Emerging evidence for a multitude of mechanisms and factors that determine amphotericin B resistance in pathogenic fungi

Candida auris is an emerging multidrug-resistant human fungal pathogen often refractory to treatment by all classes of antifungal drugs. Amphotericin B (AmB) is a fungicidal drug that, despite its toxic side effects, remains a drug of choice for the treatment of drug-resistant fungal infections, including those caused by C. auris. However, the molecular mechanisms underlying AmB resistance are poorly understood. In this study, we present data that suggests membrane lipid alterations and chromatin modifications are critical processes that may contribute to or cause adaptive AmB resistance in clinical C. auris isolates. To determine the plausible cause of increased AmB resistance, we performed RNA-seq of AmB-resistant and sensitive C. auris isolates. Remarkably, AmB-resistant strains show a pronounced enrichment of genes involved in lipid and ergosterol biosynthesis, adhesion, drug transport as well as chromatin remodeling. The transcriptomics data confirm increased adhesion and reduced lipid membrane permeability of AmB-resistant strains compared to the sensitive isolates. The AmB-resistant strains also display hyper-resistance to cell wall perturbing agents, including Congo red, calcofluor white and caffeine. Additionally, we noticed an increased phosphorylation of Mkc1 cell integrity MAP kinase upon AmB treatment. Collectively, these data identify differences in the transcriptional landscapes of AmB-resistant versus AmB-sensitive isolates and provide a framework for the mechanistic understanding of AmB resistance in C. auris.

Prolonged cultivation of certain filamentous fungi, including Aspergillus terreus, on drug-free medium leads to degeneration and morphological heterogeneity, marked by the emergence of fluffy mycelium-type sectors. This phenomenon may indicate alterations in antifungal susceptibility profiles (particularly to amphotericin B (AmB) in A. terreus), as well as reductions or losses in conidiation, sexuality, secondary metabolite production, and/or virulence. In the present study, various characteristics of an AmB-resistant wild-type strain (WT) and its AmB-susceptible sectorized derivative (ATSec) were characterized. Compared to WT, ATSec exhibited increased susceptibility to AmB, reduced sporulation, and comparable sterol contents and virulence in Galleria mellonella. To elucidate the genes involved in AmB resistance, gene expression levels were compared between WT and ATSec with and without AmB treatment. The expression of P-type ATPase-related genes, which are implicated in membrane composition changes and consequently in AmB resistance, was significantly higher in the WT strain compared to ATSec. Moreover, the up-regulation of genes involved in the biosynthesis of polyketides - a diverse group of secondary metabolites - was higher in WT compared to ATSec, with a significant number of these genes also carrying at least one mutation. The findings of this study indicate that P-type ATPases may significantly be involved in AmB susceptibility and resistance observed in ATSec and WT strains. Additionally, mutations in polyketide synthase genes in ATSec may contribute to the phenotypic alterations associated with the sectorized phenotype.

Clinical isolates of Candida auris show a high prevalence of resistance to Amphotericin B (AmB) - an uncommon trait in most Candida species. Alterations in ergosterol biosynthesis can contribute to acquired AmB resistance in C. auris laboratory strains but are rarely seen in clinical isolates. In this study, we experimentally evolved two drug-susceptible Clade II isolates of C. auris to develop AmB resistance. The evolved strains displayed a 4 to 8-fold increase in MIC50 compared to the parental cells. We analyzed changes in their karyotype, genome, lipidome, and transcriptome associated with this acquired resistance. In one lineage, AOX2 was upregulated, and its deletion reversed the AmB resistance phenotype. The aox2Δ mutant also failed to evolve AmB resistance under experimental conditions. In the same lineage, restoring the UPC2S332Rand RTG3S101T mutations to the wild-type allele restored AmB susceptibility. In another lineage, the ergosterol and sphingolipid pathways were observed to play a critical role, and upregulation of the ERG genes elevated the total sterol content, while significant downregulation of HSX11 (glucosylceramide synthase) resulted in lower levels of glucosylceramides. To our knowledge, this study is the first to show that AmB resistance in C. auris can be acquired through mechanisms both dependent or independent of sterol content modulation, highlighting Aox2 and Upc2 as Key regulators of amphotericin resistance.

Neutropenia is a common complication in the treatment of hematological diseases and the most common predisposing factor for invasion by fungi, such as Candida krusei. Recent studies have shown that C. krusei, a life-threatening pathogen, has developed resistance to amphotericin B (AMB). However, the mechanisms that led to the rapid emergence of this AMB-resistant phenotype are unclear. In this study, we found the sensitivity for AMB could be promoted by inhibiting histone acyltransferase activity and western blot analysis revealed differences in the succinylation levels of C. krusei isolated from immunocompromised patients and of the corresponding AMB-resistant mutant. By comparative succinyl-proteome analysis, we identified a total of 383 differentially expressed succinylated sites in with 344 sites in 134 proteins being upregulated in the AMB-resistant mutant, compared to 39 sites in 23 proteins in the wild-type strain. These differentially succinylated proteins were concentrated in the ribosome and cell wall. The critical pathways associated with these proteins included those involved in glycolysis, gluconeogenesis, the ribosome, and fructose and mannose metabolism. In particular, AMB resistance was found to be associated with enhanced ergosterol synthesis and aberrant amino acid and glucose metabolism. Analysis of whole-cell proteomes, confirmed by parallel reaction monitoring, showed that the key enzyme facilitating lysine acylation was significantly upregulated in the AMB-resistant strain. Our results suggest that lysine succinylation may play an indispensable role in the development of AMB resistance in C. krusei. Our study provides mechanistic insights into the development of drug resistance in fungi and can aid in efforts to stifle the emergence of AMB-resistant pathogenic fungi.

Invasive fungal infections in humans are common in people with compromised immune systems and are difficult to treat, resulting in high mortality. Amphotericin B (AmB) is one of the main antifungal drugs available to treat these infections. AmB binds with plasma membrane ergosterol, causing leakage of cellular ions and promoting cell death. The increasing use of available antifungal drugs to combat pathogenic fungal infections has led to the development of drug resistance. AmB resistance is not very common and is usually caused by changes in the amount or type of ergosterol or changes in the cell wall. Intrinsic AmB resistance occurs in the absence of AmB exposure, whereas acquired AmB resistance can develop during treatment. However, clinical resistance arises due to treatment failure with AmB and depends on multiple factors such as the pharmacokinetics of AmB, infectious fungal species, and host immune status. Candida albicans is a common opportunistic pathogen that can cause superficial infections of the skin and mucosal surfaces, thrush, to life-threatening systemic or invasive infections. In addition, immunocompromised individuals are more susceptible to systemic infections caused by Candida, Aspergillus, and Cryptococcus. Several antifungal drugs with different modes of action are used to treat systemic to invasive fungal infections and are approved for clinical use in the treatment of fungal diseases. However, C. albicans can develop a variety of defenses against antifungal medications. In fungi, plasma membrane sphingolipid molecules could interact with ergosterol, which can lead to the alteration of drug susceptibilities such as AmB. In this review, we mainly summarize the role of sphingolipid molecules and their regulators in AmB resistance.

Invasive fungal infections in humans with compromised immune systems are the primary cause of morbidity and mortality, which is becoming more widely acknowledged. Amphotericin B (AmB) is one of the antifungal drugs used to treat such infections. AmB binds with plasma membrane ergosterol, inducing cellular ions to leak and causing cell death. Reduction in ergosterol content and modification of cell walls have been described as AmB resistance mechanisms. In addition, when the sphingolipid level is decreased, the cell becomes more susceptible to AmB. Previously, PDR16, a gene that encodes phosphatidylinositol transfer protein in Saccharomyces cerevisiae, was shown to enhance AmB resistance upon overexpression. However, the mechanism of PDR16-mediated AmB resistance is not clear. Here, in this study, it was discovered that a plasma membrane proteolipid 3 protein encoded by PMP3 is essential for PDR16-mediated AmB resistance. PDR16-mediated AmB resistance does not depend on ergosterol, but a functional sphingolipid biosynthetic pathway is required. Additionally, PMP3-mediated alteration in membrane integrity abolishes PDR16 mediated AmB resistance, confirming the importance of PMP3 in the PDR16 mediated AmB resistance.

Amphotericin B (AMB) is a major fungicidal polyene agent that has a broad spectrum of action against invasive fungal infections. AMB is typically used as the last-line drug against serious and life-threatening infections when other drugs have failed to eliminate the fungal pathogens. Recently, AMB resistance in Aspergillus fumigatus has become more evident. For example, a high rate of AMB resistance (96%) was noted in the A. fumigatus population in Hamilton, Ontario, Canada. AMB-resistant strains have also been found in other countries. However, the mechanism of AMB resistance remains largely unknown. Here, we investigated the potential genes and mutations associated with AMB resistance using whole-genome sequences and examined AMB resistance distribution among genetic populations. A total of 196 whole-genome sequences representing strains from 11 countries were examined. Analyses of single nucleotide polymorphisms (SNPs) at the whole-genome level revealed that these strains belonged to three divergent genetic clusters, with the majority (90%) of AMB resistant strains located in one of the three clusters, Cluster 2. Our analyses identified over 60 SNPs significantly associated with AMB resistance. Together, these SNPs represent promising candidates from which to investigate the putative molecular mechanisms of AMB resistance and for their potential use in developing rapid diagnostic markers for clinical screening of AMB resistance in A. fumigatus.

Candida auris is a growing concern due to its resistance to antifungal drugs, particularly amphotericin B (AMB), detected in 30 to 60% of clinical isolates. However, the mechanisms of AMB resistance remain poorly understood. Here we investigated 441 in vitro- and in vivo-evolved C. auris lineages from 4 AMB-susceptible clinical strains of different clades. Genetic and sterol analyses revealed four major types of sterol alterations as a result of clinically rare variations in sterol biosynthesis genes ERG6, NCP1, ERG11, ERG3, HMG1, ERG10 and ERG12. In addition, aneuploidies in chromosomes 4 and 6 emerged during resistance evolution. Fitness trade-off phenotyping and mathematical modelling identified diverse strain- and mechanism-dependent fitness trade-offs. Variation in CDC25 rescued fitness trade-offs, thereby increasing the infection capacity. This possibly contributed to therapy-induced acquired AMB resistance in the clinic. Our findings highlight sterol-modulating mechanisms and fitness trade-off compensation as risks for AMB treatment failure in clinical settings.

Rationale: Amphotericin B (AmB), a widely used antifungal agent from the polyene macrolide class, is primarily effective against fungal infections due to its high affinity for ergosterol in fungal membranes. Recent studies suggest AmB may also influence viral infections, particularly those involving influenza (IAV) and SARS-CoV-2. However, the mechanisms by which AmB impacts these viral infections are not yet fully understood. We propose that AmB interacts with Glucosylceramidase Beta 1 (GBA1), a critical factor in endosomal maturation, potentially enhancing viral entry. Methods: We collected clinical data from hospitalized patients with concurrent fungal infections and either influenza or SARS-CoV-2 infections, focusing on ICU admissions, mechanical ventilation needs, and 28-day mortality rates following AmB treatment. To further investigate AmB's effects, we used animal models, including IAV-infected mice and SARS-CoV-2-infected hamsters, monitoring viral load, weight changes, and lung pathology. In vitro assays—including qRT-PCR, Western blotting, and immunofluorescence—were conducted to assess AmB's effects on various stages of viral entry. We also evaluated AmB's impact on endosomal composition and pH, its interaction with GBA1, and its influence on lipid nanoparticle (LNP) delivery in both in vivo and in vitro models. Results: Our clinical analysis revealed that patients treated with AmB who had co-infections exhibited significantly poorer outcomes, characterized by increased ICU admissions, a higher need for mechanical ventilation, and elevated 28-day mortality rates. In animal studies, AmB treatment led to increased viral loads, exacerbated lung pathology, and greater weight loss in both IAV-infected mice and SARS-CoV-2-infected hamsters compared to control groups. In vitro investigations showed that AmB notably enhances IAV infection during the entry stage by promoting viral escape from late endosomes via a specific interaction with GBA1, while not affecting viral attachment or endocytosis. Additionally, AmB was found to alter endosomal composition without impacting pH levels. Importantly, both in vivo and in vitro data indicated that AmB enhances LNP uptake, suggesting its potential utility in therapeutic delivery systems. Conclusions: Our findings indicate that AmB facilitates viral escape from late endosomes through its interaction with GBA1, thereby exacerbating infections caused by influenza and SARS-CoV-2. These results highlight the necessity for caution when administering AmB to patients with viral co-infections. Furthermore, the observed enhancement of LNP delivery points to a promising therapeutic application for AmB in drug delivery, underscoring its role in optimizing endosomal release for effective cargo delivery.

Invasive fungal infections in humans caused by several Candida species, increased considerably in immunocompromised or critically ill patients, resulting in substantial morbidity and mortality. Candida albicans is the most prevalent species, although the frequency of these organisms varies greatly according to geographic region. Infections with C. albicans and non-albicans Candida species have become more common, especially in the past 20 years, as a result of aging, immunosuppressive medication use, endocrine disorders, malnourishment, extended use of medical equipment, and an increase in immunogenic diseases. Despite C. albicans being the species most frequently associated with human infections, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei also have been identified. Several antifungal drugs with different modes of action are approved for use in clinical settings to treat fungal infections. However, due to the common eukaryotic structure of humans and fungi, only a limited number of antifungal drugs are available for therapeutic use. Furthermore, drug resistance in Candida species has emerged as a result of the growing use of currently available antifungal drugs against fungal infections. Amphotericin B (AmB), a polyene class of antifungal drugs, is mainly used for the treatment of serious systemic fungal infections. AmB interacts with fungal plasma membrane ergosterol, triggering cellular ion leakage via pore formation, or extracting the ergosterol from the plasma membrane inducing cellular death. AmB resistance is primarily caused by changes in the content or structure of ergosterol. This review summarizes the antifungal drug resistance exhibited by Candida species, with a special focus on AmB.

Amphotericin B (AmB) has been clinically used for serious fungal infections for over 60 years. The drug is characterized by its specific recognition of ergosterol (Erg) in the fungal cell membrane. AmB and Erg form an ion-channel assembly, which is thought to play a major role in the antibiotic activity of AmB. The precise structure of the ion channel in fungal membranes still remains unelucidated. Recently, the structure of an AmB assembly formed in artificial lipid bilayers was determined using solid-state NMR and molecular dynamics simulations. The structure elucidation was made possible by using 13C- and 19F-labelled AmBs, which were efficiently synthesized using strategies and methods established in previous studies. This review focuses on the structure of the AmB ion channel, which accounts for the antibiotic activity. Additionally, the chemical syntheses of isotope-labelled AmB and Erg used for the structural studies are also reviewed. Solid-state NMR spectra of the labelled AmBs were recorded to measure the distances between labelled sites in the AmB-Erg assembly in lipid bilayers, revealing that the ion channel consisting of seven molecules of AmB spans the bilayer with a single molecule length. Extensive molecular dynamics simulations showed that the conductance of this AmB channel is comparable with those by single-channel recording. The simulations also demonstrated that Erg stabilizes the ion-channel assemblies more efficiently than human cholesterol. The atomic-level structure of the AmB channel in the artificial bilayer will help us to understand the mechanisms of the pharmacological actions and adverse effects of AmB.

Aspergillus flavus has been frequently reported as the second cause of invasive aspergillosis (IA), as well as the leading cause in certain tropical countries. Amphotericin B (AMB) is a clinically important therapy option for a range of invasive fungal infections including invasive aspergillosis, and in vitro resistance to AMB was associated with poor outcomes in IA patients treated with AMB. Compared with the AMB-susceptible isolates of A. terreus, the AMB-resistant isolates of A. terreus showed a lower level of AMB-induced endogenous reactive oxygen species (ROS), which was an important cause of AMB resistance. In this study, we obtained one AMB-resistant isolate of A. flavus, with an AMB MIC of 32 μg/mL, which was sensitive to triazoles and echinocandins. This isolate presented elevated endogenous ROS levels, which strongly suggested that no contribution of decreased AMB-induced endogenous ROS for AMB-resistance, opposite to those observed in A. terreus. Further, we confirmed that the elevated endogenous ROS contributed to the sensitivity of the AMB-resistant A. flavus isolate to triazoles and echinocandins. Further investigation is needed to elucidate the causes of elevated endogenous ROS and the resistance mechanism to AMB in A. flavus.

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PM ObjectivesIt is notoriously difficult to prevent and treat fungal infections, however, the natural world has come up with remedies that are non-toxic, effective, and evade resistance. Here we investigate lactoferrin, an iron-binding glycoprotein found in milk, tears, and sweat, for its capacity to inhibit fungi and to synergize with commonly used antifungal drugs, with the aim of determining its mode of action.MethodsLactoferrin (LF) was obtained from a commercial supplier and two dairy companies. LF was tested on suite of pathogenic yeast and mold species for inhibition using CLSI microdilution methods. Synergy was determined with antifungal drugs amphotericin B (AMB), nystatin (NYS), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), and 5-fluorocytosine (5FC). The effect of LF on fungal cells was analyzed using scanning electron microscopy (SEM). The active peptide/s within LF were then predicted from pepsin and in silico digestion, synthesized, and tested for synergy with amphotericin B (AMB). Tethered synthetic membranes were produced and were loaded with ergosterol or cholesterol to test the nature and specificity of membrane binding by LF and the synthetic peptide.ResultsLF demonstrated antifungal activity against yeast species Cryptococcus, Candida, and Saccharomyces and was much less effective against molds. Good synergy was achieved with AMB but not azole or echinocandin drugs. While the iron-chelating capacity of LF was important for the antifungal activity it was not involved in synergy. SEM revealed cell damage suggesting an interaction between AMB, LF and the fungal membrane or cell wall. A 30-residue peptide from the C lobe of LF was synthesized and tested for activity and synergy. This peptide, dubbed lactofungin (LFG) was inactive alone but was potently synergistic with AMB, indicating a direct role in augmenting AMB activity. Synthetic membranes loaded with ergosterol but not cholesterol were disrupted by AMB + LFG, demonstrating that activity was fungal-specific and was mediated through ergosterol binding.ConclusionLF is a complex molecule that causes fungal inhibition via iron binding and when cleaved by pepsin can produce active peptides. As AMB is a highly toxic treatment, the use of LFG as a synergent could help increase activity while lowering the effective dose, thereby reducing undesirable side effects. The action of AMB + LFG appears dependent on ergosterol, suggesting inhibition will be highly fungal-specific.

The polyene antimycotic amphotericin B (AmB) and its liposomal formulation AmBisome belong to the treatment options of invasive aspergillosis caused by Aspergillus fumigatus. Increasing resistance to AmB in clinical isolates of Aspergillus species is a growing concern, but mechanisms of AmB resistance remain unclear. In this study, we conducted a proteomic analysis of A. fumigatus exposed to sublethal concentrations of AmB and AmBisome. Both antifungals induced significantly increased levels of proteins involved in aromatic acid metabolism, transmembrane transport, and secondary metabolite biosynthesis. One of the most upregulated proteins was RtaA, a member of the RTA-like protein family, which includes conserved fungal membrane proteins with putative functions as transporters or translocases. Accordingly, we found that RtaA is mainly located in the cytoplasmic membrane and to a minor extent in vacuolar-like structures. Deletion of rtaA led to increased polyene sensitivity and its overexpression resulted in modest resistance. Interestingly, rtaA expression was only induced by exposure to the polyenes AmB and nystatin, but not by itraconazole and caspofungin. Orthologues of rtaA were also induced by AmB exposure in A. lentulus and A. terreus. Deletion of rtaA did not significantly change the ergosterol content of A. fumigatus, but decreased fluorescence intensity of the sterol-binding stain filipin. This suggests that RtaA is involved in sterol and lipid trafficking, possibly by transporting the target ergosterol to or from lipid droplets. These findings reveal the contribution of RtaA to polyene resistance in A. fumigatus, and thus provide a new putative target for antifungal drug development.

Author SummaryThe evolution of drug resistance in human pathogens is considered an inevitable consequence of the selective pressures imposed by antimicrobial drugs. Yet resistance to one antifungal drug, amphotericin B (AmB), remains extremely rare despite decades of widespread use. Here we explore the biological mechanisms underlying this conundrum. By examining natural and experimental populations of Candida albicans, we identify multiple mutations that confer resistance to AmB in vitro. As with the evolution of resistance to other antifungals, we find that the chaperone protein Hsp90 is involved in enabling the evolution of resistance to AmB. We also discover, however, that mutations that confer AmB resistance impose massive costs on other aspects of fungal pathogenicity; strains that are resistant to AmB are hypersensitive to attack by the host immune system and are unable to invade and damage host tissue. Thus, the evolution of resistance to AmB is restricted by a tradeoff between tolerance of the drug and the ability to cause disease. We propose that developing new antibiotics for which resistance presents such dire tradeoffs may be a promising strategy to prevent the evolution of resistance.