An association between reduced susceptibility to echinocandins and changes in the 1,3-␤-D-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

Candida albicans is a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe a C. albicans CRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome of Candida and provides a new window into the biology of this pathogen.

The Snf1 protein kinase of Saccharomyces cerevisiae is important for many cellular responses to glucose limitation, including haploid invasive growth. We show here that Snf1 regulates transcription of FLO11, which encodes a cell surface glycoprotein required for invasive growth. We further show that Nrg1 and Nrg2, two repressor proteins that interact with Snf1, function as negative regulators of invasive growth and as repressors of FLO11. We also examined the role of Snf1, Nrg1, and Nrg2 in two other Flo11-dependent processes. Mutations affected the initiation of biofilm formation, which is glucose sensitive, but also affected diploid pseudohyphal differentiation, thereby unexpectedly implicating Snf1 in a response to nitrogen limitation. Deletion of the NRG1 and NRG2 genes suppressed the defects of a snf1 mutant in all of these processes. These findings suggest a model in which the Snf1 kinase positively regulates Flo11-dependent developmental events by antagonizing Nrg-mediated repression of the FLO11 gene.

Dectin-1, the major β-glucan receptor in leukocytes, triggers an effective immune response upon fungal recognition. Here we use sortase-mediated transpeptidation, a technique that allows placement of a variety of probes on a polypeptide backbone, to monitor the behavior of labeled functional dectin-1 in live cells with and without fungal challenge. Installation of probes on dectin-1 by sortagging permitted highly specific visualization of functional protein on the cell surface and its subsequent internalization upon ligand presentation. Retrieval of sortagged dectin-1 expressed in macrophages uncovered a unique interaction between dectin-1 and galectin-3 that functions in the proinflammatory response of macrophages to pathogenic fungi. When macrophages expressing dectin-1 are exposed to Candida albicans mutants with increased exposure of β-glucan, the loss of galectin-3 dramatically accentuates the failure to trigger an appropriate TNF-α response.pattern-recognition receptors | sortase A | polysaccharide

CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii. The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

Sip4 is a Zn 2 Cys 6 transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptional repression but also positively regulates Gal4. We show that Srb10 is required for phosphorylation of Sip4 during growth in nonfermentable carbon sources and that the catalytic activity of Srb10 stimulates the ability of LexA-Sip4 to activate transcription of a reporter. Srb10 and Sip4 coimmunoprecipitate from cell extracts and interact in two-hybrid assays, suggesting that Srb10 regulates Sip4 directly. We also present evidence that the Srb10 and Snf1 kinases interact with different regions of Sip4. These findings support the view that the Srb10 kinase not only plays negative roles in transcriptional control but also has broad positive roles during growth in carbon sources other than glucose.The transcriptional activator Sip4 of Saccharomyces cerevisiae belongs to a family of activators with a Zn 2 Cys 6 binuclear cluster DNA-binding domain, which includes Gal4, Hap1, Leu3, Put3, and others (20,22). Sip4 was identified by its two-hybrid interaction with the Snf1 protein kinase of the glucose signaling pathway (17, 37). Sip4 binds to the carbon source-responsive elements (CSRE) (28) in the promoters of gluconeogenic genes (34) and also has an inhibitory effect on glucose depletion-dependent invasive growth (6). Both the expression and the function of Sip4 are regulated in response to glucose. Transcription of SIP4 is repressed by glucose (17, 34), and RNA levels increase during the diauxic shift and sporulation (3,8).The physical interaction of Sip4 with the Snf1 protein kinase reflects a role of Snf1 in regulating Sip4 function. In response to glucose limitation, Sip4 is rapidly phosphorylated and its ability to activate transcription is rapidly upregulated; both processes depend on Snf1 kinase activity (17). Biochemical and genetic evidence indicates that a specific ␤ subunit of the Snf1 kinase, Gal83, mediates the physical and functional interaction of the kinase with Sip4 (33). These findings show that Snf1 modulates the activity of Sip4 in response to glucose, but it has not been demonstrated that Sip4 is a direct target of Snf1. Moreover, both in vitro and in vivo studies indicate that another kinase besides Snf1 contributes to phosphorylation of Sip4 (33).Several lines of evidence suggested the Srb10 (Ssn3, Ume5) kinase as a candidate. Srb10 and its cyclin, Srb11 (Ssn8), are associated with the RNA polymerase II holoenzyme, as are their mammalian homologs, cyclin-dependent kinase 8 (cdk8) and cyclin C (15,18,19). This kinase phosphorylates the carboxy-terminal domain (CTD) of the polymerase, thereby inhibiting ...

The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion. Cells contain three forms of the Snf1 kinase, each with a different ␤-subunit isoform, either Gal83, Sip1, or Sip2. We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation. The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation. Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1-and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene. In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11. Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms.