RESUMO
The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated. The present project was aimed at investigating the contribution of C. parapsilosis Als proteins by generating edited strains lacking functional Als proteins. CPAR2_404770 and CPAR2_404780, further indicated as CpALS4770 and CpALS4780, were selected for the generation of single and double edited strains using an episomal CRISPR/Cas9 technology. Phenotypic characterization of mutant strains revealed that editing of both genes had no impact on the in vitro growth of C. parapsilosis or on morphogenesis. Notably, CpALS4770-edited strain showed a reduction of biofilm formation and adhesive properties to human buccal cells (HBECs). Conversely, single CpALS4780-edited strain did not show any difference compared to the wild-type strain in all the assays performed, while the double CpALS4770-CpALS4780 mutant revealed an increased ability to produce biofilm, a hyper-adhesive phenotype to HBECs, and a marked tendency to form cellular aggregates. Murine vaginal infection experiments indicated a significant reduction in CFUs recovered from BALC/c mice infected with single and double edited strains, compared to those infected with the wild-type strain. These finding clearly indicate that CpAls4770 plays a role in adhesion to biotic and abiotic surfaces, while both CpALS4770 and CpALS4780 genes are required for C. parapsilosis ability to colonize and persist in the vaginal mucosa.
Assuntos
Candida parapsilosis , Adesão Celular/genética , Virulência/genética , Animais , Biofilmes/crescimento & desenvolvimento , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Candida parapsilosis/genética , Candida parapsilosis/patogenicidade , Candidíase , Técnicas de Cultura de Células , Feminino , Proteínas Fúngicas/genética , Inativação Gênica , Genes Fúngicos , Humanos , Camundongos , Mucosa/microbiologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0209124.].
RESUMO
Aim: In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the ALS gene family in the opportunistic pathogen Candida orthopsilosis. Materials & methods: Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both C. orthopsilosis reference and clinical strains. Results: The phenotypic characterization of the ALS triple-edited strains revealed no impact on growth in liquid or solid media. However, pseudohyphal formation and the ability to adhere to human buccal epithelial cells were significantly decreased in triple-edited clones. Conclusion: Our CRISPR/Cas9 system is a powerful tool for simultaneous editing of fungal gene families, which greatly accelerates the generation of multiple gene-edited Candida strains. Data deposition: Nucleotide sequence data are available in the GenBank databases under the accession numbers MK875971, MK875972, MK875973, MK875974, MK875975, MK875976, MK875977.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Candida parapsilosis/genética , Edição de Genes/métodos , Genes Fúngicos , Sequência de Bases , Candida parapsilosis/crescimento & desenvolvimento , Candidíase/microbiologia , Adesão Celular , Células Cultivadas , Células Epiteliais/microbiologia , Humanos , Hifas/crescimento & desenvolvimento , Boca/citologia , Família Multigênica , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Today's beer market is challenged by a decreasing consumption of traditional beer styles and an increasing consumption of specialty beers. In particular, lager-type beers (pilsner), characterized by their refreshing and unique aroma and taste, yet very uniform, struggle with their sales. The development of novel variants of the common lager yeast, the interspecific hybrid Saccharomyces pastorianus, has been proposed as a possible solution to address the need of product diversification in lager beers. Previous efforts to generate new lager yeasts through hybridization of the ancestral parental species (S. cerevisiae and S. eubayanus) yielded strains with an aromatic profile distinct from the natural biodiversity. Unfortunately, next to the desired properties, these novel yeasts also inherited unwanted characteristics. Most notably is their phenolic off-flavor (POF) production, which hampers their direct application in the industrial production processes. Here, we describe a CRISPR-based gene editing strategy that allows the systematic and meticulous introduction of a natural occurring mutation in the FDC1 gene of genetically complex industrial S. cerevisiae strains, S. eubayanus yeasts and interspecific hybrids. The resulting cisgenic POF- variants show great potential for industrial application and diversifying the current lager beer portfolio.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Saccharomyces cerevisiae/genética , Saccharomyces/genética , Cerveja/microbiologia , Biodiversidade , Edição de Genes , Saccharomyces/classificação , Saccharomyces cerevisiae/classificaçãoRESUMO
Central dogma, transformation, and genome editing are key biological concepts for which junior scientists must gain experience during training. Here we present an exercise that introduces these concepts in a single practical laboratory exercise. Our exercise utilizes CRISPR/Cas9 genome editing to introduce a stop codon into Saccharomyces cerevisiae ADE2. This edit leads to the buildup of an adenine precursor that dyes the edited cells red. As the repair template, guide RNA, and Cas9 are all encoded in our vector, transformation can be performed in 2 hours. Furthermore, since all components of the Cas9/CRISPR system are encoded by the vector, specialized equipment and reagents, such as a PCR machine or oligonucleotides, are not required to perform the experiments as designed. As such, these exercises provide an efficient cost-effective introduction to a wide variety of key molecular biology concepts and lay the foundation for more rigorous investigations in upper-level classes and independent research projects.
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This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.
Assuntos
Sistemas CRISPR-Cas , Candida albicans/genética , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Humanos , Plasmídeos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE 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.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , Fungos/genética , Edição de Genes/métodos , Mutagênese , Candida albicans/genética , Candida glabrata/genética , Quebras de DNA de Cadeia Dupla , Vetores Genéticos , RNA Guia de Cinetoplastídeos/genética , Saccharomyces cerevisiae/genéticaRESUMO
The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.
Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Genes Fúngicos , Proteínas de Choque Térmico HSP90/fisiologia , Fatores de Transcrição de Choque Térmico/metabolismo , Western Blotting , Candida albicans/genética , Imunoprecipitação da Cromatina , Fatores de Transcrição de Choque Térmico/genética , Morfogênese , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Temperatura , VirulênciaRESUMO
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.
RESUMO
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.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Proteínas de Homeodomínio/imunologia , Lectinas Tipo C/imunologia , Macrófagos Peritoneais/imunologia , Neuropeptídeos/imunologia , Fagocitose/imunologia , Proteínas Tirosina Quinases/imunologia , Actinas/genética , Actinas/imunologia , Actinas/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Candida albicans/metabolismo , Candidíase/genética , Candidíase/metabolismo , Candidíase/patologia , Linhagem Celular , Diglicerídeos/genética , Diglicerídeos/imunologia , Diglicerídeos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagocitose/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/imunologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismoRESUMO
Our recent finding that the Candida albicans RNase III enzyme CaDcr1 is an unusual, multifunctional RNase III coupled with data on the RNase III enzymes from other fungal species prompted us to seek a model that explained the evolution of RNase III's in modern budding yeast species. CaDcr1 has both dicer function (generates small RNA molecules from dsRNA precursors) and Rnt1 function, (catalyzes the maturation of 35S rRNA and U4 snRNA). Some budding yeast species have two distinct genes that encode these functions, a Dicer and RNT1, whereas others have only an RNT1 and no Dicer. As none of the budding yeast species has the canonical Dicer found in many other fungal lineages and most eukaryotes, the extant species must have evolved from an ancestor that lost the canonical Dicer, and evolved a novel Dicer from the essential RNT1 gene. No single, simple model could explain the evolution of RNase III enzymes from this ancestor because existing sequence data are consistent with two equally plausible models. The models share an architecture for RNase III evolution that involves gene duplication, loss, subfunctionalization, and neofunctionalization. This commentary explains our reasoning, and offers the prospect that further genomic data could further resolve the dilemma surrounding the budding yeast RNase III's evolution.
Assuntos
Candida albicans/genética , Evolução Molecular , Proteínas Fúngicas/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA Fúngico/genética , Ribonuclease III/genética , Candida albicans/enzimologia , Proteínas Fúngicas/metabolismo , RNA Fúngico/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonuclease III/metabolismoRESUMO
While Candida albicans is the most significant fungal pathogen for humans, Candida glabrata accounts for an increasing number of infections. Little is known about how C. glabrata interacts with the innate immune system, the first line of defense against such organisms. The C. glabrata adhesin Epa1p was previously shown to bind mammalian epithelial cells. We hypothesized that Epa1p mediates unique, nonopsonic binding to macrophages, leading to induction of immune responses. We found that Epa1p mediated adhesion by both C. glabrata (Cg) and transformed Saccharomyces cerevisiae (Sc(EPA1) ) to human macrophage-like cells, including Thp1 and U937 lines, and donor PBMCs. Adhesion was distinct from described mechanisms such as Dectin-1. Epa1p expression was necessary and sufficient for S. cerevisiae binding and phagocytosis, the latter of which was actin-mediated. Sc(EPA1) induced inflammatory cytokine production (IL-8 and TNF-α) by human PBMC-derived macrophages. Despite expressing Epa1p and binding to macrophages, Cg avoided phagocytosis and cytokine induction. In contrast to human results, in murine cell models (RAW264.7, J774A.1, and C57BL/6-derived cells), Epa1p-mediated binding was only revealed after blocking the Dectin-1 system. Recognition of Epa1p represents a novel mechanism by which human innate immune cells bind fungi, and for Sc(EPA1) results in phagocytosis and subsequent cytokine production.
Assuntos
Candida glabrata/imunologia , Adesão Celular , Citocinas/metabolismo , Proteínas Fúngicas/imunologia , Lectinas/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Fagocitose , Animais , Células Cultivadas , Expressão Gênica , Humanos , Camundongos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologia , Transformação GenéticaRESUMO
The generation of mature functional RNAs from nascent transcripts requires the precise and coordinated action of numerous RNAs and proteins. One such protein family, the ribonuclease III (RNase III) endonucleases, includes Rnt1, which functions in fungal ribosome and spliceosome biogenesis, and Dicer, which generates the siRNAs of the RNAi pathway. The recent discovery of small RNAs in Candida albicans led us to investigate the function of C. albicans Dicer (CaDcr1). CaDcr1 is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCR1 complements a Dicer knockout in Saccharomyces castellii, restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCR1 results in a severe slow-growth phenotype, whereas deletion of another core component of the RNAi pathway (CaAGO1) has little effect on growth, suggesting that CaDCR1 may have an essential function in addition to producing siRNAs. Indeed CaDcr1, the sole functional RNase III enzyme in C. albicans, has additional functions: it is required for cleavage of the 3' external transcribed spacer from unprocessed pre-rRNA and for processing the 3' tail of snRNA U4. Our results suggest two models whereby the RNase III enzymes of a fungal ancestor, containing both a canonical Dicer and Rnt1, evolved through a series of gene-duplication and gene-loss events to generate the variety of RNase III enzymes found in modern-day budding yeasts.
Assuntos
Candida albicans/enzimologia , Evolução Molecular , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , Ribonuclease III/fisiologia , Spliceossomos/genética , Candida albicans/fisiologia , Citometria de Fluxo , Regulação Fúngica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Genéticos , Filogenia , Ribonuclease III/metabolismo , Sintenia/genéticaRESUMO
Dectin-1, the major ß-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 ß-glucan, the loss of galectin-3 dramatically accentuates the failure to trigger an appropriate TNF-α response.
Assuntos
Candida albicans/fisiologia , Galectina 3/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Biotinilação , Células da Medula Óssea/citologia , Endocitose/efeitos dos fármacos , Células HEK293 , Humanos , Imunoprecipitação , Lectinas Tipo C , Camundongos , Sondas Moleculares/metabolismo , Ligação Proteica , Coloração e Rotulagem , Fator de Necrose Tumoral alfa/metabolismo , Zimosan/metabolismo , beta-Glucanas/metabolismoRESUMO
The yeast Saccharomyces cerevisiae responds to environmental stress by rapidly altering the expression of large sets of genes. We report evidence that the transcriptional repressors Nrg1 and Nrg2 (Nrg1/Nrg2), which were previously implicated in glucose repression, regulate a set of stress-responsive genes. Genome-wide expression analysis identified 150 genes that were upregulated in nrg1Delta nrg2Delta double mutant cells, relative to wild-type cells, during growth in glucose. We found that many of these genes are regulated by glucose repression. Stress response elements (STREs) and STRE-like elements are overrepresented in the promoters of these genes, and a search of available expression data sets showed that many are regulated in response to a variety of environmental stress signals. In accord with these findings, mutation of NRG1 and NRG2 enhanced the resistance of cells to salt and oxidative stress and decreased tolerance to freezing. We present evidence that Nrg1/Nrg2 not only contribute to repression of target genes in the absence of stress but also limit induction in response to salt stress. We suggest that Nrg1/Nrg2 fine-tune the regulation of a set of stress-responsive genes.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Análise por Conglomerados , Temperatura Baixa , Proteínas de Ligação a DNA , Perfilação da Expressão Gênica , Glucose/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sais/metabolismo , Dedos de ZincoRESUMO
The Nrg1 and Nrg2 repressors of Saccharomyces cerevisiae have highly similar zinc fingers and closely related functions in the regulation of glucose-repressed genes. We show that NRG1 and NRG2 are differently regulated in response to carbon source at both the RNA and protein levels. Expression of NRG1 RNA is glucose repressed, whereas NRG2 RNA levels are nearly constant. Nrg1 protein levels are elevated in response to glucose limitation or growth in nonfermentable carbon sources, whereas Nrg2 levels are diminished. Chromatin immunoprecipitation assays showed that Nrg1 and Nrg2 bind DNA both in the presence and absence of glucose. In mutant cells lacking the corepressor Ssn6(Cyc8)-Tup1, promoter-bound Nrg1, but not Nrg2, functions as an activator in a reporter assay, providing evidence that the two Nrg proteins have distinct properties. We suggest that the differences in expression and function of these two repressors, in combination with their similar DNA-binding domains, contribute to the complex regulation of the large set of glucose-repressed genes.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Glucose/metabolismo , Imunoprecipitação , Óperon Lac/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação TranscricionalRESUMO
The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.
Assuntos
Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Bioensaio , Galactose/metabolismo , Glicerol/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Saccharomyces cerevisiae/genéticaRESUMO
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 beta-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.
Assuntos
Haploidia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Proteínas Quinases Ativadas por AMP , Adesão Celular/genética , Divisão Celular/fisiologia , Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
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.