Rved in wild type (Figure 4B), tup1D/tup1D (Figure

Rved in wild type (Figure 4B), tup1D/tup1D (Figure 4D) or rim101D/rim101D (Figure 4F) cells when compared with their respective parental control strains (Figure 4A, C, E).SBTX-Title Loaded From File induced ultrastructural alterations in C. albicans cellsTEM of wild type cells revealed condensation and shrinkage of a heavily granulated cytosol and increased vacuolisation in SBTXtreated (400 mgNmL21) C. albicans cells. Structural disorganisation and loss of cytoplasmic content were also observed in SBTXtreated cells (Figure 5B, C) when compared with control cells (Figure 5A).DiscussionPreviously, we showed that SBTX inhibited morphological development in plant and human pathogenic fungi and that the presence of SBTX increased the membrane permeability of fungal cells [5]. In this work, we used TEM analysis of C. albicans cells to show that prolonged exposure to SBTX resulted in condensation and shrinkage of a heavily granulated cytosol, increased vacuolisation, loss of normal cell structure and loss of cytoplasmic content. The SBTX-induced modifications in C. albicans were even more prominent than those observed in P. membranifaciens [5]. To further investigate the transcriptional basis for the effects induced by SBTX and to shed light on its mechanism of action, gene expression analysis was performed on SBTX-treated and untreated C. albicans SC5314. Under the conditions investigated, neither culture produced hyphae and the SBTX-treated culture reached stationary phase at an OD600 that was approximately 50 of that at which untreated cells reached stationary phase. At the 18 h time point, several indicators of the transition to stationary phase were observed in the SBTX-treated cells, e.g., the downregulation of 1315463 PSF1, RIM1, HHT2, HHT21 and HHF1. As expected from the TEM analysis and previous phenotypic results, pathway analysis of differentially expressed genes during late log phase showed that several morphogenesis-related pathways and general stress responses were differentially regulated. Furthermore, nutrient sensory and uptake pathways were differentially activated in untreated and SBTX-treated cells. Our first observation was that several starvation signals were activated. Intracellular levels of glucose appeared to be low, as the high-affinity glucose transporter HGT1 [25] was activated and several other Mig1-regulated genes were derepressed. This derepression was most dramatic for enzymes of the Leloir pathway (GAL1 and GAL10). Additionally, genes involved in other metabolic pathways indicating starvation were differentially expressed: Maltose (MAL31) and glycerol import (HGT10) were activated, gluconeogenesis was induced as indicated by PCK1 derepression under low intracellular glucose levels [26], [27], glyoxylate cycle genes (ICL1 and MLS1) were activated and the gene encoding 3-hydroxyacyl-CoA Title Loaded From File epimerase (FOX2), an enzyme essential in lipid oxidation, was also induced, indicating that exposure of C. albicans to SBTX must have led to fatty acidFigure 5. Transmission electron microscopy (TEM) of C. albicans in the presence of SBTX. Representative micrographs of single cells observed by TEM of C. albicans cultured in the absence (A) or presence (B, C) of SBTX (400 mg?mL21). Asterisks indicate condensation and shrinkage of a heavily granulated cytosol and increased vacuolisation in C. albicans treated with SBTX. doi:10.1371/journal.pone.0070425.gdisplayed differential regulation at 16 h, the filamentationassociated genes TUP1, ALS4, SHA3 and ALS1 were u.Rved in wild type (Figure 4B), tup1D/tup1D (Figure 4D) or rim101D/rim101D (Figure 4F) cells when compared with their respective parental control strains (Figure 4A, C, E).SBTX-induced ultrastructural alterations in C. albicans cellsTEM of wild type cells revealed condensation and shrinkage of a heavily granulated cytosol and increased vacuolisation in SBTXtreated (400 mgNmL21) C. albicans cells. Structural disorganisation and loss of cytoplasmic content were also observed in SBTXtreated cells (Figure 5B, C) when compared with control cells (Figure 5A).DiscussionPreviously, we showed that SBTX inhibited morphological development in plant and human pathogenic fungi and that the presence of SBTX increased the membrane permeability of fungal cells [5]. In this work, we used TEM analysis of C. albicans cells to show that prolonged exposure to SBTX resulted in condensation and shrinkage of a heavily granulated cytosol, increased vacuolisation, loss of normal cell structure and loss of cytoplasmic content. The SBTX-induced modifications in C. albicans were even more prominent than those observed in P. membranifaciens [5]. To further investigate the transcriptional basis for the effects induced by SBTX and to shed light on its mechanism of action, gene expression analysis was performed on SBTX-treated and untreated C. albicans SC5314. Under the conditions investigated, neither culture produced hyphae and the SBTX-treated culture reached stationary phase at an OD600 that was approximately 50 of that at which untreated cells reached stationary phase. At the 18 h time point, several indicators of the transition to stationary phase were observed in the SBTX-treated cells, e.g., the downregulation of 1315463 PSF1, RIM1, HHT2, HHT21 and HHF1. As expected from the TEM analysis and previous phenotypic results, pathway analysis of differentially expressed genes during late log phase showed that several morphogenesis-related pathways and general stress responses were differentially regulated. Furthermore, nutrient sensory and uptake pathways were differentially activated in untreated and SBTX-treated cells. Our first observation was that several starvation signals were activated. Intracellular levels of glucose appeared to be low, as the high-affinity glucose transporter HGT1 [25] was activated and several other Mig1-regulated genes were derepressed. This derepression was most dramatic for enzymes of the Leloir pathway (GAL1 and GAL10). Additionally, genes involved in other metabolic pathways indicating starvation were differentially expressed: Maltose (MAL31) and glycerol import (HGT10) were activated, gluconeogenesis was induced as indicated by PCK1 derepression under low intracellular glucose levels [26], [27], glyoxylate cycle genes (ICL1 and MLS1) were activated and the gene encoding 3-hydroxyacyl-CoA epimerase (FOX2), an enzyme essential in lipid oxidation, was also induced, indicating that exposure of C. albicans to SBTX must have led to fatty acidFigure 5. Transmission electron microscopy (TEM) of C. albicans in the presence of SBTX. Representative micrographs of single cells observed by TEM of C. albicans cultured in the absence (A) or presence (B, C) of SBTX (400 mg?mL21). Asterisks indicate condensation and shrinkage of a heavily granulated cytosol and increased vacuolisation in C. albicans treated with SBTX. doi:10.1371/journal.pone.0070425.gdisplayed differential regulation at 16 h, the filamentationassociated genes TUP1, ALS4, SHA3 and ALS1 were u.