Ribosome quality control is a central protection mechanism for yeast exposed to deoxynivalenol and trichothecin.

BACKGROUND: The trichothecene mycotoxins deoxynivalenol (DON) and trichothecin (TTC) are inhibitors of eukaryotic protein synthesis. Their effect on cellular homeostasis is poorly understood. We report a systematic functional investigation of the effect of DON and TTC on the yeast Saccharomyces cerevisiae using genetic array, network and microarray analysis. To focus the genetic analysis on intracellular consequences of toxin action we eliminated the PDR5 gene coding for a potent pleiotropic drug efflux protein potentially confounding results. We therefore used a knockout library with a pdr5Δ strain background. RESULTS: DON or TTC treatment creates a fitness bottleneck connected to ribosome efficiency. Genes isolated by systematic genetic array analysis as contributing to toxin resistance function in ribosome quality control, translation fidelity, and in transcription. Mutants in the E3 ligase Hel2, involved in ribosome quality control, and several members of the Rpd3 histone deacetylase complex were highly sensitive to DON. DON and TTC have similar genetic profiles despite their different toxic potency. Network analysis shows a coherent and tight network of genetic interactions among the DON and TTC resistance conferring gene products. The networks exhibited topological properties commonly associated with efficient processing of information. Many sensitive mutants have a "slow growth" gene expression signature. DON-exposed yeast cells increase transcripts of ribosomal protein and histone genes indicating an internal signal for growth enhancement. CONCLUSIONS: The combination of gene expression profiling and analysis of mutants reveals cellular pathways which become bottlenecks under DON and TTC stress. These are generally directly or indirectly connected to ribosome biosynthesis such as the general secretory pathway, cytoskeleton, cell cycle delay, ribosome synthesis and translation quality control. Gene expression profiling points to an increased demand of ribosomal components and does not reveal activation of stress pathways. Our analysis highlights ribosome quality control and a contribution of a histone deacetylase complex as main sources of resistance against DON and TTC.

Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes.

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.

A histone deacetylase adjusts transcription kinetics at coding sequences during Candida albicans morphogenesis.

Despite their classical role as transcriptional repressors, several histone deacetylases, including the baker's yeast Set3/Hos2 complex (Set3C), facilitate gene expression. In the dimorphic human pathogen Candida albicans, the homologue of the Set3C inhibits the yeast-to-filament transition, but the precise molecular details of this function have remained elusive. Here, we use a combination of ChIP-Seq and RNA-Seq to show that the Set3C acts as a transcriptional co-factor of metabolic and morphogenesis-related genes in C. albicans. Binding of the Set3C correlates with gene expression during fungal morphogenesis; yet, surprisingly, deletion of SET3 leaves the steady-state expression level of most genes unchanged, both during exponential yeast-phase growth and during the yeast-filament transition. Fine temporal resolution of transcription in cells undergoing this transition revealed that the Set3C modulates transient expression changes of key morphogenesis-related genes. These include a transcription factor cluster comprising of NRG1, EFG1, BRG1, and TEC1, which form a regulatory circuit controlling hyphal differentiation. Set3C appears to restrict the factors by modulating their transcription kinetics, and the hyperfilamentous phenotype of SET3-deficient cells can be reverted by mutating the circuit factors. These results indicate that the chromatin status at coding regions represents a dynamic platform influencing transcription kinetics. Moreover, we suggest that transcription at the coding sequence can be transiently decoupled from potentially conflicting promoter information in dynamic environments.

Conventional dendritic cells mount a type I IFN response against Candida spp. requiring novel phagosomal TLR7-mediated IFN-ß signaling.

Human fungal pathogens such as the dimorphic Candida albicans or the yeast-like Candida glabrata can cause systemic candidiasis of high mortality in immunocompromised individuals. Innate immune cells such as dendritic cells and macrophages establish the first line of defense against microbial pathogens and largely determine the outcome of infections. Among other cytokines, they produce type I IFNs (IFNs-I), which are important modulators of the host immune response. Whereas an IFN-I response is a hallmark immune response to bacteria and viruses, a function in fungal pathogenesis has remained unknown. In this study, we demonstrate a novel mechanism mediating a strong IFN-ß response in mouse conventional dendritic cells challenged by Candida spp., subsequently orchestrating IFN-α/ß receptor 1-dependent intracellular STAT1 activation and IFN regulatory factor (IRF) 7 expression. Interestingly, the initial IFN-ß release bypasses the TLR 4 and TLR2, the TLR adaptor Toll/IL-1R domain-containing adapter-inducing IFN-ß and the ß-glucan/phagocytic receptors dectin-1 and CD11b. Notably, Candida-induced IFN-ß release is strongly impaired by Src and Syk family kinase inhibitors and strictly requires completion of phagocytosis as well as phagosomal maturation. Strikingly, TLR7, MyD88, and IRF1 are essential for IFN-ß signaling. Furthermore, in a mouse model of disseminated candidiasis we show that IFN-I signaling promotes persistence of C. glabrata in the host. Our data uncover for the first time a pivotal role for endosomal TLR7 signaling in fungal pathogen recognition and highlight the importance of IFNs-I in modulating the host immune response to C. glabrata.

Efg1 Controls caspofungin-induced cell aggregation of Candida albicans through the adhesin Als1.

Echinocandin drugs such as caspofungin (CASP), micafungin, and anidulafungin inhibit fungal cell wall biogenesis by blocking Fks1-mediated ß-glucan deposition into the cell surface. Candins have become suitable drugs to treat life-threatening diseases caused by several fungal species, including Candida albicans, that are pathogenic for humans. Here, we present the discovery of a novel CASP-induced flocculation phenotype of C. albicans, which formed large cell aggregates in the presence of CASP. High concentrations of sugars such as mannose or glucose inhibit CASP-induced flocculation and improve survival of C. albicans cells exposed to CASP. Notably, exposure of C. albicans cells to CASP triggers Efg1-dependent expression of the adhesin ALS1 and induces invasive growth on agar plates. Indeed, cells lacking either Efg1 or Als1 show strongly diminished CASP-induced flocculation, and the absence of Efg1 leads to marked CASP hypersensitivity. On the other hand, CASP-induced invasive growth is enhanced in cells lacking Efg1. Hence, CASP stress drives an Efg1-dependent response, indicating that this multifunctional transcriptional regulator, which is otherwise involved in filamentation, white-to-opaque switching, and virulence, also modulates cell wall remodeling upon CASP challenge. Taken together, our data suggest that CASP-induced cell wall damage activates Efg1 in parallel with the known cell integrity stress signaling pathway to coordinate cell wall remodeling.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

Host and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Type I Interferon Response Dysregulates Host Iron Homeostasis and Enhances Candida glabrata Infection.

Type I interferons (IFNs-I) fulfil multiple protective functions during pathogenic infections, but they can also cause detrimental effects and enhance immunopathology. Here, we report that IFNs-I promote the dysregulation of iron homeostasis in macrophages during systemic infections with the intracellular pathogen Candida glabrata, leading to fungal survival and persistence. By engaging JAK1, IFNs-I disturb the balance of the transcriptional activator NRF2 and repressor BACH1 to induce downregulation of the key iron exporter Fpn1 in macrophages. This leads to enhanced iron accumulation in the phagolysosome and failure to restrict fungal access to iron pools. As a result, C. glabrata acquires iron via the Sit1/Ftr1 iron transporter system, facilitating fungal intracellular replication and immune evasion. Thus, IFNs-I are central regulators of iron homeostasis, which can impact infection, and restricting iron bioavailability may offer therapeutic strategies to combat invasive fungal infections.

Impact of acute metal stress in Saccharomyces cerevisiae.

Although considered as essential cofactors for a variety of enzymatic reactions and for important structural and functional roles in cell metabolism, metals at high concentrations are potent toxic pollutants and pose complex biochemical problems for cells. We report results of single dose acute toxicity testing in the model organism S. cerevisiae. The effects of moderate toxic concentrations of 10 different human health relevant metals, Ag(+), Al(3+), As(3+), Cd(2+), Co(2+), Hg(2+), Mn(2+), Ni(2+), V(3+), and Zn(2+), following short-term exposure were analyzed by transcription profiling to provide the identification of early-on target genes or pathways. In contrast to common acute toxicity tests where defined endpoints are monitored we focused on the entire genomic response. We provide evidence that the induction of central elements of the oxidative stress response by the majority of investigated metals is the basic detoxification process against short-term metal exposure. General detoxification mechanisms also comprised the induction of genes coding for chaperones and those for chelation of metal ions via siderophores and amino acids. Hierarchical clustering, transcription factor analyses, and gene ontology data further revealed activation of genes involved in metal-specific protein catabolism along with repression of growth-related processes such as protein synthesis. Metal ion group specific differences in the expression responses with shared transcriptional regulators for both, up-regulation and repression were also observed. Additionally, some processes unique for individual metals were evident as well. In view of current concerns regarding environmental pollution our results may support ongoing attempts to develop methods to monitor potentially hazardous areas or liquids and to establish standardized tests using suitable eukaryotic a model organism.

Functional genomics of drug-induced ion homeostasis identifies a novel regulatory crosstalk of iron and zinc regulons in yeast.

Pyrrolidine dithiocarbamate (PDTC), a known inhibitor of NFκB activation, has antioxidative as well as antiviral activities. PDTC is effective against several virus families, indicating that its antiviral mechanism targets host rather than viral functions. To investigate its mode of action, we used baker's yeast as a simple eukaryotic model system and two types of genome-wide analysis. First, expression profiling using whole-genome DNA microarrays identifies more than 200 genes differentially regulated upon PDTC exposure. Interestingly, the Aft1-dependent iron regulon is a main target of PDTC, indicating a lack of iron availability. Moreover, the PDTC-caused zinc influx triggers a strong regulatory effect on zinc transporters due to the cytoplasmic zinc excess. Second, phenotypic screening the EUROSCARF collection for PDTC hypersensitivity identifies numerous mutants implicated in vacuolar maintenance, acidification as well as in transport, mitochondrial organization, and translation. Notably, the screening data indicate significant overlaps of PDTC-sensitive genes and those mediating zinc tolerance. Hence, we show that PDTC induces cytoplasmic zinc excess, eliciting vacuolar detoxification, which in turn, disturbs iron homeostasis and activates the iron-dependent regulator Aft1. Our work reveals a complex crosstalk in yeast ion homeostasis and the underlying regulatory networks.

Recognition of eukaryotic initiation factor 4G isoforms by picornaviral proteinases.

The leader proteinase (L(pro)) of foot and mouth disease virus is a papain-like cysteine proteinase. After processing itself from the polyprotein, L(pro) then cleaves the host protein eukaryotic initiation factor (eIf) 4GI, thus preventing protein synthesis from capped mRNA in the infected cell. We have investigated L(pro) interaction with eIF4GI and its isoform, eIF4GII. L(pro), expressed as a catalytically inactive fusion protein with glutathione S-transferase, binds specifically to eIF4G isomers in rabbit reticulocyte lysates. Deletion and specific mutagenesis were used to map the binding domain on L(pro) to residues 183-195 of the C-terminal extension and to residue Cys(133). These residues of the C-terminal extension and Cys(133) are adjacent in the crystal structure but lie about 25 A from the active site. The region on eIF4GI recognized by the L(pro) C-terminal extension was mapped to residues 640-669 using eIF4GI fragments generated by proteolysis or by in vitro translation. The L(pro) cleavage site at Gly(674) downward arrow Arg(675) was not necessary for binding. Similar experiments with human rhinovirus 2A proteinase (2A(pro)), a chymotrypsin-like cysteine proteinase that also cleaves eIF4G isoforms, revealed that 2A(pro) can also bind to eIF4GI fragments lacking its cleavage site. These experiments strongly suggest a novel interaction between picornaviral proteinases and eIF4G isoforms.

The processing of eIF4GI by human rhinovirus type 2 2A(pro): relationship to self-cleavage and role of zinc.

The 2A proteinase (2A(pro)) of human rhinoviruses (HRVs) is a cysteine protease containing a structurally important zinc ion. In the viral polyprotein, the enzyme cleaves between the C terminus of VP1 and its own N terminus. 2A(pro) also processes the two isoforms of the cellular protein, eukaryotic initiation factor 4G (eIF4G). We have shown that mature HRV2 2A(pro), when translated in vitro in rabbit reticulocyte lysates, efficiently cleaves eIF4GI, although the enzyme was not immediately active upon synthesis. Here, we examine the relationship between self-processing and eIF4GI cleavage. The onset of both reactions first occurred at least 10 min after initiation of protein synthesis. Furthermore, when self-processing was prevented by a specific mutation between VP1 and 2A(pro), the VP1-2A(pro) precursor was essentially unable to cleave eIF4GI, implying that self-processing is a prerequisite for eIF4GI cleavage. 2A(pro) synthesized in the presence of a potent zinc chelator is inactive; however, upon addition of excess zinc, HRV2 2A(pro) rapidly gained activity. Finally, the presence of the zinc chelator in the culture medium can protect HeLa cells from HRV infection.