Gerald R. Fink

Fungi are increasingly the cause of death as there are few effective fungal antibiotics. Fungi gain entry to the body by adhering to indwelling devices such as catheters. The adhesion is intimately linked to the β-glucan and the mannoproteins (adhesins) molecules that encase the fungal cell. The analysis of adhesion and filamentation in the model system, Saccharomyces cerevisiae guides our studies in the less tractable pathogen, Candida albicans. The genomes of both fungi encode many mannoproteins that confer unique adherence properties. These adhesins are required for interactions of fungal cells with each other (flocculation and filamentation), with inert surfaces (agar and plastic) and with mammalian cells. These cell surface molecules are the antigens recognized by the phagocytic cells of the immune system. Fungi are able to vary these cell surface molecules. Recent work has shown that antisense RNA plays a key role in the expression of these cell surface molecules. We use genetics, biochemistry and genomics to address questions such as: What mechanisms generate the diversity of cell surface molecules? What cell surface molecules are recognized by the cells of the immune system? How do macrophages and neutrophils distinguish between a pathogen, Candida albicans, and a non-pathogen, Saccharomyces cerevisia.

Long hyphal threads of the human pathogen, Candida albicans, have their underlying coat exposed by an antibiotic.Long hyphal threads of the human pathogen, Candida albicans, have their underlying coat exposed by an antibiotic.

Variation to elude the immune system

The cell surface molecules of fungi vary by both genetic and epigenetic mechanisms that confuses the immune system. The β-glucan and the mannoproteins on the surface of fungi are the signature molecules recognized by the phagocytic cells of the immune system in their attempt to destroy pathogens. But, fungi have genetic, epigenetic and regulatory mechanisms that change the ensemble of surface molecules to avoid or alter recognition. The surface mannoproteins (adhesins) of Saccharomyces cerevisiae, Candida albicans and Candida glabrata form a superfamily united by a common structure consisting of three domains (A, B, C). The amino terminal domain (A) provides much of the affinity for surfaces. This domain is followed by a segment of variable length (B) that is extremely rich in serines and threonines and contains many tandem repeats. The carboxyterminal region (C), links the mannoprotein to the β-glucan.

A. Genetic Mechanisms create variation

Most of the genes in the genome with internal tandem repeat sequences encode cell wall proteins and the length of the tandem repeats are highly variable from strain to strain. To identify the S. cerevisiae open reading frames (ORFs) that contain intragenic tandem repeats, we scanned all 6591 open reading frames of Saccharomyces for the presence of long (>40 nt) tandem repeats. The search yielded 29 ORFs with repeats longer than 40 nt. 22 of the 29 ORFs (75 %) with conserved long repeats encode cell surface proteins, whereas only 1.3 % of all ~6000 S. cerevisiae ORFs are cell surface proteins. A comparison of lab and wild strains for the size of the repeat sequences in each of the 22 cell surface genes using PCR primers flanking the repeats showed that all 22cell surface genes with internal tandem repeats showed size variation from one strain to another.

Gene expansion and contraction of intragenic repeats is a frequent event in real time. The FLO1 gene is 4.6 kb long and contains 18 repeats of about 100 nt, separated by a less conserved 45-nt sequence. A colony grown from a single cell generates progeny that have increased or decreased numbers of repeats in FLO1 at a frequency of 10-4. The new FLO1 genes generated during mitotic growth were as large as 8 kb and as small as 2.5 kb, the sizes varying by an integral number of repeat units. The change in gene size has profound phenotypic consequences: There is a striking, linear correlation between gene size and the extent of adhesion: as the FLO1 proteins become longer (carrying more repeats), theadhesion properties gradually become stronger. Flocculation (i.e. adhesion to other yeast cells) shows the same relationship to the repeat number: the more repeats, the greater the fraction of flocculating cells.

Taken together, the recombination frequencies observed in the various mutants indicate that recombination between the cell wall gene intra-genic repeats is caused by a replication slippage process similar to that observed in inter-genic repeats. Loss of the RAD27-encoded flap endonuclease, which causes the formation of double-stranded breaks during replication, increases the instability of FLO1 repeats almost 40-fold. Furthermore, the decrease in recombination observed in rad50Δ, rad52Δ and rad1Δ rad52Δ mutants suggests that the process depends on break repair by single-strand annealing, a conclusion further supported by the decrease in FLO1 recombination in the rad59Δ mutant, which is known to be deficient in this type of DNA repair.

B. Epigenetic mechanisms

Many of the cell surface genes that show repeat-size variation also vary epigenetically. Our previous work had shown that FLO11, one of the 22 cell surface genes with repeat-size variation, also varies epigenetically, so that some cells express FLO11 ("on" ) and others did not ("off").

Sherwin Chan, a postdoctoral fellow, made promoter fusions to GFP and URA3 of all 78 genes in the yeast genome that are annotated in SGD as producing a "cell wall localized protein". These are fusions to the initiating ATG in each gene and have only the coding region of the reporter. As controls, we have made promoter fusions to many other housekeeping genes randomly chosen to act as controls. The fusions are then tested to determine whether they variegate by three criteria:

  1. Promoter fusions to URA3 variegate
  2. Promoter fusions to GFP--Clonal analysis by fluorescence microscopy and FACS documents: Heritable change in phenotype transmitted from generation to generation despite identical genotype.
  3. Abolition of variegation by trans-acting chromatin factors e.g. histone deacetylases. Remarkably, unlike the controls (genes encoding non-cell wall proteins) many of the genes with internal repeats that we had shown have genetic variation in repeat length, also show epigenetic switching by these criteria.

The variegation of these cell surface proteins is under the control of chromatin binding proteins. Previous work showed that one of these, FLO11, is under the control of a number of trans-acting chromatin binding factors. Knockouts of these factors prevents epigenetic switching and locks the cells into an "on" or "off" expression state.

Antisense Transcription Controls Cell Fate in Saccharomyces cerevisiae

Many organisms make an antisense RNA in addition to the sense transcript that encodes a protein, but the function of these antisense transcripts has been unexplored. We have discovered that the key developmental event in Saccharomyces cerevisiae, the switch from mitotic (vegetative) growth to meiosis, is under the control of IME4 antisense RNA. The IME4 gene makes both an antisense and sense transcript, but each is restricted to a cell type – haploid cells make an antisense IME4 transcript and diploids make only the sense. Haploid cells of opposite mating type (MAT a and MAT α) mate to produce MAT a/α diploid cells. In these diploids, haploid functions are repressed by the a1/α2 protein heterodimer.

In MATa/α diploids, a1/α2 repression of IME4 antisense transcription allows transcription of the sense transcript and subsequent synthesis of the Ime4 protein which, promotes the switch from mitotic to meiotic cell division. In haploids (lacking a1/α2), antisense transcription proceeds and prevents sense transcription.

By altering the antisense expression haploid cells can be made to behave like diploids and diploids like haploids. Artificial overexpression of the IME4 sense transcript in haploids turns on the entire meiotic program, albeit an abortive one. Conversely, IME4 antisense transcription in MAT a/α diploids, obtained by preventing a1/α2 binding, blocks meiosis and restricts the cells to a vegetative growth cycle. In fact, diploid cells that express the antisense RNA show haploid features such as increased adhesion and inability to turn on the meiotic program. IME4 orthologues exist in many metazoans including humans, but their function is unknown.

A drug-sensitive genetic network masks fungi from the immune system. Our data suggest that effective antibiotics may act not only by killing the pathogen but, at lower concentrations, by revealing otherwise hidden signatures recognized by the immune system. Many fungal pathogens are recognized by the immune system by virtue of their β-glucan, a potent pro-inflammatory molecule, which is normally hidden underneath a mannoprotein coat. We found that the underlying β-glucan in the cell wall of Candida albicans is unmasked by sub-inhibitory doses of the anti-fungal drug caspofungin, causing the exposed fungi to elicit more pro-inflammatory cytokines. Using a genome-wide library of bakers’ yeast, we identified a conserved genetic network that is required for concealing β-glucan from the immune system and limiting the pro-inflammatory response of macrophages exposed to fungi. Several genes identified in this screen have C. albicans homologs, which are also important for masking β-glucan in this pathogen. These unmasked C. albicans mutants cause an increased elicitation of key pro-inflammatory cytokines from primary mouse macrophages that is dependent on the β-glucan receptor. The potent antifungal agent, caspofungin, also unmasks the β-glucan at non-inhibitory concentrations and makes the organism accessible to the immune system. The β-glucan unmasking activities of caspofungin may contribute to its effectiveness against a broad spectrum of fungi that share this β-glucan masking gene network.

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. We have shown 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. These are manifestations of neutrophil recognition of a pathogen. Remarkably, 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.

Carey BW, Markoulaki S, Hanna JH, Faddah DA, Buganim Y, Kim J, Ganz K, Steine EJ, Cassady JP, Creyghton MP, Welstead GG, Gao Q, Jaenisch R. Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Cell Stem Cell l9, 588-598 (2011).

Chen, H, and Fink, G. R. (2006). Feedback control of morphogenesis in fungi by aromatic alcohols. Genes Dev. 20, 1150-61.

Wheeler, R.T. and Fink, G.R. (2006). A drug-sensitive genetic network masks fungi from the immune system. PLoS Pathog 2, e35.

Voynov, V., Verstrepen, K. Jansen, A., and Fink, G. (2006). Genes with internal repeats require the THO complex for transcription. Proc. Nat. Acad. Sci. 103, 14423-14428

Hongay CF, Grisafi PL, Galitski T, Fink GR.Antisense transcription controls cell fate in Saccharomyces cerevisiae. (2006) Cell. 127:735-45.

Cowen LE, Carpenter AE, Matangkasombut O, Fink GR, Lindquist S. (2006) Genetic architecture of Hsp90-dependent drug resistance Eukaryot Cell. (12):2184-8.

Alper, H., Moxley J, Nevoigt E,,  Fink GR, Stephanopoulos G. (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314:1565-8.

Verstrepen, K.J., Jansen, A., Lewitter, F., Fink, G.R. (2005). Intragenic tandem repeats generate functional variability. Nature Genetics. 37, 986-990