Reshaping Echinocandin Antifungal Drugs To Circumvent Glucan Synthase Point-Mutation-Mediated Resistance.

Echinocandins are a class of antifungal drugs that inhibit the activity of the ß-(1,3)-glucan synthase complex, which synthesizes fungal cell wall ß-(1,3)-glucan. Echinocandin resistance is linked to mutations in the FKS gene, which encodes the catalytic subunit of the glucan synthase complex. We present a molecular-docking-based model that provides insight into how echinocandins interact with the target Fks protein: echinocandins form a ternary complex with both Fks and membrane lipids. We used reductive dehydration of alcohols to generate dehydroxylated echinocandin derivatives and evaluated their potency against a panel of Candida pathogens constructed by introducing resistance-conferring mutations in the FKS gene. We found that removing the hemiaminal alcohol, which drives significant conformational alterations in the modified echinocandins, reduced their efficacy. Conversely, eliminating the benzylic alcohol of echinocandins enhanced potency by up to two orders of magnitude, in a manner dependent upon the resistance-conferring mutation. Strains that have developed resistance to either rezafungin, the most recently clinically approved echinocandin, or its dehydroxylated derivative RZF-1, exhibit high resistance to rezafungin while demonstrating moderate resistance to RZF-1. These findings provide valuable insight for combating echinocandin resistance through chemical modifications.

Multiple phosphorylation sites regulate the activity of the repressor Mig1 in Candida albicans.

IMPORTANCE: The SNF1 protein kinase signaling pathway, which is highly conserved in eukaryotic cells, is important for metabolic adaptations in the pathogenic yeast Candida albicans. However, so far, it has remained elusive how SNF1 controls the activity of one of its main effectors, the repressor protein Mig1 that inhibits the expression of genes required for the utilization of alternative carbon sources when glucose is available. In this study, we have identified multiple phosphorylation sites in Mig1 that contribute to its inactivation. Mutation of these sites strongly increased Mig1 repressor activity in the absence of SNF1, but SNF1 could still sufficiently inhibit the hyperactive Mig1 to enable growth on alternative carbon sources. These findings reveal features of Mig1 that are important for controlling its repressor activity. Furthermore, they demonstrate that both SNF1 and additional protein kinases regulate Mig1 in this pathogenic yeast.

Candida albicans exhibits heterogeneous and adaptive cytoprotective responses to antifungal compounds.

Candida albicans, an opportunistic human pathogen, poses a significant threat to human health and is associated with significant socio-economic burden. Current antifungal treatments fail, at least in part, because C. albicans can initiate a strong drug tolerance response that allows some cells to grow at drug concentrations above their minimal inhibitory concentration. To better characterize this cytoprotective tolerance program at the molecular single-cell level, we used a nanoliter droplet-based transcriptomics platform to profile thousands of individual fungal cells and establish their subpopulation characteristics in the absence and presence of antifungal drugs. Profiles of untreated cells exhibit heterogeneous expression that correlates with cell cycle stage with distinct metabolic and stress responses. At 2 days post-fluconazole exposure (a time when tolerance is measurable), surviving cells bifurcate into two major subpopulations: one characterized by the upregulation of genes encoding ribosomal proteins, rRNA processing machinery, and mitochondrial cellular respiration capacity, termed the Ribo-dominant (Rd) state; and the other enriched for genes encoding stress responses and related processes, termed the Stress-dominant (Sd) state. This bifurcation persists at 3 and 6 days post-treatment. We provide evidence that the ribosome assembly stress response (RASTR) is activated in these subpopulations and may facilitate cell survival.

Many drugs currently used to treat fungal diseases are becoming less effective. This is partly due to the rise of antifungal resistance, where certain fungal cells acquire mutations that enable them to thrive and proliferate despite the medication. Antifungal tolerance also contributes to this problem, wherein certain cells can continue to grow and multiply, while other ­ genetically identical ones ­ cannot. This variability is partly due to differences in gene expression within the cells. The specific nature of these differences has remained elusive, mainly because their study requires the use of expensive and challenging single-cell technologies. To address this challenge, Dumeaux et al. adapted an existing technique to perform single-cell transcriptomics in the pathogenic yeast Candida albicans. Their approach was cost effective and made it possible to examine the gene expression in thousands of individual cells within a population that had either been treated with antifungal drugs or were left untreated. After two to three days following exposure to the antifungal treatment, C. albicans cells commonly exhibited one of two states: one subgroup, the 'Ribo-dominant' cells, predominantly expressed genes for ribosomal proteins, while the other group, the 'Stress-dominant' cells, upregulated their expression of stress-response genes. This suggests that drug tolerance may be related to different gene expression patterns in growing cell subpopulations compared with non-growing subpopulations. The findings also indicate that the so-called 'ribosome assembly stress response' known to help baker's yeast cells to survive, might also aid C. albicans in surviving exposure to antifungal treatments. The innovative use of single-cell transcriptomics in this study could be applied to other species of fungi to study differences in cell communication under diverse growth conditions. Moreover, the unique gene expression patterns in C. albicans identified by Dumeaux et al. may help to design new antifungal treatments that target pathways linked to drug resistance.

The Metabolism of Susceptibility: Clearing the FoG Between Tolerance and Resistance in Candida albicans.

Purpose of Review: Failure of antifungal treatment is alarmingly common in patients infected with Candida albicans isolates that test as susceptible in vitro. This means that clinical susceptibility tests have limited predictive value for treatment success. To guide the improvement of patient outcomes, we must understand the effects of environmental and metabolic states on drug responses. Recent Findings: Lab conditions often deviate from host environments, and current susceptibility testing standards ignore slow-growing, tolerant phenotypes; both factors may contribute to antifungal treatment failure. Metabolomic studies reveal that strain background, nutrient availability, and drug exposure influence the metabolic state of C. albicans cells; similarly, the metabolic state influences drug susceptibility. Summary: Identifying tolerant strains in the clinic may improve patient outcomes. Studies that analyze the effects of essential but limited nutrients have the potential to improve the avoidance of persistent candidiasis and to reduce the frequency of antifungal treatment failures. Here, we highlight literature that explores the effect of drug exposure and antifungal drug resistance status on the C. albicans metabolome. Similar analyses need to be carried out relative to antifungal drug tolerance. Additionally, we focus on the biological relevance of four essential small molecules-iron, zinc, phosphate, and sphingolipids-to antifungal tolerance and resistance.

A Suppressor Mutation in the ß-Subunit Kis1 Restores Functionality of the SNF1 Complex in Candida albicans snf4Δ Mutants.

The heterotrimeric protein kinase SNF1 is a key regulator of metabolic adaptation in the pathogenic yeast Candida albicans, and mutants with a defective SNF1 complex cannot grow on carbon sources other than glucose. We identified a novel type of suppressor mutation in the ß-subunit Kis1 that rescued the growth defects of cells lacking the regulatory γ-subunit Snf4 of the SNF1 complex. Unlike wild-type Kis1, the mutated Kis1A396T could bind to the catalytic α-subunit Snf1 in the absence of Snf4. Binding of Kis1A396T did not enhance phosphorylation of Snf1 by the upstream activating kinase Sak1, which is impaired in snf4Δ mutants. Nevertheless, the mutated Kis1A396T reestablished SNF1-dependent gene expression, confirming that SNF1 functionality was restored. The repressor proteins Mig1 and Mig2 were phosphorylated even in the absence of Snf1, but their phosphorylation patterns were altered, indicating that SNF1 regulates Mig1 and Mig2 activity indirectly. In contrast to wild-type cells, mutants lacking Snf4 were unable to reduce the amounts of Mig1 and Mig2 when grown on alternative carbon sources, and this deficiency was also remediated by the mutated Kis1A396T. These results provide novel insights into the regulation of SNF1 and the repressors Mig1 and Mig2 in the metabolic adaptation of C. albicans. IMPORTANCE The highly conserved protein kinase SNF1 plays a key role in the metabolic adaptation of the pathogenic yeast Candida albicans, but it is not clear how it regulates its downstream targets in this fungus. We show that the repressor proteins Mig1 and Mig2 are phosphorylated also in cells lacking the catalytic α-subunit Snf1 of the SNF1 complex, but the amounts of both proteins were reduced in wild-type cells when glucose was replaced by alternative carbon sources, pointing to an indirect mechanism of regulation. Mutants lacking the regulatory γ-subunit Snf4 of the SNF1 complex, which cannot grow on alternative carbon sources, were unable to downregulate Mig1 and Mig2 levels. We identified a novel type of suppressor mutation, an amino acid substitution in the ß-subunit Kis1, which enabled Kis1 to bind to Snf1 in the absence of Snf4, thereby restoring Mig1 and Mig2 downregulation, SNF1-dependent gene expression, and growth on alternative carbon sources. These results provide new insights into the SNF1 signaling pathway in C. albicans.

The zinc cluster transcription factor Czf1 regulates cell wall architecture and integrity in Candida albicans.

The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C. albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C. albicans czf1Δ mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C. albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall.

Generation of Viable Candida albicans Mutants Lacking the "Essential" Protein Kinase Snf1 by Inducible Gene Deletion.

The protein kinase Snf1, a member of the highly conserved AMP-activated protein kinase family, is a central regulator of metabolic adaptation. In the pathogenic yeast Candida albicans, Snf1 is considered to be essential, as previous attempts by different research groups to generate homozygous snf1Δ mutants were unsuccessful. We aimed to elucidate why Snf1 is required for viability in C. albicans by generating snf1Δ null mutants through forced, inducible gene deletion and observing the terminal phenotype before cell death. Unexpectedly, we found that snf1Δ mutants were viable and could grow, albeit very slowly, on rich media containing the preferred carbon source glucose. Growth was improved when the cells were incubated at 37°C instead of 30°C, and this phenotype enabled us to isolate homozygous snf1Δ mutants also by conventional, sequential deletion of both SNF1 alleles in a wild-type C. albicans strain. All snf1Δ mutants could grow slowly on glucose but were unable to utilize alternative carbon sources. Our results show that, under optimal conditions, C. albicans can live and grow without Snf1. Furthermore, they demonstrate that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicansIMPORTANCE Essential genes are those that are indispensable for the viability and growth of an organism. Previous studies indicated that the protein kinase Snf1, a central regulator of metabolic adaptation, is essential in the pathogenic yeast Candida albicans, because no homozygous snf1 deletion mutants of C. albicans wild-type strains could be obtained by standard approaches. In order to investigate the lethal consequences of SNF1 deletion, we generated conditional mutants in which SNF1 could be deleted by forced, inducible excision from the genome. Unexpectedly, we found that snf1 null mutants were viable and could grow slowly under optimal conditions. The growth phenotypes of the snf1Δ mutants explain why such mutants were not recovered in previous attempts. Our study demonstrates that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans.

An Intragenic Recombination Event Generates a Snf4-Independent Form of the Essential Protein Kinase Snf1 in Candida albicans.

The heterotrimeric protein kinase SNF1 plays a key role in the metabolic adaptation of the pathogenic yeast Candida albicans It consists of the essential catalytic α-subunit Snf1, the γ-subunit Snf4, and one of the two ß-subunits Kis1 and Kis2. Snf4 is required to release the N-terminal catalytic domain of Snf1 from autoinhibition by the C-terminal regulatory domain, and snf4Δ mutants cannot grow on carbon sources other than glucose. In a screen for suppressor mutations that restore growth of a snf4Δ mutant on alternative carbon sources, we isolated a mutant in which six amino acids between the N-terminal kinase domain and the C-terminal regulatory domain of Snf1 were deleted. The deletion was caused by an intragenic recombination event between two 8-bp direct repeats flanking six intervening codons. In contrast to truncated forms of Snf1 that contain only the kinase domain, the Snf4-independent Snf1Δ311 - 316 was fully functional and could replace wild-type Snf1 for normal growth, because it retained the ability to interact with the Kis1 and Kis2 ß-subunits via its C-terminal domain. Indeed, the Snf4-independent Snf1Δ311 - 316 still required the ß-subunits of the SNF1 complex to perform its functions and did not rescue the growth defects of kis1Δ mutants. Our results demonstrate that a preprogrammed in-frame deletion event within the SNF1 coding region can generate a mutated form of this essential kinase which abolishes autoinhibition and thereby overcomes growth deficiencies caused by a defect in the γ-subunit Snf4.IMPORTANCE Genomic alterations, including different types of recombination events, facilitate the generation of genetically altered variants and enable the pathogenic yeast Candida albicans to adapt to stressful conditions encountered in its human host. Here, we show that a specific recombination event between two 8-bp direct repeats within the coding sequence of the SNF1 gene results in the deletion of six amino acids between the N-terminal kinase domain and the C-terminal regulatory domain and relieves this essential kinase from autoinhibition. This preprogrammed deletion allowed C. albicans to overcome growth defects caused by the absence of the regulatory subunit Snf4 and represents a built-in mechanism for the generation of a Snf4-independent Snf1 kinase.

The Snf1-activating kinase Sak1 is a key regulator of metabolic adaptation and in vivo fitness of Candida albicans.

The metabolic flexibility of the opportunistic fungal pathogen Candida albicans is important for colonisation and infection of different host niches. Complex regulatory networks, in which protein kinases play central roles, link metabolism and other virulence-associated traits, such as filamentous growth and stress resistance, and thereby control commensalism and pathogenicity. By screening a protein kinase deletion mutant library that was generated in the present work using an improved SAT1 flipper cassette, we found that the previously uncharacterised kinase Sak1 is a key upstream activator of the protein kinase Snf1, a highly conserved regulator of nutrient stress responses that is essential for viability in C. albicans. The sak1Δ mutants failed to grow on many alternative carbon sources and were hypersensitive to cell wall/membrane stress. These phenotypes were mirrored in mutants lacking other subunits of the SNF1 complex and partially compensated by a hyperactive form of Snf1. Transcriptional profiling of sak1Δ mutants showed that Sak1 ensures basal expression of glyoxylate cycle and gluconeogenesis genes even in glucose-rich media and thereby contributes to the metabolic plasticity of C. albicans. In a mouse model of gastrointestinal colonisation, sak1Δ mutants were rapidly outcompeted by wild-type cells, demonstrating that Sak1 is essential for the in vivo fitness of C. albicans.