Project description:Analyzing culture supernatants of yeast and hyphal cells of Candida albicans by mass spectrometry, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome of this human pathogen. By sequence homology, we assigned three yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, in addition to Rbe1p and Rbt4p to a novel family of proteins. Correspondent with our secretome analysis RBE1 was expressed in blastospores and opaque cells, whereas transcription was down-regulated in hyphae. On the contrary, RBT4 was up-regulated in hyphae and down-regulated in opaque cells. Remarkably, transcription of RBT4 and RBE1 was each up-regulated in blastospores of M-bM-^HM-^Frbe1 or hyphae of M-bM-^HM-^Frbt4 deletion strains, respectively, indicating a compensatory function of both proteins. In a M-bM-^HM-^Frbe1/M-bM-^HM-^Frbt4 double deletion strain, genome-wide transcriptional analysis showed differential transcription of a limited set of genes that are also implicated in virulence and oxidative stress response. In this context, deletion of RBE1 or RBT4 in a clinical C. albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in the M-bM-^HM-^Frbe1/M-bM-^HM-^Frbt4 double deletion strain, where virulence was strongly affected. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leukocytes (neutrophils). Therfore, our data suggest that the crucial contribution of both C. albicans pathogenesis-related proteins for in vivo virulence results at least partially from reduced survival in phagocytes. Experiments were performed under blastospore (YPD) as well as hyphae (alpha-MEM)-inducing conditions. In total, three biological replicates were performed for each condition. All experiments were performed as dye swaps. Thus, in total six arrays have been hybridzed for each comparison (alpha-MEM and YPD, respectively). Hybridization experiments included a reference strain (C. albicans SC5314) and a double deletion strain (C. albicans MRC27). The array included one technical replicate of each probe.

Project description:The yeast protein kinases Sat4/Hal4 and Hal5 are required for the plasma membrane stability of the K+ transporter Trk1 and some amino acid and glucose permeases. The transcriptomic analysis presented here indicates alterations in the general control of both nitrogen and carbon metabolism. Accordingly, we observed reduced uptake of methionine and leucine in the hal4 hal5 mutant. This decrease correlates with activation of the Gcn2-Gcn4 pathway, as measured by expression of the lacZ gene under the control of the Gcn4 promoter. However, with the exception of methionine biosynthetic genes, few amino acid biosynthetic genes are induced in the hal4 hal5 mutant, whereas several genes involved in amino acid catabolism are repressed. Concerning glucose metabolism, we found that this mutant exhibits derepression of respiratory genes in the presence of glucose, leading to an increased activity of mitochondrial enzymes, as measured by SDH activity. In addition, the reduced glucose consumption in the hal4 hal5 mutant correlates with a more acidic intracellular pH and with low activity of the plasma membrane H+-ATPase. As a compensatory mechanism for the low glycolytic rate, the hal4 hal5 mutant overexpresses the HXT4 high affinity glucose transporter and the hexokinase genes. These results indicate that the hal4 hal5 mutant presents defects in the general control of nitrogen and carbon metabolism, which correlate with reduced transport of amino acids and glucose, respectively. A more acidic intracellular pH may contribute to some defects of this mutant. Four biological replicates were used to assess diferentially expression between wild type yeast strain and the hal4hal5 mutant strain for three sample sets: 1) cells grown in YPD pH 4.5 and BY4741 genetic background, 2) cells grown in YPD pH 4.5 and W303 genetic background, 3) cells grown in YPD pH 6.0 and W303 genetic background. Differentially expressed genes were identified using one-class significant analysis of microarrays (SAM; Tusher et al, 2004)

Project description:Fungal infections are a serious health problem in clinics especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes and echinocandines. Due to emerging resistance to standard therapy and significant side effects and high costs for several antifungals.,there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities we previously screened a compound library, using a new type of activity-selectivity (AS) assay analysing both the antifungal activity and the compatibility with human cells at the same time. One compound, ((S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole (EMC120B12)), showed high antifungal activity against several species of pathogenic yeasts including C. glabrata and C. krusei, species which are highly refractory to antifungals, especially to the commonly used azoles. Here we could show by transcriptional profiling and sterol analysis that the target of this new antifungal compound is the ergosterol pathway. The effects of EMC120B12 on sterol biosynthesis mimic those of fluconazole, strongly indicating that EMC120B12 also targets ERG11 like the azols. But not only the marker sterol 14 methylergosta 8,24(28) dien 3β,6α diol accumulated in C. krusei under EMC120B12 treatment, but also hitherto unknown related sterols. The novel sterols have a 3β,6α diol structure. Furthermore, this is the first time that a benzimidazole structure has been shown to result in a block of the sterol pathway by accumulating marker sterols connected to ERG11 inactivation. In total, three biological replicates were performed. All experiments were performed as dye swaps. Thus, in total 18 arrays have been hybridzed. Hybridization experiments included an untreated reference sample and a sample of cells treated with either ((1S)-1-[1-(3-chlorobenzyl)-1H-benzimidazol-2-yl]-2-methylpropyl-amine) (EMC120B12), Fluconazole or Nocodazole. The array included one technical replicate of each probe.

Project description:Background: Minimal change nephrotic syndrome (MCNS) is considered to be associated with T cell dysfunction, via unknown mechanisms. Experimental observations suggest that some humoral factors alter the permeability of glomerular filtration barrier. However, the nature of such factors remains still uncertain. Methods: Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy. Results: Out of 24,446 genes screened, 33 genes were up-regulated (at least 1.5-fold) in PBMC from these MCNS patients during the nephrosis phase. Up-regulated genes mainly encoded proteins involved in signal transduction and cytokine response. For further examination, we selected two genes encoding provable secretary proteins, chemokine (C-C) ligand 13 (also known as monocyte chemotactic protein-4) (CCL13) and a novel galectin-related protein (HSPC159). The results of RT-PCR showed that expressions of CCL13 and HSPC159 mRNA in nephrosis PBMC samples are higher than those in remission PBMC samples from all 20 MCNS patients examined. On the other hand, these mRNA expression patterns were variable among six patients with membranous nephropathy. Conclusions: We conclude that CCL13 and HSPC159 mRNA expressions in PBMC is up-regulated in MCNS patients during the nephrosis phase. These expression changes in PBMC might be involved in the pathophysiologic processes of MCNS. Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy.

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Yeast in blood

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