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1.  High-resolution mapping reveals a conserved, widespread, dynamic meiotically regulated mRNA methylation program 
Cell  2013;155(6):1409-1421.
N6-methyladenosine (m6A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m6A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated 8/8 methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time-course, and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminates a conserved, dynamically regulated methylation program in yeast meiosis, and provides an important resource for studying the function of this epitranscriptomic modification.
PMCID: PMC3956118  PMID: 24269006
2.  Compatibility with Killer explains the Rise of RNAi-deficient fungi 
Science (New York, N.Y.)  2011;333(6049):1592.
The RNA interference (RNAi) pathway is found in most eukaryotic lineages but curiously is absent in others, including that of Saccharomyces cerevisiae. Here, we show that reconstituting RNAi in S. cerevisiae causes loss of a beneficial dsRNA virus, known as killer virus. Incompatibility between RNAi and killer viruses extends to other fungal species, in that RNAi is absent in all species known to possess dsRNA killer viruses, whereas killer viruses are absent in closely related species that retained RNAi. Thus, the advantage imparted by acquiring and retaining killer viruses explains the persistence of RNAi-deficient species during fungal evolution.
PMCID: PMC3790311  PMID: 21921191
3.  Compartmentalization of metabolic pathways in yeast mitochondria improves production of branched chain alcohols 
Nature biotechnology  2013;31(4):335-341.
Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeted expression of metabolic pathways to mitochondria can increase production levels compared with expression of the same pathways in the cytoplasm. Compartmentalisation of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overexpressing only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains reveals that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves higher local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion, and reducing the loss of intermediates to competing pathways.
PMCID: PMC3659820  PMID: 23417095
4.  RNAi in budding yeast 
Science (New York, N.Y.)  2009;326(5952):544-550.
RNAi, a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and Y’ subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y’ mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.
PMCID: PMC3786161  PMID: 19745116
5.  Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of Candida albicans in Macrophages 
PLoS Pathogens  2013;9(6):e1003446.
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.
Author Summary
The opportunistic yeast Candida albicans is a commensal organism of the human digestive tract, but also the most common cause of human fungal infections. Phagocytosis, the process by which innate immune cells engulf pathogens, is vital to prevent C. albicans infections. The major phagocytic receptor involved in anti-fungal immunity is Dectin-1. We identify two new interacting proteins of Dectin-1 in macrophages: Bruton's Tyrosine Kinase (BTK) and Vav1. In the course of phagocytosis, different phosphoinositides (PIs) are formed in the phagosomal membrane to allow the recruitment of proteins equipped with specialized lipid-interaction domains. We show that BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 at the phagocytic cup, but not with diacylglycerol (DAG), which marks more mature phagosomal membranes. Inhibition of BTK affects the production of DAG and the recruitment of DAG-interacting proteins. BTK and Vav1 are essential for C. albicans immune responses, as BTK- or Vav1-deficient macrophages show reduced uptake of C. albicans and BTK- or Vav1-deficient deficient mice are more susceptible to systemic C. albicans infection. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.
PMCID: PMC3694848  PMID: 23825946
6.  Single-cell analysis reveals that noncoding RNAs contribute to clonal heterogeneity by modulating transcription factor recruitment 
Molecular Cell  2012;45(4):470-482.
Mechanisms through which long intergenic noncoding RNAs (ncRNAs) exert regulatory effects on eukaryotic biological processes remain largely elusive. Most studies of these phenomena rely on methods that measure average behaviors in cell populations, lacking resolution to observe the effects of ncRNA transcription on gene expression in a single cell. Here, we combine quantitative single-molecule RNA FISH experiments with yeast genetics and computational modeling to gain mechanistic insights into the regulation of the Saccharomyces cerevisiae protein-coding gene FLO11 by two intergenic ncRNAs, ICR1 and PWR1. Direct detection of FLO11 mRNA and these ncRNAs in thousands of individual cells revealed alternative expression states and provides evidence that ICR1 and PWR1 contribute to FLO11’s variegated transcription, resulting in Flo11-dependent phenotypic heterogeneity in clonal cell populations by modulating recruitment of key transcription factors to the FLO11 promoter.
PMCID: PMC3288511  PMID: 22264825
7.  Genetic Variation in Saccharomyces cerevisiae: Circuit Diversification in a Signal Transduction Network 
Genetics  2012;192(4):1523-1532.
The connection between genotype and phenotype was assessed by determining the adhesion phenotype for the same mutation in two closely related yeast strains, S288c and Sigma, using two identical deletion libraries. Previous studies, all in Sigma, had shown that the adhesion phenotype was controlled by the filamentation mitogen-activated kinase (fMAPK) pathway, which activates a set of transcription factors required for the transcription of the structural gene FLO11. Unexpectedly, the fMAPK pathway is not required for FLO11 transcription in S288c despite the fact that the fMAPK genes are present and active in other pathways. Using transformation and a sensitized reporter, it was possible to isolate RPI1, one of the modifiers that permits the bypass of the fMAPK pathway in S288c. RPI1 encodes a transcription factor with allelic differences between the two strains: The RPI1 allele from S288c but not the one from Sigma can confer fMAPK pathway-independent transcription of FLO11. Biochemical analysis reveals differences in phosphorylation between the alleles. At the nucleotide level the two alleles differ in the number of tandem repeats in the ORF. A comparison of genomes between the two strains shows that many genes differ in size due to variation in repeat length.
PMCID: PMC3512157  PMID: 23051644
budding yeast; natural variation; repeat length polymorphisms; agar adhesion
8.  Genes come and go 
RNA Biology  2012;9(9):1123-1128.
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.
PMCID: PMC3579876  PMID: 23018782
Candida; DCR1; RNT1; RNase III; bifunctional dicer; evolution; yeast
9.  Ruler Arrays Reveal Haploid Genomic Structural Variation 
PLoS ONE  2012;7(8):e43210.
Despite the known relevance of genomic structural variants to pathogen behavior, cancer, development, and evolution, certain repeat based structural variants may evade detection by existing high-throughput techniques. Here, we present ruler arrays, a technique to detect genomic structural variants including insertions and deletions (indels), duplications, and translocations. A ruler array exploits DNA polymerase’s processivity to detect physical distances between defined genomic sequences regardless of the intervening sequence. The method combines a sample preparation protocol, tiling genomic microarrays, and a new computational analysis. The analysis of ruler array data from two genomic samples enables the identification of structural variation between the samples. In an empirical test between two closely related haploid strains of yeast ruler arrays detected 78% of the structural variants larger than 100 bp.
PMCID: PMC3428316  PMID: 22952647
10.  RNA Methylation by the MIS Complex Regulates a Cell Fate Decision in Yeast 
PLoS Genetics  2012;8(6):e1002732.
For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program. After this point, cells, returned to nutrient-rich medium, entered a form of synchronous PH development that was morphologically and genetically indistinguishable from starvation-induced PH growth. We show that lineage restriction during this time was, in part, dependent on the mRNA methyltransferase activity of Ime4, which played separable roles in meiotic induction and suppression of the PH program. Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4. This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions.
Author Summary
Cellular differentiation involves the limitation of cellular potential in response to developmental cues. Budding yeast cells differentiate in response to nutrient availability. In the presence of nutrients, cells divide mitotically by producing round, yeast-form buds. Under nutrient limitation, cells can either divide under a pseudo-hyphal (PH) foraging program or undergo meiosis to form protective spores. We show here that developmental commitment occurs in two distinct phases. When nutrients were removed, cells first became committed to a starvation response, during which they entered the meiotic program. If nutrient limitation persisted, cells became committed to meiosis and sporulation. By contrast, if nutrients were returned at this point, cells synchronously initiated PH foraging growth. We found that both sporulation and PH growth were governed by RNA methylation, and we identified an mRNA–methyltransferase complex comprising Mum2, Ime4, and Slz1 as a central regulator of these developmental trajectories. Our results indicate that the yeast starvation response is an extended developmental process and reveal a fundamental role for post-transcriptional RNA modification in controlling cell fate.
PMCID: PMC3369947  PMID: 22685417
11.  FASTER MT: Isolation of Pure Populations of a and α Ascospores from Saccharomyces cerevisiae 
G3: Genes|Genomes|Genetics  2012;2(4):449-452.
The budding yeast Saccharomyces cerevisiae has many traits that make it useful for studies of quantitative inheritance. Genome-wide association studies and bulk segregant analyses often serve as first steps toward the identification of quantitative trait loci. These approaches benefit from having large numbers of ascospores pooled by mating type without contamination by vegetative cells. To this end, we inserted a gene encoding red fluorescent protein into the MATa locus. Red fluorescent protein expression caused MATa and a/α diploid vegetative cells and MATa ascospores to fluoresce; MATα cells without the gene did not fluoresce. Heterozygous diploids segregated fluorescent and nonfluorescent ascospores 2:2 in tetrads and bulk populations. The two populations of spores were separable by fluorescence-activated cell sorting with little cross contamination or contamination with diploid vegetative cells. This approach, which we call Fluorescent Ascospore Technique for Efficient Recovery of Mating Type (FASTER MT), should be applicable to laboratory, industrial, and undomesticated, strains.
PMCID: PMC3337473  PMID: 22540036
budding yeast; red fluorescent protein; MATa; fluorescence-activated cell sorting; hygromycin resistance; BUD5-TAF2
12.  A Prototype Antifungal Contact Lens 
Econazole-eluting contact lenses with a novel design provided extended antifungal activity against the Candida albicans fungus. This drug-eluting contact lens could be used to treat and prevent fungal ocular infections.
To design a contact lens to treat and prevent fungal ocular infections.
Curved contact lenses were created by encapsulating econazole-impregnated poly(lactic-co-glycolic) acid (PLGA) films in poly(hydroxyethyl methacrylate) (pHEMA) by ultraviolet photopolymerization. Release studies were conducted in phosphate-buffered saline at 37°C with continuous shaking. The contact lenses and their release media were tested in an antifungal assay against Candida albicans. Cross sections of the pre- and postrelease contact lenses were characterized by scanning electron microscopy and by Raman spectroscopy.
Econazole-eluting contact lenses provided extended antifungal activity against Candida albicans fungi. Fungicidal activity varied in duration and effectiveness depending on the mass of the econazole-PLGA film encapsulated in the contact lens.
An econazole-eluting contact lens could be used as a treatment for fungal ocular infections.
PMCID: PMC3176015  PMID: 21527380
13.  Distal chromatin structure influences local nucleosome positions and gene expression 
Nucleic Acids Research  2012;40(9):3870-3885.
The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called ‘antinucleosomal elements’ like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression.
PMCID: PMC3351160  PMID: 22241769
Biomaterials  2009;31(6):1444.
Invasive fungal infections can be devastating, particularly in immunocompromised patients, and difficult to treat with systemic drugs. Furthermore, systemic administration of those medications can have severe side effects. We have developed an injectable local antifungal treatment for direct administration into existing or potential sites of fungal infection. Amphotericin B (AmB), a hydrophobic, potent, and broad-spectrum antifungal agent, was rendered water-soluble by conjugation to a dextran-aldehyde polymer. The dextran-aldehyde-AmB conjugate retained antifungal efficacy against C. albicans. Mixing carboxymethylcellulose-hydrazide with dextran-aldehyde formed a gel that cross-linked in situ by formation of hydrazone bonds. The gel provided in vitro release of antifungal activity for 11 days, and contact with the gel killed Candida for three weeks. There was no apparent tissue toxicity in the murine peritoneum and the gel caused no adhesions. Gels produced by entrapment of a suspension of AmB in CMC-dextran without conjugation of drug to polymers did not release fungicidal activity, but did kill on contact. Injectable systems of these types, containing soluble or insoluble drug formulations, could be useful for treatment of local antifungal infections, with or without concurrent systemic therapy.
PMCID: PMC2813953  PMID: 19942285
15.  Control of Transcription by Cell Size 
PLoS Biology  2010;8(11):e1000523.
The demonstration of a causal, regulatory relationship between cell size and gene expression in yeast suggests that cells maintain size in order to maintain transcriptional homeostasis.
Cell size increases significantly with increasing ploidy. Differences in cell size and ploidy are associated with alterations in gene expression, although no direct connection has been made between cell size and transcription. Here we show that ploidy-associated changes in gene expression reflect transcriptional adjustment to a larger cell size, implicating cellular geometry as a key parameter in gene regulation. Using RNA-seq, we identified genes whose expression was altered in a tetraploid as compared with the isogenic haploid. A significant fraction of these genes encode cell surface proteins, suggesting an effect of the enlarged cell size on the differential regulation of these genes. To test this hypothesis, we examined expression of these genes in haploid mutants that also produce enlarged size. Surprisingly, many genes differentially regulated in the tetraploid are identically regulated in the enlarged haploids, and the magnitude of change in gene expression correlates with the degree of size enlargement. These results indicate a causal relationship between cell size and transcription, with a size-sensing mechanism that alters transcription in response to size. The genes responding to cell size are enriched for those regulated by two mitogen-activated protein kinase pathways, and components in those pathways were found to mediate size-dependent gene regulation. Transcriptional adjustment to enlarged cell size could underlie other cellular changes associated with polyploidy. The causal relationship between cell size and transcription suggests that cell size homeostasis serves a regulatory role in transcriptome maintenance.
Author Summary
Cells of the same type, whether microbial, plant, or metazoan in origin, exhibit remarkable uniformity in size. This uniformity arises from control mechanisms that respond to internal cellular changes as well as external environmental factors. Although precise control of cell size is a universal phenomenon, its relationship to cellular physiology is underexplored. In this study using yeast we show a causal relationship between cell size and gene regulation: changes in cell size correlate with changes in the expression of a set of genes. Hence, the maintenance of uniformity in cell size could be a homeostatic mechanism for the maintenance of gene expression in a cell or in a population of cells within a tissue. The relationship between cell size and gene expression uncovered in this study may have fundamental implications in evolution, in the development of multicellular organisms, and in the formation of tumors, as these processes often involve genome duplication accompanied by enlarged cell size.
PMCID: PMC2970550  PMID: 21072241
16.  FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast 
Cell  2008;135(4):726-737.
The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1-expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1+ cells avoid exploitation by non-expressing flo1 cells by self/non-self recognition: FLO1+ cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known “green beard genes”, which direct cooperation towards other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly-evolving social trait.
PMCID: PMC2703716  PMID: 19013280
Flocculation; FLO1; drug resistance; evolution; ethanol; selfish gene; Darwin; Dawkins; Hamilton; green beard gene
17.  Dynamic, Morphotype-Specific Candida albicans β-Glucan Exposure during Infection and Drug Treatment 
PLoS Pathogens  2008;4(12):e1000227.
Candida albicans, a clinically important dimorphic fungal pathogen that can evade immune attack by masking its cell wall β-glucan from immune recognition, mutes protective host responses mediated by the Dectin-1 β-glucan receptor on innate immune cells. Although the ability of C. albicans to switch between a yeast- or hyphal-form is a key virulence determinant, the role of each morphotype in β-glucan masking during infection and treatment has not been addressed. Here, we show that during infection of mice, the C. albicans β-glucan is masked initially but becomes exposed later in several organs. At all measured stages of infection, there is no difference in β-glucan exposure between yeast-form and hyphal cells. We have previously shown that sub-inhibitory doses of the anti-fungal drug caspofungin can expose β-glucan in vitro, suggesting that the drug may enhance immune activity during therapy. This report shows that caspofungin also mediates β-glucan unmasking in vivo. Surprisingly, caspofungin preferentially unmasks filamentous cells, as opposed to yeast form cells, both in vivo and in vitro. The fungicidal activity of caspofungin in vitro is also filament-biased, as corroborated using yeast-locked and hyphal-locked mutants. The uncloaking of filaments is not a general effect of anti-fungal drugs, as another anti-fungal agent does not have this effect. These results highlight the advantage of studying host–pathogen interaction in vivo and suggest new avenues for drug development.
Author Summary
Candida is a common human commensal but disseminated candidiasis is a serious clinical problem, especially among immunocompromised patients. The innate immune system controls Candida infection, in part through the germline-encoded β-glucan receptor Dectin-1. However, during in vitro growth, Candida albicans mutes Dectin-1 recognition by cloaking its β-glucan underneath a layer of mannan. Bridging these two seemingly contradictory observations, we demonstrate that C. albicans masks β-glucan early during infection, but it becomes exposed later, allowing Dectin-1 to recognize the fungi and mediate immunity. Remarkably, treatment of mice with sub-therapeutic doses of the antifungal drug caspofungin causes exposure of β-glucan on C. albicans even when it would not be exposed naturally. We introduce a new technique for monitoring of epitope exposure during infection, which can be used to monitor the availability of any epitope for immune recognition. This technique allowed us to show that natural unmasking of β-glucan is not morphotype-specific, but drug-mediated unmasking is biased towards the invasive filamentous form of C. albicans. These results highlight the unexplored area of dynamic epitope exposure during infection and therapy, which might be targetable to enhance immune recognition and fungal clearance.
PMCID: PMC2587227  PMID: 19057660
18.  Phagocytosis by Human Neutrophils is Stimulated by a Unique Fungal Cell Wall Component 
Cell host & microbe  2007;2(1):55-67.
Innate immunity depends upon recognition of surface features common to a broad group of pathogens. In gram negative bacteria, lipopolysaccharide recognition by immune cells leads to inflammation. In fungi, the glucose polymer β-glucan has been implicated in fungal recognition. Fungal walls have two kinds of β-glucan: β-1,3-glucan and β-1,6-glucan. The predominance of β-1,3-glucan in the wall and its recognition by macrophages has led to the presumption that β-1,3-glucan is the key immunological determinant for both macrophages and neutrophils. Here we show that in human neutrophils, β-1,6-glucan mediates engulfment, production of reactive oxygen species, and expression of HSPs more efficiently than β-1,3-glucan. Neutrophils rapidly ingest beads coated with β-1,6-glucan, while ignoring those coated with β-1,3-glucan. Complement factors C3b/C3d are deposited on β-1,6-glucan more readily than on β-1,3-glucan, recognized by CR3. β-1,6-glucan is also important for efficient engulfment of Candida albicans. These unique stimulatory effects could have useful medical applications.
PMCID: PMC2083279  PMID: 18005717
19.  Mat Formation in Saccharomyces cerevisiae Requires Nutrient and pH Gradients▿  
Eukaryotic Cell  2007;7(1):122-130.
The ability of Saccharomyces cerevisiae to form morphologically complex colony-like structures called mats requires expression of the cell surface glycoprotein Flo11p and growth on a semisolid surface. As the mat grows, it forms two visually distinct populations called the rim (edge of the mat) and the hub (interior of the mat), which can be physically separated from one another based on their agar adherence properties. Here, we show that growth of the mat on a semisolid agar surface creates concentric glucose and pH gradients in the medium that are required for the differentiation of the hub and rim. Disruption of the pathways that respond to changing levels of glucose block mat formation by decreasing FLO11 expression. However, in wild-type cells, Flo11p is expressed in both portions of the structure. The difference in adherence between the rim and hub appears to be a consequence of the reduced adherence of Flo11p at the elevated pH of the rim.
PMCID: PMC2224160  PMID: 17951523
20.  Intragenic tandem repeats generate functional variability 
Nature genetics  2005;37(9):986-990.
Tandemly repeated DNA sequences are highly dynamic components of genomes1. Most repeats are in intergenic regions, but some are found within coding sequences or pseudogenes2. In humans, expansion of intragenic triplet repeats is associated with various diseases, including Huntington’s chorea and fragile X syndrome3,4. The persistence of intragenic repeats in genomes argues in favor of a compensating benefit. Here we show that in the Saccharomyces genome, the majority of the genes containing intragenic repeats encode cell wall proteins. The repeats trigger frequent recombination events within the gene or between the gene and a pseudogene, causing expansion and contraction in the gene size. This size variation creates quantitative alterations in phenotypes (e.g. adhesion, flocculation, biofilm formation). We propose that variation in intragenic repeat number provides the functional diversity of cell surface antigens that, in fungi and other pathogens, allows rapid adaptation to the environment and/or elusion of the host immune system.
PMCID: PMC1462868  PMID: 16086015
21.  Coding Tandem Repeats Generate Diversity in Aspergillus fumigatus Genes▿ †  
Eukaryotic Cell  2007;6(8):1380-1391.
Genes containing multiple coding mini- and microsatellite repeats are highly dynamic components of genomes. Frequent recombination events within these tandem repeats lead to changes in repeat numbers, which in turn alters the amino acid sequence of the corresponding protein. In bacteria and yeasts, the expansion of such coding repeats in cell wall proteins is associated with alterations in immunogenicity, adhesion, and pathogenesis. We hypothesized that identification of repeat-containing putative cell wall proteins in the human pathogen Aspergillus fumigatus may reveal novel pathogenesis-related elements. Here, we report that the genome of A. fumigatus contains as many as 292 genes with internal repeats. Fourteen of 30 selected genes showed size variation of their repeat-containing regions among 11 clinical A. fumigatus isolates. Four of these genes, Afu3g08990, Afu2g05150 (MP-2), Afu4g09600, and Afu6g14090, encode putative cell wall proteins containing a leader sequence and a glycosylphosphatidylinositol anchor motif. All four genes are expressed and produce variable-size mRNA encoding a discrete number of repeat amino acid units. Their expression was altered during development and in response to cell wall-disrupting agents. Deletion of one of these genes, Afu3g08990, resulted in a phenotype characterized by rapid conidial germination and reduced adherence to extracellular matrix suggestive of an alteration in cell wall characteristics. The Afu3g08990 protein was localized to the cell walls of dormant and germinating conidia. Our findings suggest that a subset of the A. fumigatus cell surface proteins may be hypervariable due to recombination events in their internal tandem repeats. This variation may provide the functional diversity in cell surface antigens which allows rapid adaptation to the environment and/or elusion of the host immune system.
PMCID: PMC1951137  PMID: 17557878
22.  Genetic Architecture of Hsp90-Dependent Drug Resistance▿  
Eukaryotic Cell  2006;5(12):2184-2188.
Hsp90 potentiates the evolution of azole resistance in the model yeast Saccharomyces cerevisiae and the opportunistic pathogen Candida albicans via calcineurin. Here, we explored effectors downstream of calcineurin regulating this Hsp90-dependent trait. Using S. cerevisiae erg3 mutants as a model, we determined that both Crz1 and Hph1 modulate azole resistance.
PMCID: PMC1694807  PMID: 17056742
23.  A Drug-Sensitive Genetic Network Masks Fungi from the Immune System 
PLoS Pathogens  2006;2(4):e35.
Fungal pathogens can be recognized by the immune system via their β-glucan, a potent proinflammatory molecule that is present at high levels but is predominantly buried beneath a mannoprotein coat and invisible to the host. To investigate the nature and significance of “masking” this molecule, we characterized the mechanism of masking and consequences of unmasking for immune recognition. We found that the underlying β-glucan in the cell wall of Candida albicans is unmasked by subinhibitory doses of the antifungal drug caspofungin, causing the exposed fungi to elicit a stronger immune response. Using a library of bakers' yeast (Saccharomyces cerevisiae) mutants, we uncovered a conserved genetic network that is required for concealing β-glucan from the immune system and limiting the host response. Perturbation of parts of this network in the pathogen C. albicans caused unmasking of its β-glucan, leading to increased β-glucan receptor-dependent elicitation of key proinflammatory cytokines from primary mouse macrophages. By creating an anti-inflammatory barrier to mask β-glucan, opportunistic fungi may promote commensal colonization and have an increased propensity for causing disease. Targeting the widely conserved gene network required for creating and maintaining this barrier may lead to novel broad-spectrum antimycotics.
Opportunistic fungal pathogens such as Candida albicans often cause fatal infections in patients with a compromised immune system. Unfortunately, current drugs often fail to halt fungal disease, are ineffective against drug-resistant strains, and have severe side effects. Despite the clear clinical significance of fungal infections, it is still not understood how fungi are recognized by the immune system. Candida has high levels of the structural molecule β-glucan in its cell wall, but the majority of its β-glucan is masked by a mannoprotein coat and is therefore invisible to the immune system. Masking of β-glucan may be a fungal virulence factor, because exposed β-glucan provokes a proinflammatory response that is important for mounting an effective immune response against the fungus and clearing the infection. By surveying the genome of the model fungus Saccharomyces cerevisiae (bakers' yeast), the authors discovered a genetic network required for masking β-glucan from the immune system. Mutation of genes in this network in C. albicans caused unmasking of β-glucan and an increased immune response to the fungus. The authors also found that sublethal doses of the antifungal drug caspofungin cause unmasking and lead to a greater immune response. Drugs targeting this fungally conserved masking network may provide new tools to fight fungal infections.
PMCID: PMC1447670  PMID: 16652171
24.  Identification of the Proteins Required for Biosynthesis of Diphthamide, the Target of Bacterial ADP-Ribosylating Toxins on Translation Elongation Factor 2†  
Molecular and Cellular Biology  2004;24(21):9487-9497.
Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 were previously identified. Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals. We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature. We show that Dph1 and Dph2 are homologous in sequence but functionally independent. The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1. We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing DnaJ-like protein. Other features of these genes are also discussed. The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes.
PMCID: PMC522255  PMID: 15485916
25.  Transcriptional Response of Candida albicans upon Internalization by Macrophages 
Eukaryotic Cell  2004;3(5):1076-1087.
The opportunistic fungal pathogen Candida albicans is both a benign gut commensal and a frequently fatal systemic pathogen. The interaction of C. albicans with the host's innate immune system is the primary factor in this balance; defects in innate immunity predispose the patient to disseminated candidiasis. Because of the central importance of phagocytic cells in defense against fungal infections, we have investigated the response of C. albicans to phagocytosis by mammalian macrophages using genomic transcript profiling. This analysis reveals a dramatic reprogramming of transcription in C. albicans that occurs in two successive steps. In the early phase cells shift to a starvation mode, including gluconeogenic growth, activation of fatty acid degradation, and downregulation of translation. In a later phase, as hyphal growth enables C. albicans to escape from the macrophage, cells quickly resume glycolytic growth. In addition, there is a substantial nonmetabolic response imbedded in the early phase, including machinery for DNA damage repair, oxidative stress responses, peptide uptake systems, and arginine biosynthesis. Further, a surprising percentage of the genes that respond specifically to macrophage contact have no known homologs, suggesting that the organism has undergone substantial evolutionary adaptations to the commensal or pathogen lifestyle. This transcriptional reprogramming is almost wholly absent in the related, but nonpathogenic, yeast Saccharomyces cerevisiae, suggesting that these large-scale and coordinated changes contribute significantly to the ability of this organism to survive and cause disease in vivo.
PMCID: PMC522606  PMID: 15470236

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