No abstract

The opportunistic fungal pathogen Candida albicans is a part of the normal flora but it also causes systemic candidiasis if it reaches the bloodstream. Upon being phagocytized by macrophages, an important component of innate immunity, C. albicans rapidly upregulates a set of arginine biosynthetic genes. Arginine, urea, and CO 2 induced hyphae in a density-dependent manner in wild-type, cph1/cph1, and rim101/rim101 strains but not in efg1/efg1 or cph1/cph1 efg1/efg1 strains. Arginase (Car1p) converts arginine to urea, which in turn is degraded by urea amidolyase (Dur1,2p) to produce CO 2 , a signal for hyphal switching. We used a dur1,2/dur1,2 mutant (KWN6) and the complemented strain, KWN8 (dur1,2/dur1,2::DUR1,2/DUR1,2) to study germ tube formation. KWN6 could not make germ tubes in the presence of arginine or urea but did in the presence of 5% CO 2 , which bypasses Dur1,2p. We also tested the effect of arginine on the interaction between the macrophage line RAW 264.7 and several strains of C. albicans. Arginine activated an Efg1p-dependent yeast-to-hypha switch, enabling wild-type C. albicans and KWN8 to escape from macrophages within 6 h, whereas KWN6 was defective in this regard. Additionally, two mutants that cannot synthesize arginine, BWP17 and SN152, were defective in making hyphae inside the macrophages, whereas the corresponding arginine prototrophs, DAY286 and SN87, formed germ tubes and escaped from macrophages. Therefore, metabolism of arginine by C. albicans controls hyphal switching and provides an important mechanism for escaping host defense.

Concentrations of (E,E)-farnesol needed to inhibit germ tube formation were determined for Candida albicans strains A72 and SC5314 by using six different conditions known to trigger germination. For defined media, 1 to 2 M farnesol was sufficient. However, with serum at 2 to 20%, up to 250 M farnesol was required. Farnesol blocked germ tube formation but did not block elongation of existing germ tubes.

In Saccharomyces cerevisiae the UPF1 protein is required for nonsense-mediated mRNA decay, the accelerated turnover of mRNAs containing a nonsense mutation. Several lines of evidence suggest that translation plays an important role in the mechanism of nonsense mRNA decay, including a previous report that nonsense mRNAs assemble in polyribosomes. In this study we show that UPF1 and ribosomal protein L1 co-localize in the cytoplasm and that UPF1 co-sediments with polyribosomes. To detect UPF1, three copies of the influenza hemagglutinin epitope were placed at the C-terminus. The tagged protein, UPF1-3EP, retains 86% (+/- 5%) of function. Using immunological detection, we found that UPF1-3EP is primarily cytoplasmic and was not detected either in the nucleus or in the mitochondrion. UPF1-3EP and L1 co-distributed with polyribosomes fractionated in a 7-47% sucrose gradient. The sucrose sedimentation profiles for UPF1-3EP and L1 exhibited similar changes using three different sets of conditions that altered the polyribosome profile. When polyribosomes were disaggregated, UPF1-3EP and L1 accumulated in fractions coincident with 80S ribosomal particles. These results suggest that UPF1-3EP associates with polyribosomes. L3 and S3 mRNAs, which code for ribosomal proteins of the 60S and 40S ribosomal subunits, respectively, were on average about 100-fold more abundant than UPF1 mRNA. Assuming that translation rates for L3, S3, and UPF1 mRNA are similar, this result suggests that there are far fewer UPF1 molecules than ribosomes per cell. Constraints imposed by the low UPF1 abundance on the functional relationships between UPF1, polyribosomes, and nonsense mRNA turnover are discussed.

The nonsense-mediated mRNA decay (NMD) pathway, present in most eukaryotic cells, is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons and, importantly, some wild-type mRNAs. Earlier studies demonstrated that aberrant mRNAs with artificially extended 3′-untranslated regions (3′-UTRs) are degraded by NMD. However, the extent to which wild-type mRNAs with long 3′-UTRs are degraded by NMD is not known. We used a global approach to identify wild-type mRNAs in Saccharomyces cerevisiae that have longer than expected 3′-UTRs, and of these mRNAs tested, 91% were degraded by NMD. We demonstrate for the first time that replacement of the natural, long 3′-UTR from wild-type PGA1 mRNA, which encodes a protein that is important for cell wall biosynthesis, with a short 3′-UTR renders it immune to NMD. The natural PGA1 3′-UTR is sufficient to target a NMD insensitive mRNA for decay by the NMD pathway. Finally, we show that nmd mutants are sensitive to Calcofluor White, which suggests that the regulation of PGA1 and other cell wall biosynthesis proteins by NMD is physiologically significant.

Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17-and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in C. albicans.

In yeast, the accelerated rate of decay of nonsense mutant mRNAs, called nonsense-mediated mRNA decay, requires three proteins, Upf1p, Upf2p, and Upf3p. Single, double, and triple disruptions of the UPF genes had nearly identical effects on nonsense mRNA accumulation, suggesting that the encoded proteins function in a common pathway. We examined the distribution of epitope-tagged versions of Upf proteins by sucrose density gradient fractionation of soluble lysates and found that all three proteins co-distributed with 80 S ribosomal particles and polyribosomes. Treatment of lysates with RNase A caused a coincident collapse of polyribosomes and each Upf protein into fractions containing 80 S ribosomal particles, as expected for proteins that are associated with polyribosomes. Mutations in the cysteine-rich (zinc finger) and RNA helicase domains of Upf1p caused loss of function, but the mutant proteins remained polyribosome-associated. Density gradient profiles for Upf1p were unchanged in the absence of Upf3p, and although similar, were modestly shifted to fractions lighter than those containing polyribosomes in the absence of Upf2p. Upf2p shifted toward heavier polyribosome fractions in the absence of Upf1p and into fractions containing 80 S particles and lighter fractions in the absence of Upf3p. Our results suggest that the association of Upf2p with polyribosomes typically found in a wild-type strain depends on the presence and opposing effects of Upf1p and Upf3p.The notion of a global pathway for eukaryotic mRNA decay suggested by early work in animal cells has recently been greatly advanced by studies in the yeast Saccharomyces cerevisiae (1-4). Using an in vivo transcriptional pulse, the temporal fate of newly synthesized mRNA was established by monitoring poly(A) tail length, loss of the m 7 Gppp cap, disappearance of the mRNA, and the appearance of degradation intermediates. mRNAs with shorter half-lives were generally subject to faster rates of deadenylation and decapping. Once the poly(A) tails were reduced to a short oligo(A) length (10 -12 nucleotides), the mRNAs were decapped and digested from the 5Ј end. Decapping requires Dcp1p (5). Processive degradation from the 5Ј end requires the product of the XRN1 gene, which is known to encode a 5Ј 3 3Ј exoribonuclease (6). Deadenylation-dependent decapping followed by 5Ј 3 3Ј exonucleolytic decay is likely to be the global default pathway for the degradation of most eukaryotic mRNAs.Yeast mRNAs containing a premature stop codon decay more rapidly than their wild-type counterparts (7). This accelerated decay, called nonsense-mediated mRNA decay (NMD), 1 requires cis-acting elements in the mRNA in addition to a premature stop codon (8). Premature translational termination triggers decapping at the 5Ј end of nonsense mRNAs with kinetics that are independent of deadenylation (9). Following decapping, decay proceeds through the Xrn1p-mediated nucleolysis that is common to intrinsic decay. These results support the view that when translation is prematurely terminat...

Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.