Mechanistic Basis of pH-Dependent 5-Flucytosine Resistance in Aspergillus fumigatus.

The antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is licensed for the treatment of fungal diseases; however, it is rarely used as a monotherapeutic to treat infection. Despite being potent against other fungal pathogens, 5FC has limited activity against when standard assays are used to determine susceptibility. However, in modified assays where the pH is set to pH 5, the activity of 5FC increases significantly.

Correction: Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex.

[This corrects the article DOI: 10.1371/journal.ppat.

Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex.

Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase.

Where antibiotic resistance mutations meet quorum-sensing.

We do not need to rehearse the grim story of the global rise of antibiotic resistant microbes. But what if it were possible to control the rate with which antibiotic resistance evolves by mutation? It seems that some bacteria may already do exactly that: they modify the rate at which they mutate to antibiotic resistance dependent on their biological environment. In our recent study [Krašovec, Nat.

Mutation rate plasticity in rifampicin resistance depends on Escherichia coli cell-cell interactions.

Variation of mutation rate at a particular site in a particular genotype, in other words mutation rate plasticity (MRP), can be caused by stress or ageing. However, mutation rate control by other factors is less well characterized. Here we show that in wild-type Escherichia coli (K-12 and B strains), the mutation rate to rifampicin resistance is plastic and inversely related to population density: lowering density can increase mutation rates at least threefold.

Yeast cells with impaired drug resistance accumulate glycerol and glucose.

Multiple drug resistance (MDR) in yeast is effected by two major superfamilies of membrane transporters: the major facilitator superfamily (MFS) and the ATP-binding cassette (ABC) superfamily. In the present work, we investigated the cellular responses to disruptions in both MFS (by deleting the transporter gene, QDR3) and ABC (by deleting the gene for the Pdr3 transcription factor) transporter systems by growing diploid homozygous deletion yeast strains in glucose- or ammonium-limited continuous cultures. The transcriptome and the metabolome profiles of these strains, as well as the flux distributions in the optimal solution space, reveal novel insights into the underlying mechanisms of action of QDR3 and PDR3.

Synergistic effects of TOR and proteasome pathways on the yeast transcriptome and cell growth.

The proteasome has been implicated in gene transcription through a variety of mechanisms. How the proteasome regulates genome-wide transcription in relation to nutrient signalling pathways is largely unknown. Using chemical inhibitors to compromise the functions of the proteasome and/or TORC1, we reveal that the proteasome and TORC1 synergistically promote the expression of de novo purine and amino acid biosynthetic genes, and restrict the transcription of those associated with proteolysis, starvation and stress responses.

The genetic control of growth rate: a systems biology study in yeast.

Background: Control of growth rate is mediated by tight regulation mechanisms in all free-living organisms since long-term survival depends on adaptation to diverse environmental conditions. The yeast, Saccharomyces cerevisiae, when growing under nutrient-limited conditions, controls its growth rate via both nutrient-specific and nutrient-independent gene sets. At slow growth rates, at least, it has been found that the expression of the genes that exert significant control over growth rate (high flux control or HFC genes) is not necessarily regulated by growth rate itself.

How yeast re-programmes its transcriptional profile in response to different nutrient impulses.

Background: A microorganism is able to adapt to changes in its physicochemical or nutritional environment and this is crucial for its survival. The yeast, Saccharomyces cerevisiae, has developed mechanisms to respond to such environmental changes in a rapid and effective manner; such responses may demand a widespread re-programming of gene activity. The dynamics of the re-organization of the cellular activities of S.

Absolute and relative quantification of mRNA expression (transcript analysis).

In this protocol, we describe a pipeline for transcript analysis in yeast via the quantification of mRNA expression levels. In the first section, we consider the well-established, proprietary Affymetrix GeneChip® approach to generating transcriptomics data. In the next section, we concentrate on providing a detailed protocol for the validation of these data using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).

Phenomic and transcriptomic analyses reveal that autophagy plays a major role in desiccation tolerance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae can survive extreme desiccation, but the molecular mechanisms are poorly understood. To define genes involved in desiccation tolerance, two complementary genome-wide approaches, phenomics and transcriptomics, have been used, together with a targeted analysis of gene deletion mutants tested individually for their ability to survive drying. Genome-wide phenotypic analyses carried out on a pooled library of single-gene deletion mutants subjected to three cycles of desiccation and re-growth to post-diauxic phase identified about 650 genes that contributed to strain survival in the drying process.

Gene duplication and environmental adaptation within yeast populations.

Population-level differences in the number of copies of genes resulting from gene duplication and loss have recently been recognized as an important source of variation in eukaryotes. However, except for a small number of cases, the phenotypic effects of this variation are unknown. Data from the Saccharomyces Genome Resequencing Project permit the study of duplication in genome sequences from a set of individuals within the same population.

Conditional cell-wall mutants of Saccharomyces cerevisiae as delivery vehicles for therapeutic agents in vivo to the GI tract.

Strains of Saccharomyces cerevisiae capable of lysis upon conditional down-regulation of cell-wall biogenesis genes (SRB1 and PKC1) have been reported. Here, we show that they lyse and release recombinant protein not only under laboratory conditions, but (more importantly) under conditions found in the human stomach and duodenum. These findings provide proof that, in principle, such conditional lysis strains could be used as an integral part of a system for the oral delivery of therapeutic proteins.

Identification and characterization of high-flux-control genes of yeast through competition analyses in continuous cultures.

Using competition experiments in continuous cultures grown in different nutrient environments (glucose limited, ammonium limited, phosphate limited and white grape juice), we identified genes that show haploinsufficiency phenotypes (reduced growth rate when hemizygous) or haploproficiency phenotypes (increased growth rate when hemizygous). Haploproficient genes (815, 1,194, 733 and 654 in glucose-limited, ammonium-limited, phosphate-limited and white grape juice environments, respectively) frequently show that phenotype in a specific environmental context. For instance, genes encoding components of the ubiquitination pathway or the proteasome show haploproficiency in nitrogen-limited conditions where protein conservation may be beneficial.

Global phenotype screening and transcript analysis outlines the inhibitory mode(s) of action of two amphibian-derived, alpha-helical, cationic peptides on Saccharomyces cerevisiae.

Dermaseptin S3(1-16) [DsS3(1-16)] and magainin 2 (Mag 2) are two unrelated, amphibian-derived cationic peptides that adopt an alpha-helical structure within microbial membranes and have been proposed to kill target organisms via membrane disruption. Using a combination of global deletion mutant library phenotypic screening, expression profiling, and physical techniques, we have carried out a comprehensive in vitro analysis of the inhibitory action of these two peptides on the model fungus Saccharomyces cerevisiae. Gene ontology profiling (of biological processes) was used to identify both common and unique effects of each peptide.