Alternative Oxidase: From Molecule and Function to Future Inhibitors.

In the respiratory chain of the majority of aerobic organisms, the enzyme alternative oxidase (AOX) functions as the terminal oxidase and has important roles in maintaining metabolic and signaling homeostasis in mitochondria. AOX endows the respiratory system with flexibility in the coupling among the carbon metabolism pathway, electron transport chain (ETC) activity, and ATP turnover. AOX allows electrons to bypass the main cytochrome pathway to restrict the generation of reactive oxygen species (ROS). The inhibition of AOX leads to oxidative damage and contributes to the loss of adaptability and viability in some pathogenic organisms. Although AOXs have recently been identified in several organisms, crystal structures and major functions still need to be explored. Recent work on the trypanosome alternative oxidase has provided a crystal structure of an AOX protein, which contributes to the structure-activity relationship of the inhibitors of AOX. Here, we review the current knowledge on the development, structure, and properties of AOXs, as well as their roles and mechanisms in plants, animals, algae, protists, fungi, and bacteria, with a special emphasis on the development of AOX inhibitors, which will improve the understanding of respiratory regulation in many organisms and provide references for subsequent studies of AOX-targeted inhibitors.

Understanding fluconazole tolerance in Candida albicans: implications for effective treatment of candidiasis and combating invasive fungal infections.

OBJECTIVES: Fluconazole (FLC) tolerant phenotypes in Candida species contribute to persistent candidemia and the emergence of FLC resistance. Therefore, making FLC fungicidal and eliminating FLC tolerance are important for treating invasive fungal diseases (IFDs) caused by Candida species. However, the mechanisms of FLC tolerance in Candida species remain to be fully explored. METHODS: This review discusses the high incidence of FLC tolerance in Candida species and the importance of successfully clearing FLC tolerance in treating candidiasis. We further define and characterize FLC tolerance in C. albicans. RESULTS: This review identifies global factors affecting FLC tolerance and suggest that FLC tolerance is a strategy of C. albicans response to FLC damage whose mechanism differs from FLC resistance. CONCLUSIONS: This review highlights the significance of the cell membrane and cell wall integrity in FLC tolerance, guiding approaches to combat IFDs caused by Candida species..

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 Adr1 transcription factor directs regulation of the ergosterol pathway and azole resistance in Candida albicans.

IMPORTANCE: Research often relies on well-studied orthologs within related species, with researchers using a well-studied gene or protein to allow prediction of the function of the ortholog. In the opportunistic pathogen Candida albicans, orthologs are usually compared with Saccharomyces cerevisiae, and this approach has been very fruitful. Many transcription factors (TFs) do similar jobs in the two species, but many do not, and typically changes in function are driven not by modifications in the structures of the TFs themselves but in the connections between the transcription factors and their regulated genes. This strategy of changing TF function has been termed transcription factor rewiring. In this study, we specifically looked for rewired transcription factors, or Candida-specific TFs, that might play a role in drug resistance. We investigated 30 transcription factors that were potentially rewired or were specific to the Candida clade. We found that the Adr1 transcription factor conferred resistance to drugs like fluconazole, amphotericin B, and terbinafine when activated. Adr1 is known for fatty acid and glycerol utilization in Saccharomyces, but our study reveals that it has been rewired and is connected to ergosterol biosynthesis in Candida albicans.

Genome sequencing of 15 acid-tolerant yeasts.

We report draft genome sequences for 15 non-conventional Saccharomycotina yeast strains obtained from public culture repositories. Included in our collection are eight strains of Pichia with broad tolerance to dicarboxylic acids. The genome sequences of these strains will enable comparative genomics of acid-tolerant phenotypes and strain engineering of non-conventional hosts.

Screening non-conventional yeasts for acid tolerance and engineering Pichia occidentalis for production of muconic acid.

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L-1 adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L-1), yield (0.134 g g-1 glucose) and productivity (0.511 g L-1 h-1) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.

Candidiasis: From cutaneous to systemic, new perspectives of potential targets and therapeutic strategies.

Candidiasis is an infection caused by fungi from a Candida species, most commonly Candida albicans. C. albicans is an opportunistic fungal pathogen typically residing on human skin and mucous membranes of the mouth, intestines or vagina. It can cause a wide variety of mucocutaneous barrier and systemic infections; and becomes a severe health problem in HIV/AIDS patients and in individuals who are immunocompromised following chemotherapy, treatment with immunosuppressive agents or after antibiotic-induced dysbiosis. However, the immune mechanism of host resistance to C. albicans infection is not fully understood, there are a limited number of therapeutic antifungal drugs for candidiasis, and these have disadvantages that limit their clinical application. Therefore, it is urgent to uncover the immune mechanisms of the host protecting against candidiasis and to develop new antifungal strategies. This review synthesizes current knowledge of host immune defense mechanisms from cutaneous candidiasis to invasive C. albicans infection and documents promising insights for treating candidiasis through inhibitors of potential antifungal target proteins.

A Small Molecule Inhibitor of Erg251 Makes Fluconazole Fungicidal by Inhibiting the Synthesis of the 14α-Methylsterols.

Fluconazole (FLC) is widely used to prevent and treat invasive fungal infections. However, FLC is a fungistatic agent, allowing clinical FLC-susceptible isolates to tolerate FLC. Making FLC fungicidal in combination with adjuvants is a promising strategy to avoid FLC resistance and eliminate the persistence and recurrence of fungal infections. Here, we identify a new small molecule compound, CZ66, that can make FLC fungicidal. The mechanism of action of CZ66 is targeting the C-4 sterol methyl oxidase, encoded by the ERG251 gene, resulting in decreased content of sterols with the 14α-methyl group and ultimately eliminating FLC tolerance of Candida albicans. CZ66 most likely interacts with Erg251 through residues Glu195, Gly206, and Arg241. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance. IMPORTANCE Fluconazole (FLC) tolerance increases the frequency of acquired FLC resistance, and a high FLC tolerance level is associated with persistent candidemia. Multiple functional proteins, such as calcineurin, heat shock protein 90 (Hsp90), and ADP ribosylation factor, are essential for the survival of C. albicans exposed to FLC, but how these factors increase the fungicidal activity of FLC remains to be determined. In this study, we found that 14α-methylsterols replace ergosterol to allow C. albicans to survive FLC, but Erg251 inactivated by CZ66 results in loss of 14α-methylsterol synthesis and cell death of C. albicans treated with FLC. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance.

Rosemary essential oil and its components 1,8-cineole and α-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albicans.

The essential oil from Rosmarinus officinalis L., a composite mixture of plant-derived secondary metabolites, exhibits antifungal activity against virulent candidal species. Here we report the impact of rosemary oil and two of its components, the monoterpene α-pinene and the monoterpenoid 1,8-cineole, against Candida albicans, which induce ROS-dependent cell death at high concentrations and inhibit hyphal morphogenesis and biofilm formation at lower concentrations. The minimum inhibitory concentrations (100% inhibition) for both rosemary oil and 1,8-cineole were 4500 µg/ml and 3125 µg/ml for α-pinene, with the two components exhibiting partial synergy (FICI = 0.55 ± 0.07). At MIC and 1/2 MIC, rosemary oil and its components induced a generalized cell wall stress response, causing damage to cellular and organelle membranes, along with elevated chitin production and increased cell surface adhesion and elasticity, leading to complete vacuolar segregation, mitochondrial depolarization, elevated reactive oxygen species, microtubule dysfunction, and cell cycle arrest mainly at the G1/S phase, consequently triggering cell death. Interestingly, the same oils at lower fractional MIC (1/8-1/4) inhibited virulence traits, including reduction of mycelium (up to 2-fold) and biofilm (up to 4-fold) formation, through a ROS-independent mechanism.

A Deep Learning Approach to Capture the Essence of Candida albicans Morphologies.

We present deep learning-based approaches for exploring the complex array of morphologies exhibited by the opportunistic human pathogen Candida albicans. Our system, entitled Candescence, automatically detects C. albicans cells from differential image contrast microscopy and labels each detected cell with one of nine morphologies. This ranges from yeast white and opaque forms to hyphal and pseudohyphal filamentous morphologies. The software is based upon a fully convolutional one-stage (FCOS) object detector, a deep learning technique that uses an extensive set of images that we manually annotated with the location and morphology of each cell. We developed a novel cumulative curriculum-based learning strategy that stratifies our images by difficulty from simple yeast forms to complex filamentous architectures. Candescence achieves very good performance (~85% recall; 81% precision) on this difficult learning set, where some images contain hundreds of cells with substantial intermixing between the predicted classes. To capture the essence of each C. albicans morphology and how they intermix, we used a second technique from deep learning entitled generative adversarial networks. The resultant models allow us to identify and explore technical variables, developmental trajectories, and morphological switches. Importantly, the model allows us to quantitatively capture morphological plasticity observed with genetically modified strains or strains grown in different media and environments. We envision Candescence as a community meeting point for quantitative explorations of C. albicans morphology. IMPORTANCE The fungus Candida albicans can "shape shift" between 12 morphologies in response to environmental variables. The cytoprotective capacity provided by this polymorphism makes C. albicans a formidable pathogen to treat clinically. Microscopy images of C. albicans colonies can contain hundreds of cells in different morphological states. Manual annotation of images can be difficult, especially as a result of densely packed and filamentous colonies and of technical artifacts from the microscopy itself. Manual annotation is inherently subjective, depending on the experience and opinion of annotators. Here, we built a deep learning approach entitled Candescence to parse images in an automated, quantitative, and objective fashion: each cell in an image is located and labeled with its morphology. Candescence effectively replaces simple rules based on visual phenotypes (size, shape, and shading) with neural circuitry capable of capturing subtle but salient features in images that may be too complex for human annotators.

Transcriptional Profiling of the Candida albicans Response to the DNA Damage Agent Methyl Methanesulfonate.

The infection of a mammalian host by the pathogenic fungus Candida albicans involves fungal resistance to reactive oxygen species (ROS)-induced DNA damage stress generated by the defending macrophages or neutrophils. Thus, the DNA damage response in C. albicans may contribute to its pathogenicity. Uncovering the transcriptional changes triggered by the DNA damage-inducing agent MMS in many model organisms has enhanced the understanding of their DNA damage response processes. However, the transcriptional regulation triggered by MMS remains unclear in C. albicans. Here, we explored the global transcription profile in response to MMS in C. albicans and identified 306 defined genes whose transcription was significantly affected by MMS. Only a few MMS-responsive genes, such as MGT1, DDR48, MAG1, and RAD7, showed potential roles in DNA repair. GO term analysis revealed that a large number of induced genes were involved in antioxidation responses, and some downregulated genes were involved in nucleosome packing and IMP biosynthesis. Nevertheless, phenotypic assays revealed that MMS-induced antioxidation gene CAP1 and glutathione metabolism genes GST2 and GST3 showed no direct roles in MMS resistance. Furthermore, the altered transcription of several MMS-responsive genes exhibited RAD53-related regulation. Intriguingly, the transcription profile in response to MMS in C. albicans shared a limited similarity with the pattern in S. cerevisiae, including COX17, PRI2, and MGT1. Overall, C. albicans cells exhibit global transcriptional changes to the DNA damage agent MMS; these findings improve our understanding of this pathogen's DNA damage response pathways.

Baicalein Acts against Candida albicans by Targeting Eno1 and Inhibiting Glycolysis.

Baicalein (BE) is a promising antifungal small-molecule compound with an extended antifungal spectrum, good synergy with fluconazole, and low toxicity, but its target protein and antifungal mechanism remain elusive. In this study, we found that BE can function against Candida albicans by disrupting glycolysis through targeting Eno1 and inhibiting its function. Eno1 acts as a key therapeutic target of the drug, as BE had no antifungal activity against the eno1 null mutant in a Galleria mellonella model of C. albicans infection. To investigate the mechanism of action, we solved the crystal structure of C. albicans Eno1(CaEno1) and then compared the difference between this structure and that of Eno1 from humans. The predicted primary binding site of BE on CaEno1 is between amino acids D261 and W274, with D263, S269, and K273 playing critical roles in the interaction with BE. Both positions S269 and K273 have different residues in the human Eno1 (hEno1). This finding suggests that BE may bind selectively to CaEno1, which would limit the potential for side effects in humans. Our findings demonstrate that Eno1 is a target protein of BE and thus may serve as a novel target for the development of antifungal therapeutics acting through the inhibition of glycolysis. IMPORTANCE Baicalein (BE) is a promising antifungal agent which has been well characterized, but its target protein is still undiscovered. The protein Eno1 plays a crucial role in the survival of Candida albicans. However, there are few antifungal agents which inhibit the functions of Eno1. Here, we found that BE can function against Candida albicans by disrupting glycolysis through targeting Eno1 and inhibiting its function. We further solved the crystal structure of C. albicans Eno1(CaEno1) and predicted that the primary binding site of BE on CaEno1 is between amino acids D261 and W274, with D263, S269, and K273 playing critical roles in the interaction with BE. Our findings will be helpful to get specific small-molecule inhibitors of CaEno1 and open the way for the development of new antifungal therapeutics targeted at inhibiting glycolysis.

The MyLO CRISPR-Cas9 toolkit: a markerless yeast localization and overexpression CRISPR-Cas9 toolkit.

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-associated protein 9 (Cas9)-based genome editing tools, we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with 3 components: (1) a set of optimized Streptococcus pyogenes Cas9-guide RNA expression vectors with 5 selectable markers and the option to either preclone or cotransform the gRNAs; (2) vectors for the one-step construction of integration cassettes expressing an untagged or green fluorescent protein/red fluorescent protein/hemagglutinin-tagged gene of interest at one of 3 levels, supporting localization and overexpression studies; and (3) integration cassettes containing moderately expressed green fluorescent protein- or red fluorescent protein-tagged compartmental markers for colocalization experiments. These components allow rapid, high-efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to colabel all target subcellular compartments with green fluorescent protein and red fluorescent protein. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle that allows the stable genomic integration of DNA with the ease of use approaching that of transforming plasmids.

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.

Genetic Screening of Candida albicans Inactivation Mutants Identifies New Genes Involved in Macrophage-Fungal Cell Interactions.

Candida albicans, an important fungal pathogen of humans, displays different morphologies, such as yeast, pseudo-hyphae and hyphae, which are recognized unequally by phagocytic cells of the innate immune response. Once C. albicans cells invade host tissues, immune cells such as macrophages are attracted to the site of infection and activated to recognize, engulf and kill the pathogen. We have investigated this fungal cell-macrophage interface by using high-throughput screening of the C. albicans GRACE library to identify genes that can influence this interaction and modify the kinetics of engulfment. Compared with the wild-type (WT) strain, we identified generally faster rates of engulfment for those fungal strains with constitutive pseudo-hyphal and hyphal phenotypes, whereas yeast-form-locked strains showed a reduced and delayed recognition and internalization by macrophages. We identified a number of GRACE strains that showed normal morphological development but exhibited different recognition and engulfment kinetics by cultured macrophages and characterized two mutants that modified interactions with the murine and human-derived macrophages. One mutant inactivated an uncharacterized C. albicans open reading frame that is the ortholog of S. cerevisiae OPY1, the other inactivated CaKRE1. The modified interaction was monitored during a 4 h co-culture. Early in the interaction, both opy1 and kre1 mutant strains showed reduced recognition and engulfment rates by macrophages when compared with WT cells. At fungal germ tube initiation, the engulfment kinetics increased for both mutants and WT cells, however the WT cells still showed a higher internalization by macrophages up to 2 h of interaction. Subsequently, between 2 and 4 h of the interaction, when most macrophages contain engulfed fungal cells, the engulfment kinetics increased for the opy1 mutant and further decreased for the kre1 mutant compared with Ca-WT. It appears that fungal morphology influences macrophage association with C. albicans cells and that both OPY1 and KRE1 play roles in the interaction of the fungal cells with phagocytes.

SAGA Complex Subunits in Candida albicans Differentially Regulate Filamentation, Invasiveness, and Biofilm Formation.

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a highly conserved, multiprotein co-activator complex that consists of five distinct modules. It has two enzymatic functions, a histone acetyltransferase (HAT) and a deubiquitinase (DUB) and plays a central role in processes such as transcription initiation, elongation, protein stability, and telomere maintenance. We analyzed conditional and null mutants of the SAGA complex module components in the fungal pathogen Candida albicans; Ngg1, (the HAT module); Ubp8, (the DUB module); Tra1, (the recruitment module), Spt7, (the architecture module) and Spt8, (the TBP interaction unit), and assessed their roles in a variety of cellular processes. We observed that spt7Δ/Δ and spt8Δ/Δ strains have a filamentous phenotype, and both are highly invasive in yeast growing conditions as compared to the wild type, while ngg1Δ/Δ and ubp8Δ/Δ are in yeast-locked state and non-invasive in both YPD media and filamentous induced conditions compared to wild type. RNA-sequencing-based transcriptional profiling of SAGA mutants reveals upregulation of hyphal specific genes in spt7Δ/Δ and spt8Δ/Δ strains and downregulation of ergosterol metabolism pathway. As well, spt7Δ/Δ and spt8Δ/Δ confer susceptibility to antifungal drugs, to acidic and alkaline pH, to high temperature, and to osmotic, oxidative, cell wall, and DNA damage stresses, indicating that these proteins are important for genotoxic and cellular stress responses. Despite having similar morphological phenotypes (constitutively filamentous and invasive) spt7 and spt8 mutants displayed variation in nuclear distribution where spt7Δ/Δ cells were frequently binucleate and spt8Δ/Δ cells were consistently mononucleate. We also observed that spt7Δ/Δ and spt8Δ/Δ mutants were quickly engulfed by macrophages compared to ngg1Δ/Δ and ubp8Δ/Δ strains. All these findings suggest that the SAGA complex modules can have contrasting functions where loss of Spt7 or Spt8 enhances filamentation and invasiveness while loss of Ngg1 or Ubp8 blocks these processes.

The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans.

Zinc cluster transcription factors (TFs) are essential fungal regulators of gene expression. In the pathogen Candida albicans, the gene orf19.1604 encodes a zinc cluster TF regulating filament development. Hyperactivation of orf19.1604, which we have named RHA1 for Regulator of Hyphal Activity, generates wrinkled colony morphology under nonhyphal growth conditions, triggers filament formation, invasiveness, and enhanced biofilm formation and causes reduced virulence in the mouse model of systemic infection. The strain expressing activated Rha1 shows up-regulation of genes required for filamentation and cell-wall-adhesion-related proteins. Increased expression is also seen for the hyphal-inducing TFs Brg1 and Ume6, while the hyphal repressor Nrg1 is downregulated. Inactivation of RHA1 reduces filamentation under a variety of filament-inducing conditions. In contrast to the partial effect of either single mutant, the double rha1 ume6 mutant strain is highly defective in both serum- and Spider-medium-stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization even in brg1 ume6 double mutants. Thus, in response to external signals, Rha1 functions with other morphogenesis regulators including Brg1 and Ume6, to mediate filamentation.

Corrigendum to "Loss of Arp1, a putative actin-related protein, triggers filamentous and invasive growth and impairs pathogenicity in Candida albicans" (Computational and Structural Biotechnology Journal, 2020, 18: 4002-4015).

[This corrects the article DOI: 10.1016/j.csbj.2020.11.034.].

Signal-mediated localization of Candida albicans pheromone response pathway components.

A MAPK cascade consists of three kinases, (MEKK, MEK and MAPK), that are sequentially activated in response to a stimulus and serve to transmit signals. In C. albicans and in yeast, an MAPK cascade is linked to the pheromone pathway through a scaffold protein (Cst5 and Ste5, respectively). Cst5 is much shorter and lacks key domains compared to Ste5, so in C. albicans, other elements, in particular the MEKK Ste11, play key roles in controlling the associations and localizations of network components. ABSTRACT: Candida albicans opaque cells release pheromones to stimulate cells of opposite mating type to activate their pheromone response pathway. Although this fungal pathogen shares orthologous proteins involved in the process with Saccharomyces cerevisiae, the pathway in each organism has unique characteristics. We have used GFP-tagged fusion proteins to investigate the localization of the scaffold protein Cst5, as well as the MAP kinases Cek1 and Cek2, during pheromone response in C. albicans. In wild-type cells, pheromone treatment directed Cst5-GFP to surface puncta concentrated at the tips of mating projections. These puncta failed to form in cells defective in either the Gα or ß subunits. However, they still formed in response to pheromone in cells missing Ste11, but with the puncta distributed around the cell periphery in the absence of mating projections. These puncta were absent from hst7Δ/Δ cells, but could be detected in the ste11Δ/Δ hst7Δ/Δ double mutant. Cek2-GFP showed a strong nuclear localization late in the response, consistent with a role in adaptation, while Cek1-GFP showed a weaker, but early increase in nuclear localization after pheromone treatment. Activation loop phosphorylation of both Cek1 and Cek2 required the presence of Ste11. In contrast to Cek2-GFP, which showed no localization signal in ste11Δ/Δ cells, Cek1-GFP showed enhanced nuclear localization that was pheromone independent in the ste11Δ/Δ mutant. The results are consistent with CaSte11 facilitating Hst7-mediated MAP kinase phosphorylation and also playing a potentially critical role in both MAP kinase and Cst5 scaffold localization.

Modulation of the complex regulatory network for methionine biosynthesis in fungi.

The assimilation of inorganic sulfate and the synthesis of the sulfur-containing amino acids methionine and cysteine is mediated by a multibranched biosynthetic pathway. We have investigated this circuitry in the fungal pathogen Candida albicans, which is phylogenetically intermediate between the filamentous fungi and Saccharomyces cerevisiae. In S. cerevisiae, this pathway is regulated by a collection of five transcription factors (Met4, Cbf1, Met28, and Met31/Met32), while in the filamentous fungi the pathway is controlled by a single Met4-like factor. We found that in C. albicans, the Met4 ortholog is also a core regulator of methionine biosynthesis, where it functions together with Cbf1. While C. albicans encodes this Met4 protein, a Met4 paralog designated Met28 (Orf19.7046), and a Met31 protein, deletion, and activation constructs suggest that of these proteins only Met4 is actually involved in the regulation of methionine biosynthesis. Both Met28 and Met31 are linked to other functions; Met28 appears essential, and Met32 appears implicated in the regulation of genes of central metabolism. Therefore, while S. cerevisiae and C. albicans share Cbf1 and Met4 as central elements of the methionine biosynthesis control, the other proteins that make up the circuit in S. cerevisiae are not members of the C. albicans control network, and so the S. cerevisiae circuit likely represents a recently evolved arrangement.