Principal Investigator Gerald Fink
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/a diploid cells. In these diploids, haploid functions are repressed by the a1/a2 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 βbeta-glucan receptor. The potent antifungal agent, caspofungin, also unmasks the beta-glucan at non-inhibitory concentrations and makes the organism accessible to the immune system. The beta-glucan unmasking activities of caspofungin may contribute to its effectiveness against a broad spectrum of fungi that share this beta-glucan masking gene network.
Fungal walls have two kinds of β-glucan: beta-1,3-glucan and beta-1,6-glucan. The predominance of beta-1,3-glucan in the wall and its recognition by macrophages has led to the presumption that beta-1,3-glucan is the key immunological determinant for both macrophages and neutrophils. We have shown that in human neutrophils, beta-1,6-glucan mediates engulfment, production of reactive oxygen species, and expression of HSPs more efficiently than beta-1,3-glucan. These are manifestations of neutrophil recognition of a pathogen. Remarkably, neutrophils rapidly ingest beads coated with beta-1,6-glucan, while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan, recognized by CR3. beta-1,6-glucan is also important for efficient engulfment of Candida albicans. These unique stimulatory effects could have useful medical applications.