Fungal Sterol Research Articles

Design, Synthesis, and Evaluation of Novel 4-Chloropyrazole-Based Pyridines as Potent Fungicide Candidates.

A rational molecular design approach was developed in our laboratory to guide the discovery of novel sterol biosynthesis inhibitors. Based on the application of bioactivities of heterocyclic rings and molecular docking targeting the sterol biosynthesis 14α-demethylase, a series of 4-chloropyrazole-based pyridine derivatives were rationally designed, synthesized, and characterized and their fungicidal activities were also evaluated. Bioassay results showed that 7e, 7f, and 7m exhibited commendable, diverse antifungal actions that are comparable to those of the positive controls imazalil and triadimefon. The active compounds' mode of action was further studied by microscopy observations, Q-PCR, and enzyme inhibition assay and discovered that target compounds affect fungal sterol biosynthesis via disturbing RcCYP51 enzyme system. These findings support that their fungicidal mode of action still targets the cytochrome P450-dependent 14α-demethylase as the molecular design did at first. The above results strongly suggest that our rational molecular design protocol is not only practical but also efficient.

Comparison of Safety, Efficacy, and Cost-Effectiveness of Miconazole 2% Versus Sertaconazole 2% in the Treatment of TineaCorporis: A Randomized Control Trial

Tineacorporis is a dermatophytic infection of the body, which involves the keratin layer of the skin. These lesions are present as an annular plaque with an advancing border along with central clearing. Miconazole, a topical antifungal drug and has good efficacy, in anti dermatophyte. Sertaconazole is a highly selective inhibitor of fungal cytochrome P-450 sterol C-14 α-demethylation via the inhibition of the enzyme cytochrome P450 14α-demethylase.It is claimed to be superior to other old topical imidazoles in tineacorporis. .The present study was aimed to compare the safety, efficacy and cost-effectiveness of topical antifungals, where we have compared Miconazole and Sertaconazole 2% creams in the treatment of fungal infection caused by tineacorporis, by assessing the ADR’s and therapeutic outcomes, using comparative, unicentered, randomized, non-blinded trial with 2 parallel treatment arms of one-month duration. In this study 106 patients were randomly assigned into 2 groups of Miconazole 2% cream and Sertaconazole 2% with 54 and 52 patients in each group respectively. Measurement is carried out at baseline, 1st, 2nd and 3rd follow-up for desired effects like itching, erythema, pain and lesions, physician’s global assessment (PGA), safety and cost-effectiveness. The results showed that sertaconazole 2% cream is efficacious and superior to Miconazole 2% cream in the improvement of clinical parameters and PGA. At the end of the follow-up phase, both groups of drugs are effective and well-tolerated in patients with no recurrence of tineacorporis. Effectiveness of SSertaconazole is early and superior with minor side effects. However, Miconazole is cost effective and safe.

Design, Synthesis, Molecular Docking and Antifungal Evaluation of Mixed Heterocyclic Moieties Containing Pyridine, 1,3,4-Oxadiazole and 1,2,3-Triazol Rings

In the current study, derivatives containing pyridine, 1,3,4-oxadiazole and 1,2,3-triazole heterocycles in their structures have been synthesized, 1,2,3-triazole moiety was synthesized using click chemistry, a powerful modular synthesis approach, and evaluated for in-vitro antifungal activity. When compared to the regular medicine Fluconazole, one derivative (5a) showed superior fungicidal action. Prior to synthesis, an in silico computational analysis was conducted to determine the binding interactions of these synthesized compounds in the active sites of the fungal enzyme sterol 14-demethylase (CYP51) through molecular docking via Genetic Optimization for Ligand Docking (GOLD) suite v.5.6.2. Most of the tested compounds in molecular docking showed significant activities compared with fluconazole as reference compound due to their hydrogen bonding interaction with key amino acids in Sterol 14-demethylase enzyme Tyr116 and iron metal, and these results are compatible with their in vitro anti-fungal study.

Steroidal Antimetabolites Protect Mice against Trypanosoma brucei.

Trypanosoma brucei, the causative agent for human African trypanosomiasis, is an emerging ergosterol-dependent parasite that produces chokepoint enzymes, sterol methyltransferases (SMT), not synthesized in their animal hosts that can regulate cell viability. Here, we report the lethal effects of two recently described natural product antimetabolites that disrupt Acanthamoeba sterol methylation and growth, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT) that can equally target T. brucei. We found that CHT/ERGT inhibited cell growth in vitro, yielding EC50 values in the low nanomolar range with washout experiments showing cidal activity against the bloodstream form, consistent with their predicted mode of suicide inhibition on SMT activity and ergosterol production. Antimetabolite treatment generated altered T. brucei cell morphology and death rapidly within hours. Notably, in vivo ERGT/CHT protected mice infected with T. brucei, doubling their survival time following daily treatment for 8–10 days at 50 mg/kg or 100 mg/kg. The current study demonstrates a new class of lead antibiotics, in the form of common fungal sterols, for antitrypanosomal drug development.

Plant Host Traits Mediated by Foliar Fungal Symbionts and Secondary Metabolites.

Fungal symbionts living inside plant leaves ("endophytes") can vary from beneficial to parasitic, but the mechanisms by which the fungi affect the plant host phenotype remain poorly understood. Chemical interactions are likely the proximal mechanism of interaction between foliar endophytes and the plant, as individual fungal strains are often exploited for their diverse secondary metabolite production. Here, we go beyond single strains to examine commonalities in how 16 fungal endophytes shift plant phenotypic traits such as growth and physiology, and how those relate to plant metabolomics profiles. We inoculated individual fungi on switchgrass, Panicum virgatum L. This created a limited range of plant growth and physiology (2-370% of fungus-free controls on average), but effects of most fungi overlapped, indicating functional similarities in unstressed conditions. Overall plant metabolomics profiles included almost 2000 metabolites, which were broadly correlated with plant traits across all the fungal treatments. Terpenoid-rich samples were associated with larger, more physiologically active plants and phenolic-rich samples were associated with smaller, less active plants. Only 47 metabolites were enriched in plants inoculated with fungi relative to fungus-free controls, and of these, Lasso regression identified 12 metabolites that explained from 14 to 43% of plant trait variation. Fungal long-chain fatty acids and sterol precursors were positively associated with plant photosynthesis, conductance, and shoot biomass, but negatively associated with survival. The phytohormone gibberellin, in contrast, was negatively associated with plant physiology and biomass. These results can inform ongoing efforts to develop metabolites as crop management tools, either by direct application or via breeding, by identifying how associations with more beneficial components of the microbiome may be affected.

Amphotericin B assembles into seven-molecule ion channels: An NMR and molecular dynamics study.

Amphotericin B, an antifungal drug with a long history of use, forms fungicidal ion-permeable channels across cell membranes. Using solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we experimentally elucidated the three-dimensional structure of the molecular assemblies formed by this drug in membranes in the presence of the fungal sterol ergosterol. A stable assembly consisting of seven drug molecules was observed to form an ion conductive channel. The structure is somewhat similar to the upper half of the barrel-stave model proposed in the 1970s but substantially different in the number of molecules and in their arrangement. The present structure explains many previous findings, including structure-activity relationships of the drug, which will be useful for improving drug efficacy and reducing adverse effects.

Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols

The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of cholesterol. Here we engineer yeast to produce cholesterol and introduce diverse mu, delta, and kappa opioid receptors to create sensitive opioid biosensors that recapitulate agonist binding profiles and antagonist inhibition. Additionally, human mu opioid receptor variants, including those with clinical relevance, largely display expected phenotypes. By testing mu opioid receptor-based biosensors with systematically adjusted cholesterol biosynthetic intermediates, we relate sterol profiles to biosensor sensitivity. Finally, we apply sterol-modified backgrounds to other human receptors revealing sterol influence in SSTR5, 5-HTR4, FPR1, and NPY1R signaling. This work provides a platform for generating human G protein-coupled receptor-based biosensors, facilitating receptor deorphanization and high-throughput screening of receptors and effectors.

Investigation of Novel Mucorales Fungal Inhibitors: Synthesis, In‐Silico Study and Anti‐Fungal Potency of Novel Class of Coumarin‐6‐Sulfonamides‐Thiazole and Thiadiazole Hybrids

AbstractA series of coumarin‐6‐sulfonamides derivatives bearing a thiazole and thiadiazole scaffold were synthesized, their chemical structures were inferred from correct spectral and microanalytical data. All the newly synthesized compounds have been screened for their ability to prevent the proliferation of Mucor racemosus, Syncephalastrum racemosum in vitro. The most potent compounds were chosen to determine the MIC value in compared to the standard antifungal agent (Posaconazole). Remarkably, an excellent anti‐mucormycosis was obtained by compounds known as 8 d, and 8 d with MIC value close to that observed by the standard Posaconazole. Amolecular. Docking study was performed where compounds 3, 5 a, 5 d, 8 d, 8 e, 10 c and 10 e were docked against the fungal enzyme sterol 14‐alpha demethylase (CYP51) “PDB id: 5FSA”. In silico, compounds 8 d & 8 e showed the best docking by forming 3H bonds and free binding energy (12.2 & 12 K cal/mol) close to the binding energy (12.2 K cal/mol) of the co‐crystalized inhibitor (posconazol. From wet lab and dry lab results, one can recommend compounds 8 d & 8 e for further antifungal drug development.

Structure and Biological Activity of Ergostane-Type Steroids from Fungi.

Mushrooms are known not only for their taste but also for beneficial effects on health attributed to plethora of constituents. All mushrooms belong to the kingdom of fungi, which also includes yeasts and molds. Each year, hundreds of new metabolites of the main fungal sterol, ergosterol, are isolated from fungal sources. As a rule, further testing is carried out for their biological effects, and many of the isolated compounds exhibit one or another activity. This study aims to review recent literature (mainly over the past 10 years, selected older works are discussed for consistency purposes) on the structures and bioactivities of fungal metabolites of ergosterol. The review is not exhaustive in its coverage of structures found in fungi. Rather, it focuses solely on discussing compounds that have shown some biological activity with potential pharmacological utility.

Application of Pressurized Liquid Extractions to Obtain Bioactive Compounds from Tuber aestivum and Terfezia claveryi

A PLE (pressurized liquid extraction) method was adjusted following a full-factorial experimental design to obtain bioactive-enriched fractions from Tuber aestivum and Terfezia claveryi. Temperature, time and solvent (water, ethanol and ethanol–water 1:1) parameters were investigated. The response variables investigated were: obtained yield and the levels of total carbohydrate (compounds, β-glucans, chitin, proteins, phenolic compounds and sterols). Principal component analysis indicated water solvent and high temperatures as more adequate parameters to extract polysaccharide-rich fractions (up to 68% of content), whereas ethanol was more suitable to extract fungal sterols (up to 12.5% of content). The fractions obtained at optimal conditions (16.7 MPa, 180 °C, 30 min) were able to protect Caco2 cells from free radical exposure, acting as antioxidants, and were able to reduce secretion of pro-inflammatory cytokines in vitro: IL-6 (50%), and TNFα (80% only T. claveryi ethanol extract), as well as reduce high inhibitory activity (T. aestivum IC50: 9.44 mG/mL).

Azole-Resistant Alleles of ERG11 in Candida glabrata Trigger Activation of the Pdr1 and Upc2A Transcription Factors.

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata, these azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance alleles from C. albicans ERG11. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.

Recent advances in fungal serine protease inhibitors

Four types of antifungal drugs are available that include inhibitors of ergosterol synthesis, of fungal RNA biosynthesis, and of cell wall biosynthesis as well as physiochemical regulators of fungal membrane sterols. Increasing resistance to antifungal drugs can severely limit treatment options of fungal nail infections, vaginal candidiasis, ringworm, blastomycosis, histoplasmosis, and Candida infections of the mouth, throat, and esophagus, among other infections. Development of strategies focused on new fungicides can effectively help tackle troublesome fungal diseases. The virulence and optimal growth of fungi depend on various extracellular secreted factors, among which proteases, such as serine proteases, are of particular interest. A specific extracellular proteolytic system enables fungi to survive and penetrate the tissues. Given the role of fungal proteases in infection, any molecule capable of selectively and specifically inhibiting their activity can lead to the development of potential drugs. Owing to their specific mode of action, fungal protease inhibitors can avoid fungal resistance observed with currently available treatments. Although fungal secreted proteases have been extensively studied as potential virulence factors, our understanding of the substrate specificity of such proteases remains poor. In this review, we summarize the recent advances in the design and development of specific serine protease inhibitors and provide a brief history of the compounds that inhibit fungal serine protease activity.

Exploration of novel TOSMIC tethered imidazo[1,2-a]pyridine compounds for the development of potential antifungal drug candidate.

New candidates of imidazo[1,2-a]pyridine were designed by combining 2-amino pyridine, TOSMIC and various assorted aldehydes to explore their antioxidant and antifungal potential. The design of these derivatives was based on utilizing the antifungal potential of azoles and TOSMIC moiety. These derivatives were synthesized by adopting multi-component reaction methodology, as it serves as a rapid and efficient tool to target structurally diverse heterocyclic compounds in quantitative yield. The resulting imidazo[1,2-a]pyridine derivatives were structurally verified by 1 HNMR, 13 CNMR, HRMS, and HPLC. The compounds were analyzed for their antioxidant and fluorescent properties and it was observed that compound 15 depicted highest potential. The compounds were evaluated for their antifungal potential to highlight their medical application in the area of Invasive Fungal Infections (IFI). Compound 12 gave the highest antifungal inhibition against Aspergillus fumigatus 3007 and Candida albicans 3018. To elucidate the antifungal mechanism, confocal images of treated fungi were analyzed, which depicted porous nature of fungal membrane. Estimation of fungal membrane sterols by UPLC indicated decrease in ergosterol component of fungal membrane. In silico studies further corroborated with the in vitro results as docking studies depicted interaction of synthesized heterocyclic compounds with amino acids present in the active site of target enzyme (lanosterol 14 alpha demethylase). Absorption, distribution, metabolism, and excretion (ADME) analysis was indicative of drug-likeliness of the synthesized compounds.

An Updated Review of What, When and How of Sertaconazole: A Potent Antifungal Agent

Sertaconazole is an imidazole/triazole type antifungal agent which selectively inhibit fungal cytochrome P450 sterol C-14 α-demethylase and act as potent antifungal agent. This enzyme is essential for fungal cell wall synthesis as it converts lanosterol to ergosterol. The antifungal spectrum of drug includes Candida, Malassezia, Cryptococcus, Aspergillus, Scopulariopsis and Scedosporium. In addition, sertaconazole has been reported to exhibit an antimicrobial activity against staphylococci, streptococci, and protozoa. The drug is practically insoluble in water and has an apparent half-life of approximately 60 hrs. It is prescribed for the treatment of interdigital tinea pedis (topical), mycoses of skin and mucosa in dermatology and gynaecology. Sertaconazole penetrates the horny layer of skin and the therapeutic concentrations may be found after a long period of time, avoiding the potential risk of systemic absorption. It is available in the market as cream, lotion and tablet formulations. This review summarises the physiochemical characterization of drug, involved pharmacokinetic, pharmacodynamics, safety profile along with the reported preclinical, clinical and formulation studies and patents thereof and the paradigm shift in the use of sertaconazole.

Roles for Structural Biology in the Discovery of Drugs and Agrochemicals Targeting Sterol 14α-Demethylases.

Antifungal drugs and antifungal agrochemicals have significant limitations. These include several unintended consequences of their use including the growing importance of intrinsic and acquired resistance. These problems underpin an increasingly urgent need to improve the existing classes of antifungals and to discover novel antifungals. Structural insights into drug targets and their complexes with both substrates and inhibitory ligands increase opportunity for the discovery of more effective antifungals. Implementation of this promise, which requires multiple skill sets, is beginning to yield candidates from discovery programs that could more quickly find their place in the clinic. This review will describe how structural biology is providing information for the improvement and discovery of inhibitors targeting the essential fungal enzyme sterol 14α-demethylase.

Sterol 14α-Demethylase Ligand-Binding Pocket-Mediated Acquired and Intrinsic Azole Resistance in Fungal Pathogens.

The fungal cytochrome P450 enzyme sterol 14α-demethylase (SDM) is a key enzyme in the ergosterol biosynthesis pathway. The binding of azoles to the active site of SDM results in a depletion of ergosterol, the accumulation of toxic intermediates and growth inhibition. The prevalence of azole-resistant strains and fungi is increasing in both agriculture and medicine. This can lead to major yield loss during food production and therapeutic failure in medical settings. Diverse mechanisms are responsible for azole resistance. They include amino acid (AA) substitutions in SDM and overexpression of SDM and/or efflux pumps. This review considers AA affecting the ligand-binding pocket of SDMs with a primary focus on substitutions that affect interactions between the active site and the substrate and inhibitory ligands. Some of these interactions are particularly important for the binding of short-tailed azoles (e.g., voriconazole). We highlight the occurrence throughout the fungal kingdom of some key AA substitutions. Elucidation of the role of these AAs and their substitutions may assist drug design in overcoming some common forms of innate and acquired azole resistance.

Gas chromatography with mass spectrometry detection and characterization of 27 sterols in two truffle (Tuber) species

Fungal sterols (mycosterols) were investigated in seven samples of both Tuber aestivum and Tuber borchii truffles from Italy, Spain and Romania by means of gas chromatography with mass spectrometry (GC/MS). Sterol contents varied from 130 to 590 mg/100 g dry weight (T. borchii) and 110−420 mg/100 g dry weight (T. aestivum). The sterol pattern of both truffle species was dominated by ergosterol (60–85 %) and brassicasterol (4–33 %). In addition, 25 minor sterols were detected with 27 (n = 3), 28 (n = 17), 29 (n = 3), 30 (n = 2) and 31 (n = 2) carbon atoms (Limit of detection: 2 μg/100 g dry weight). Fourteen minor sterols were described for the first time in Tuber species. Ten minor compounds were detected in all samples in varying abundances, while the others were (i) exclusively detected in all samples of one species (T. borchii: fecosterol, ergosta-8-enol; T. aestivum: 24-methyleneergosta-5,22,24-trienol) or (ii) varied strongly in abundance (≥90 %). Variations in main and selected minor sterols could be partly related to different harvest times. In addition, differences in fungisterol and ergosta-5,7-dienol between the both species were attributed to different pathways in fungal sterol biosynthesis (oneway ANOVA p ≤ 0.01). Our study indicates that sterol pattern analysis could be used to differentiate different Tuber species.

Undervirilized male infant with in utero exposure to maternal use of high dose antifungal therapy

BackgroundAntifungals act on fungal sterols structurally similar to human cholesterol. Ketoconazole reversibly suppresses steroidogenesis by inhibiting cytochrome P450 enzymes and interferes with dihydrotestosterone (DHT) activity by binding to the androgen receptor. Hypospadias was reported in infants exposed to nystatin in utero.Case presentationA male infant exposed to antepartum nystatin presented with severe under-undervirilization and transient adrenal corticosteroid abnormalities. He was born in USA at 31 weeks gestation to a mother treated with vaginal Polygynax capsules (nystatin-100,000 international units, neomycin sulphate-35,000 international units and polymyxin B-35,000 international units) for vaginal discharge in the Ivory Coast. She used approximately 60 capsules between the first trimester until delivery. The infant was born with micropenis, chordee, perineo-scrotal hypospadias and bifid scrotum with bilaterally palpable gonads. The karyotype was 46,XY. No Mullerian structures were seen on ultrasound. Serum 17-hydroxyprogesterone (17 OHP) on newborn screening was high (304 ng/ml, normal < 35). Cortisol response to cosyntropin on the 3rd day of life (DOL) was 10 mcg/ml; the subnormal cortisol response may have resulted from prematurity and the predelivery treatment with betamethasone. The elevation of several adrenal corticosteroids was not consistent with any specific enzymatic defect. Hydrocortisone and fludrocortisone were initiated at another hospital for suspected mild glucocorticoid and mineralocorticoid deficiencies. Genetic screening for adrenal and gonadal developmental defects performed when transferred to our care were normal. All medications were gradually discontinued over 5–8 months. Adrenal and testicular responses to cosyntropin and human chorionic gonadotropin (hCG) were normal at 8 months.ConclusionsWe report severe undervirilization in a 46,XY infant born to a mother treated with prolonged and high dose nystatin during pregnancy. This presentation suggests that prolonged antepartum use of high dose nystatin could lead to severe but transient defects in androgen synthesis and/or action possibly by acting as an endocrine disruptor. Further studies are warranted to confirm this finding. Thus, endocrine disruptors should be considered in male newborns with atypical genitalia not explained by common pathologies.

Metabolic Reconfiguration in C. elegans Suggests a Pathway for Widespread Sterol Auxotrophy in the Animal Kingdom

Cholesterol is one of the hallmarks of animals. In vertebrates, the cholesterol synthesis pathway (CSP) is the primary source of cholesterol that has numerous structural and regulative roles [1]. Nevertheless, the few invertebrates tested for cholesterol synthesis show complete sterol auxotrophy [2-6], raising questions about how animals thrive without cholesterol synthesis and about the prevalence of sterol auxotrophy in animals. In the nematode Caenorhabditis elegans (C.elegans), sterols are the precursors of the steroid hormone dafachronic acid that coordinates development to adulthood [7, 8]; thus, sterol-deprived C.elegans arrest at the diapause "dauer" larval stage [9]. Using this system, we have identified a pathway that converts plant and fungal sterols into cholesterol through the activity of enzymes with sequence similarity to specific human CSP enzymes. Based on this finding, we propose that two critical steps shaped the evolution of animal sterol auxotrophy: (1) the loss of the orthologs of the first three enzymes of the CSP and (2) the co-opting of other downstream enzymes of the CSP for the utilization of dietary sterols. Using this mechanistic signature, we studied the evolution of cholesterol auxotrophy across the animal kingdom. Complete sets of CSP enzymes in basal animals suggest that the loss of cholesterol synthesis occurred during animal evolution. A sterol auxothropy signature in the genomes of many invertebrates, including nematodes and most arthropods, suggests widespread cholesterol auxotrophy in animals. Thus, we propose that this co-opted pathway supports widespread cholesterol auxotrophy by interkingdom interactions between cholesterol-auxotrophic animals and sterol-producing fungi and plants.

Sterol-Response Pathways Mediate Alkaline Survival in Diverse Fungi.

The ability for cells to maintain homeostasis in the presence of extracellular stress is essential for their survival. Stress adaptations are especially important for microbial pathogens to respond to rapidly changing conditions, such as those encountered during the transition from the environment to the infected host. Many fungal pathogens have acquired the ability to quickly adapt to changes in extracellular pH to promote their survival in the various microenvironments encountered during a host infection. For example, the fungus-specific Rim/Pal alkaline response pathway has been well characterized in many fungal pathogens, including Cryptococcus neoformans However, alternative mechanisms for sensing and responding to host pH have yet to be extensively studied. Recent observations from a genetic screen suggest that the C. neoformans sterol homeostasis pathway is required for growth at elevated pH. This work explores interactions among mechanisms of membrane homeostasis, alkaline pH tolerance, and Rim pathway activation. We find that the sterol homeostasis pathway is necessary for growth in an alkaline environment and that an elevated pH is sufficient to induce Sre1 activation. This pH-mediated activation of the Sre1 transcription factor is linked to the biosynthesis of ergosterol but is not dependent on Rim pathway signaling, suggesting that these two pathways are responding to alkaline pH independently. Furthermore, we discover that C. neoformans is more susceptible to membrane-targeting antifungals under alkaline conditions, highlighting the impact of microenvironmental pH on the treatment of invasive fungal infections. Together, these findings further connect membrane integrity and composition with the fungal pH response and pathogenesis.IMPORTANCE The work described here further elucidates how microorganisms sense and adapt to changes in their environment to establish infections in the human host. Specifically, we uncover a novel mechanism by which an opportunistic human fungal pathogen, Cryptococcus neoformans, responds to increases in extracellular pH in order to survive and thrive within the relatively alkaline environment of the human lung. This mechanism, which is intimately linked with fungal membrane sterol homeostasis, is independent of the previously well-studied alkaline response Rim pathway. Furthermore, this ergosterol-dependent alkaline pH response is present in Candida albicans, indicating that this mechanism spans diverse fungal species. These results are also relevant for novel antimicrobial drug development as we show that currently used ergosterol-targeting antifungals are more active in alkaline environments.