conditions to severe systemic diseases that result in substantial morbidity and mortality. This pathogen frequently forms biofilms resistant to antifungal drugs and the host immune system, leading to treatment failures. Recent research has demonstrated the potential of nanorobots to penetrate biological barriers and disrupt fungal biofilms. In this perspective paper, we provide a brief overview of recent breakthroughs in nanorobots for candidiasis treatment and discuss current challenges and prospects.

Sirtulin-Ypk1 regulation axis governs the TOR signaling pathway and fungal pathogenicity in Cryptococcus neoformans.

Cryptococcus neoformans is an important opportunistic fungal pathogen in humans. While there are currently few effective antifungal treatments, the absence of novel molecular targets in fungal pathogenicity hinders the development of new drugs. There is increasing evidence that protein post-translational modifications (PTMs) can modulate the pathogenicity of fungi. In this study, we discovered that the pathogenicity of C. neoformans was significantly impacted by the dynamic acetylation changes of Ypk1, the immediate downstream target of the TOR complex. We discovered that Ypk1 is acetylated at lysines 315 and 502, both of which are within kinase functional domains. Deacetylation of Ypk1 is necessary for formation of the capsule structure, the response to the TOR pathway inhibitor rapamycin, nutrient utilization, and host infection. We also demonstrate that the sirtuin protein family is involved in the Ypk1 deacetylation mechanism. We anticipate that the sirtuin-Ypk1 regulation axis could be used as a potential target for the development of antifungal medications.

Unveiling the antifungal mechanisms of CTP, a new copper(II)-theophylline/1,10-phenanthroline complex, on drug-resistant non-albicans Candida species.

Candida species undeniably rank as the most prevalent opportunistic human fungal pathogens worldwide, with Candida albicans as the predominant representative. However, the emergence of non-albicans Candida species (NACs) has marked a significant shift, accompanied by rising incidence rates and concerning trends of antifungal resistance. The search for new strategies to combat antifungal-resistant Candida strains is of paramount importance. Recently, our research group reported the anti-Candida activity of a coordination compound containing copper(II) complexed with theophylline (theo) and 1,10-phenanthroline (phen), known as "CTP" - Cu(theo)2phen(H2O).5H2O. In the present work, we investigated the mechanisms of action of CTP against six medically relevant, antifungal-resistant NACs, including C. auris, C. glabrata, C. haemulonii, C. krusei, C. parapsilosis and C. tropicalis. CTP demonstrated significant efficacy in inhibiting mitochondrial dehydrogenases, leading to heightened intracellular reactive oxygen species production. CTP treatment resulted in substantial damage to the plasma membrane, as evidenced by the passive incorporation of propidium iodide, and induced DNA fragmentation as revealed by the TUNEL assay. Scanning electron microscopy images of post-CTP treatment NACs further illustrated profound alterations in the fungal surface morphology, including invaginations, cavitations and lysis. These surface modifications significantly impacted the ability of Candida cells to adhere to a polystyrene surface and to form robust biofilm structures. Moreover, CTP was effective in disassembling mature biofilms formed by these NACs. In conclusion, CTP represents a promising avenue for the development of novel antifungals with innovative mechanisms of action against clinically relevant NACs that are resistant to antifungals commonly used in clinical settings.

The Putative Cytochrome b5 Domain-Containing Protein CaDap1 Homologue Is Involved in Antifungal Drug Tolerance, Cell Wall Chitin Maintenance, and Virulence in Candida albicans.

Candida albicans (Ca), a prominent opportunistic fungal pathogen in humans, has garnered considerable attention due to its infectious properties. Herein, we have identified and characterized CaCDAP1 (Ca orf19.1034), a homolog of ScDAP1 found in Saccharomyces cerevisiae. CaCDAP1 encodes a 183-amino acid protein with a conserved cytochrome b5-like heme-binding domain. The deletion of CaDAP1 renders Ca cells susceptible to caspofungin and terbinafine. CaDAP1 deletion confers resistance to Congo Red and Calcofluor White, and sensitivity to sodium dodecyl sulfate. The deletion of CaDAP1 results in a 50% reduction in chitin content within the cell wall, the downregulation of phosphorylation levels in CaMkc1, and the upregulation of phosphorylation levels in CaCek1. Notably, CaDAP1 deletion results in the abnormal hyphal development of Ca cells and diminishes virulence in a mouse systemic infection model. Thus, CaDAP1 emerges as a critical regulator governing cellular responses to antifungal drugs, the synthesis of cell wall chitin, and virulence in Ca.

Thamnostylum piriforme, a novel etiological agent of superficial mycosis

Fungi are opportunistic eukaryotic entities often taking advantage of susceptibilities offered by a host due to its immunocompromised status, changed microbiome, or ruptured physical barriers and eventually cause infections. They either invade the skin superficially or are deep-seated. Superficial mycosis affects the skin, hair, and nails inhabiting the outermost layer, stratum corneum. In the present study, we report a case of superficial mycosis (onychomycosis in particular) in a 45-year-old immunocompetent man who was an ex-defense personnel and presently serving as a security guard at the University of Jammu, District Jammu, Jammu and Kashmir, India. The infection evolved 17 years ago and negatively affected the quality of life of the patient. For the identification of the causal agent, direct microscopy, cultural, micro-morphological, molecular characterization (ITS sequencing), and phylogenetic analysis were taken into account. A mucoralean fungal species, Thamnostylum piriforme, was isolated from the fingernails (left hand) of the investigated patient, which represents a new global report as the causal agent of superficial mycosis. In vitro antifungal susceptibility testing showed T. piriforme sensitivity to itraconazole, amphotericin B and ketoconazole while resistance to fluconazole. Careful selection of optimal therapy for fungal infection based primarily on correct identification and antifungal susceptibility testing could provide effective results during treatment against these opportunistic human fungal pathogens.

Extracellular Vesicles from a Biofilm of a Clinical Isolate of Candida albicans Negatively Impact on Klebsiella pneumoniae Adherence and Biofilm Formation.

The opportunistic human fungal pathogen Candida albicans produces and releases into the surrounding medium extracellular vesicles (EVs), which are involved in some processes as communication between fungal cells and host-pathogen interactions during infection. Here, we have conducted the isolation of EVs produced by a clinical isolate of C. albicans during biofilm formation and proved their effect towards the ability of the Gram-negative bacterial pathogen Klebsiella pneumoniae to adhere to HaCaT cells and form a biofilm in vitro. The results represent the first evidence of an antagonistic action of fungal EVs against bacteria.

Antimicrobial peptide AMP-17 induces protection against systemic candidiasis and interacts synergistically with fluconazole against Candida albicans biofilm.

Candida albicans, a common commensal and opportunistic fungal pathogen in humans, can occasionally progress to disseminated candidiasis which is a serious condition with a high morbidity and fatality rate. The emergence of drug-resistant fungal strains compels us to look for an efficient treatment solution. Our earlier studies have demonstrated that the unique antimicrobial peptide AMP-17 from Musca domestica has a strong antifungal impact on C. albicans in vitro. Here, we verified the therapeutic effects of AMP-17 on systemic candidiasis in vivo and the peptide interacts with fluconazole, a common antifungal medication, to treat systemic candidiasis. In the disseminated candidiasis model of Galleria mellonella and mice challenged with C. albicans, AMP-17 increased the survival rates of infected larvae and mice to 66.7 and 75%, respectively. Furthermore, the peptide lowered the load of C. albicans in the infected larvae and the kidneys of the mice by nearly 90%. Additional histological examination and measurements of plasma cytokines showed that the injection of AMP-17 markedly reduced the inflammatory response and balanced cytokine expression. Furthermore, checkerboard micro dilution experiments demonstrated that AMP-17 and fluconazole worked in synergy to inhibit C. albicans in the biofilm mode. According to morphological studies, AMP-17 and fluconazole together decreased the production of hyphae throughout the C. albicans biofilm formation process, loosening the mature biofilms' structure and lowering the amount of carbohydrates in the extracellular matrix (ECM) of the biofilms. Taken together, these results showed that AMP-17 would be a viable treatment for systemic candidiasis and might be a different approach to combating Candida biofilm, either by itself or in conjunction with fluconazole.

Measuring Stress Phenotypes in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human fungal pathogen capable of surviving in a wide range of environments and hosts. It has been developed as a model organism to study fungal pathogenesis due to its fully sequenced haploid genome and optimized gene deletion and mutagenesis protocols. These methods have greatly aided indetermining the relationship between Cryptococcus genotype and phenotype. Furthermore, the presence of congenic mata and matα strains associated with a defined sexual cycle has helped further understand cryptococcal biology. Several in vitro stress conditions have been optimized to closely mimic the stress that yeast encounter in the environment or within the infected host. These conditions have proven to be extremely useful in elucidating the role of several genes in allowing yeast to adapt and survive in hostile external environments. This chapter describes various in vitro stress conditions that could be used to test the sensitivity of different mutant strains, as well as the protocol for preparing them. We have also included a list of mutants that could be used as a positive control strain when testing the sensitivity of the desired strain to a specific stress.

Systematic identification and characterization of five transcription factors mediating the oxidative stress response in Candida albicans

Candida albicans is an opportunistic human fungal pathogen that causes superficial and systemic infections, particularly in immunocompromised individuals. In response to C. albicans infection, innate immune cells of the host produce and accumulate reactive oxygen species (ROS), which can lead to irreversible damage and apoptosis of fungal cells. Several transcription factors involved in this oxidative stress response have been identified; however, a systematic study to identify the transcription factors that mediate the oxidative stress response has not yet been conducted. Here, we screened a comprehensive transcription factor mutant library consisting of 211 transcription factor deletion mutant strains in the presence and absence of hydrogen peroxide (H2O2), a potent ROS inducer, and identified five transcription factors (Skn7, Dpb4, Cap1, Dal81, and Stp2) that are sensitive to H2O2. Genome-wide transcriptional profiling revealed that H2O2 induces a discrete set of differentially regulated genes among the five identified transcription factor mutant strains. Functional enrichment analysis identified KEGG pathways pertaining to glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, and ribosome synthesis as the most enriched pathways. GO term analysis of the top common differentially expressed genes among the transcription factor mutant strains identified hexose catabolism and iron transport as the most enriched GO terms upon exposure to H2O2.This study is the first to systematically identify and characterise the transcription factors involved in the response to H2O2. Based on our transcriptional profiling results, we found that exposure to H2O2 modulates several downstream genes involved in fungal virulence. Overall, this study sheds new light on the metabolism, physiological functions, and cellular processes involved in the H2O2-induced oxidative stress response in C. albicans.

Morpho-anatomical attributes of the Egyptian Conocarpus erectus L. (Combretaceae R.Br.) with its phytochemicals and fungal-endophytes

Egyptian Conocarpus erectus L., or the buttonwood, was studied in different terms. This study used light and scanning electron microscopy, HPLC, and GC–MS analyses. Foliar morphology, anatomy of stems and leaves, pollen morphology, and phytochemical and endofungal attributes were investigated. It was an evergreen shrub with alternate leaves, a pair of nectary glands on the leaf base, and a few indumentums. Trichomes of both non- and glandular hairs were found. Stomata were sized, reaching 37.5 × 25.0 µm. They appeared on both abaxial and adaxial surfaces. The stem and leaf interior structure was typically like most dicots. Their internal structure revealed the mycelium of Aspergillus flavus throughout. Similarly, the accumulation of phenolic compounds as colored particles and integrated phytochemicals in crystalloid druses are observed internally. The endophytic fungus A. flavus, isolated from the leaves of C. erectus, contained the highest concentration of kojic acid. As a result, we could consider this A. flavus strain as a source of kojic acid for application in industry. Fungal and C. erectus extracts showed broad-range antifungal activity against three opportunistic human pathogenic fungi (Geotrichum candidum OL960606, Neoscytalidium dimidiatum OL960610, and Scopulariopsis coprophila OL960621). The major bioactive compounds of C. erectus leaf extracts were gallic acid, rutin, and rosmarinic acid. In conclusion, the endophytic A. flavus isolated from C. erectus has a variety of biological and medicinal uses that make it a potential commercial resource.

DYRK-family kinases regulate Candida albicans morphogenesis and virulence through the Ras1/PKA pathway.

Candida albicans is an opportunistic human fungal pathogen that frequently causes life-threatening infections in immunocompromised individuals. To cause disease, the fungus employs several virulence traits, including its ability to transition between yeast and filamentous states. Previous work identified a role for the kinase Yak1 in regulating C. albicans filamentation. Here, we demonstrate that Yak1 regulates morphogenesis through the canonical cAMP/PKA pathway and that this regulation is environmentally contingent, as host-relevant concentrations of CO2 bypass the requirement of Yak1 for C. albicans morphogenesis. We show a related kinase, Pom1, is important for filamentation in the absence of Yak1 under these host-relevant conditions, as deletion of both genes blocked filamentous growth under all conditions tested. Finally, we demonstrate that Yak1 is required for filamentation in a mouse model of C. albicans dermatitis using genetic and pharmacological approaches. Overall, our results expand our understanding of how Yak1 regulates an important virulence trait in C. albicans.

The MYST Family Histone Acetyltransferase SasC Governs Diverse Biological Processes in Aspergillus fumigatus.

The conserved MYST proteins form the largest family of histone acetyltransferases (HATs) that acetylate lysines within the N-terminal tails of histone, enabling active gene transcription. Here, we have investigated the biological and regulatory functions of the MYST family HAT SasC in the opportunistic human pathogenic fungus Aspergillus fumigatus using a series of genetic, biochemical, pathogenic, and transcriptomic analyses. The deletion (Δ) of sasC results in a drastically reduced colony growth, asexual development, spore germination, response to stresses, and the fungal virulence. Genome-wide expression analyses have revealed that the ΔsasC mutant showed 2402 significant differentially expressed genes: 1147 upregulated and 1255 downregulated. The representative upregulated gene resulting from ΔsasC is hacA, predicted to encode a bZIP transcription factor, whereas the UV-endonuclease UVE-1 was significantly downregulated by ΔsasC. Furthermore, our Western blot analyses suggest that SasC likely catalyzes the acetylation of H3K9, K3K14, and H3K29 in A. fumigatus. In conclusion, SasC is associated with diverse biological processes and can be a potential target for controlling pathogenic fungi.

Deubiquitination module is critical for oxidative stress response and biofilm formation in Candida glabrata.

Candidiasis is one of the most important fungal diseases and generally refers to diseases of the skin or mucosal tissues caused by Candida species. Candida glabrata is an opportunistic human fungal pathogen. Infection with C. glabrata has significantly increased due to innate antifungal drug tolerance and the ability to adhere to mucocutaneous surfaces. Spt-Ada-Gcn5 acetyltransferase complex contains two different post-translational modifications, histone acetylation (HAT) module and deubiquitination (DUB) module, which are decisive in gene regulation and highly conserved in eukaryotes. Previous research in our laboratory found that the HAT module ADA2 could regulate C. glabrata oxidative stress tolerance, drug tolerance, cell wall integrity, and virulence. However, the roles of the DUB module that is comprised of UBP8, SGF11, SGF73, and SUS1 genes in those phenotypes are not yet understood. In this study, we found that DUB module genes UBP8, SGF11, and SUS1, but not SGF73 positively regulate histone H2B DUB. Furthermore, ubp8, sgf11, and sus1 mutants exhibited decreased biofilm formation and sensitivity to cell wall-perturbing agent sodium dodecyl sulfate and antifungal drug amphotericin B. In addition, the sgf73 mutant showed increased biofilm formation but was susceptible to oxidative stresses, antifungal drugs, and cell wall perturbing agents. The ubp8, sgf11, and sus1 mutants showed marginal hypovirulence, whereas the sgf73 mutant exhibited virulence similar to the wild type in a murine systemic infection model. In conclusion, the C. glabrata DUB module plays distinct roles in H2B ubiquitination, oxidative stress response, biofilm formation, cell wall integrity, and drug tolerance, but exhibits minor roles in virulence.

Synthesis and Antimicrobial Analysis of High Surface Area Strontium-Substituted Calcium Phosphate Nanostructures for Bone Regeneration.

Continuous microwave-assisted flow synthesis has been used as a simple, more efficient, and low-cost route to fabricate a range of nanosized (<100 nm) strontium-substituted calcium phosphates. In this study, fine nanopowder was synthesized via a continuous flow synthesis with microwave assistance from the solutions of calcium nitrate tetrahydrate (with strontium nitrate as Sr2+ ion source) and diammonium hydrogen phosphate at pH 10 with a time duration of 5 min. The morphological characterization of the obtained powder has been carried out by employing techniques such as transmission electron microscopy, X-ray diffraction, and Brunauer-Emmett-Teller surface area analysis. The chemical structural analysis to evaluate the surface properties was made by using X-ray photoelectron spectroscopy. Zeta potential analysis was performed to evaluate the colloidal stability of the particles. Antimicrobial studies were performed for all the compositions using four bacterial strains and an opportunistic human fungal pathogen Macrophomina phaseolina. It was found that the nanoproduct with high strontium content (15 wt% of strontium) showed pronounced antibacterial potential against M. luteus while it completely arrested the fungal growth after 48 h by all of its concentrations. Thus the synthesis strategy described herein facilitated the rapid production of nanosized Sr-substituted CaPs with excellent biological performance suitable for a bone replacement application.

The human fungal pathogen Aspergillus fumigatus can produce the highest known number of meiotic crossovers.

Sexual reproduction involving meiosis is essential in most eukaryotes. This produces offspring with novel genotypes, both by segregation of parental chromosomes as well as crossovers between homologous chromosomes. A sexual cycle for the opportunistic human pathogenic fungus Aspergillus fumigatus is known, but the genetic consequences of meiosis have remained unknown. Among other Aspergilli, it is known that A. flavus has a moderately high recombination rate with an average of 4.2 crossovers per chromosome pair, whereas A. nidulans has in contrast a higher rate with 9.3 crossovers per chromosome pair. Here, we show in a cross between A. fumigatus strains that they produce an average of 29.9 crossovers per chromosome pair and large variation in total map length across additional strain crosses. This rate of crossovers per chromosome is more than twice that seen for any known organism, which we discuss in relation to other genetic model systems. We validate this high rate of crossovers through mapping of resistance to the laboratory antifungal acriflavine by using standing variation in an undescribed ABC efflux transporter. We then demonstrate that this rate of crossovers is sufficient to produce one of the common multidrug resistant haplotypes found in the cyp51A gene (TR34/L98H) in crosses among parents harboring either of 2 nearby genetic variants, possibly explaining the early spread of such haplotypes. Our results suggest that genomic studies in this species should reassess common assumptions about linkage between genetic regions. The finding of an unparalleled crossover rate in A. fumigatus provides opportunities to understand why these rates are not generally higher in other eukaryotes.

Inflammatory ER stress responses dictate the immunopathogenic progression of systemic candidiasis.

Recognition of pathogen-associated molecular patterns can trigger the inositol-requiring enzyme 1 α (IRE1α) arm of the endoplasmic reticulum (ER) stress response in innate immune cells. This process maintains ER homeostasis and also coordinates diverse immunomodulatory programs during bacterial and viral infections. However, the role of innate IRE1α signaling in response to fungal pathogens remains elusive. Here, we report that systemic infection with the human opportunistic fungal pathogen Candida albicans induced proinflammatory IRE1α hyperactivation in myeloid cells that led to fatal kidney immunopathology. Mechanistically, simultaneous activation of the TLR/IL-1R adaptor protein MyD88 and the C-type lectin receptor dectin-1 by C. albicans induced NADPH oxidase-driven generation of ROS, which caused ER stress and IRE1α-dependent overexpression of key inflammatory mediators such as IL-1β, IL-6, chemokine (C-C motif) ligand 5 (CCL5), prostaglandin E2 (PGE2), and TNF-α. Selective ablation of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, mitigated kidney inflammation and prolonged the survival of mice with systemic C. albicans infection. Therefore, controlling IRE1α hyperactivation may be useful for impeding the immunopathogenic progression of disseminated candidiasis.

Redefining pleiotropic drug resistance in a pathogenic yeast: Pdr1 functions as a sensor of cellular stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (CYB5, SSK1, SSK2, HOG1, TRP1). A bZIP transcription repressor of mitochondrial function (CIN5) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata. Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. IMPORTANCE Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1-a key determinant of fluconazole resistance-is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.