<i>Saccharomyces cerevisiae</i> biofilm tolerance towards systemic antifungals depends on growth phase

BACKGROUND: Biofilm-forming Candida species cause infections that can be difficult to eradicate, possibly because of antifungal drug tolerance mechanisms specific to biofilms. In spite of decades of research, the connection between biofilm and drug tolerance is not fully understood. RESULTS: We used Saccharomyces cerevisiae as a model for drug susceptibility of yeast biofilms. Confocal laser scanning microscopy showed that S. cerevisiae and C. glabrata form similarly structured biofilms and that the viable cell numbers were significantly reduced by treatment of mature biofilms with amphotericin B but not voriconazole, flucytosine, or caspofungin. We showed that metabolic activity in yeast biofilm cells decreased with time, as visualized by FUN-1 staining, and mature, 48-hour biofilms contained cells with slow metabolism and limited growth. Time-kill studies showed that in exponentially growing planktonic cells, voriconazole had limited antifungal activity, flucytosine was fungistatic, caspofungin and amphotericin B were fungicidal. In growth-arrested cells, only amphotericin B had antifungal activity. Confocal microscopy and colony count viability assays revealed that the response of growing biofilms to antifungal drugs was similar to the response of exponentially growing planktonic cells. The response in mature biofilm was similar to that of non-growing planktonic cells. These results confirmed the importance of growth phase on drug efficacy. CONCLUSIONS: We showed that in vitro susceptibility to antifungal drugs was independent of biofilm or planktonic growth mode. Instead, drug tolerance was a consequence of growth arrest achievable by both planktonic and biofilm populations. Our results suggest that efficient strategies for treatment of yeast biofilm might be developed by targeting of non-dividing cells

The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner.

The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids

A common mechanism involving the TORC1 pathway can lead to amphotericin B-persistence in biofilm and planktonic <i>Saccharomyces cerevisiae</i> populations populations

Fungal infections are an increasing clinical problem. Decreased treatment effectiveness is associated with biofilm formation and drug recalcitrance is thought to be biofilm specific. However, no systematic investigations have tested whether resistance mechanisms are shared between biofilm and planktonic populations. We performed multiplexed barcode sequencing (Bar-seq) screening of a pooled collection of gene-deletion mutants cultivated as biofilm and planktonic cells. Screening for resistance to the ergosterol-targeting fungicide amphotericin B (AmB) revealed that the two growth modes had significant overlap in AmB-persistent mutants. Mutants defective in sterol metabolism, ribosome biosynthesis, and the TORC1 and Ras pathways showed increased persistence when treated with AmB. The ras1, ras2 and tor1 mutants had a high-persister phenotype similar to wild-type biofilm and planktonic cells exposed to the TORC1 pathway inhibitor rapamycin. Inhibition of TORC1 with rapamycin also increased the proportion of persisters in Candida albicans and Candida glabrata. We propose that decreased TORC1-mediated induction of ribosome biosynthesis via Ras can lead to formation of AmB-persister cells regardless of whether the cells are in planktonic or biofilm growth mode. Identification of common pathways leading to growth mode-independent persister formation is important for developing novel strategies for treating fungal infections

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types

Genetic basis for <i>Saccharomyces cerevisiae</i> biofilm in liquid medium

Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the Σ1278b background and found 71 genes that were essential for biofilm development. Quantitative northern blots further revealed that AIM1, ASG1, AVT1, DRN1, ELP4, FLO8, FMP10, HMT1, KAR5, MIT1, MRPL32, MSS11, NCP1, NPR1, PEP5, PEX25, RIM8, RIM101, RGT1, SNF8, SPC2, STB6, STP22, TEC1, VID24, VPS20, VTC3, YBL029W, YBL029C-A, YFL054C, YGR161W-C, YIL014C-A, YIR024C, YKL151C, YNL200C, YOR034C-A, and YOR223W controlled biofilm through FLO11 induction. Almost all deletion mutants that were unable to form biofilms in liquid medium also lost the ability to form surface-spreading biofilm colonies (mats) on agar and 69% also lost the ability to grow invasively. The protein kinase A isoform Tpk3p functioned specifically in biofilm and mat formation. In a tpk3 mutant, transcription of FLO11 was induced three-fold compared with wild-type, but biofilm development and cell–cell adhesion was absent, suggesting that Tpk3p regulates FLO11 positive posttranscriptionally and negative transcriptionally. The study provides a resource of biofilm-influencing genes for additional research on biofilm development and suggests that the regulation of FLO11 is more complex than previously anticipated

Genetic Basis for Saccharomyces cerevisiae Biofilm in Liquid Medium

Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the Σ1278b background and found 71 genes that were essential for biofilm development. Quantitative northern blots further revealed that AIM1, ASG1, AVT1, DRN1, ELP4, FLO8, FMP10, HMT1, KAR5, MIT1, MRPL32, MSS11, NCP1, NPR1, PEP5, PEX25, RIM8, RIM101, RGT1, SNF8, SPC2, STB6, STP22, TEC1, VID24, VPS20, VTC3, YBL029W, YBL029C-A, YFL054C, YGR161W-C, YIL014C-A, YIR024C, YKL151C, YNL200C, YOR034C-A, and YOR223W controlled biofilm through FLO11 induction. Almost all deletion mutants that were unable to form biofilms in liquid medium also lost the ability to form surface-spreading biofilm colonies (mats) on agar and 69% also lost the ability to grow invasively. The protein kinase A isoform Tpk3p functioned specifically in biofilm and mat formation. In a tpk3 mutant, transcription of FLO11 was induced three-fold compared with wild-type, but biofilm development and cell–cell adhesion was absent, suggesting that Tpk3p regulates FLO11 positive posttranscriptionally and negative transcriptionally. The study provides a resource of biofilm-influencing genes for additional research on biofilm development and suggests that the regulation of FLO11 is more complex than previously anticipated

Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production

Glucagon is secreted from pancreatic a cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in b cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion