Additional file 5: Figure SI-15. of Ribosomal subunit protein typing using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification and discrimination of Aspergillus species

Detailed experimental protocols. Detailed sample preparation procedures. Figure SI-16. Detailed construction procedure of the ribosomal protein biomarker list together with peak assignments. (PPTX 61 kb

Additional file 1: Table SI-1. of Ribosomal subunit protein typing using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification and discrimination of Aspergillus species

The accession number (TrEMBL) of ribosomal protein biomarkers of genome-sequenced strains used in this study. (DOCX 22 kb

Additional file 2: Table SI-2. of Ribosomal subunit protein typing using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification and discrimination of Aspergillus species

Corrected amino acid sequences and relating information of RSPs. (DOCX 80 kb

Additional file 1: of Chronic Kirschsteiniothelia infection superimposed on a pre-existing non-infectious bursitis of the ankle: the first case report of human infection

Nucleoid sequence of the internal transcribed spacer region. (PPTX 45 kb

Additional file 2: of Chronic Kirschsteiniothelia infection superimposed on a pre-existing non-infectious bursitis of the ankle: the first case report of human infection

The result of molecular analysis with GenBank database using the Basic Local Alignment Search Tool (BLAST) algorithm (lcl|Query_85557 is the specimen from the presenting patient). (PDF 100 kb

Cell wall damage followed by remodeling occurs during adaptation to 5-flucytosine stress.

<p>A) <i>C</i>. <i>glabrata</i> KUE100 cells exposed to lyticase stress were harvested in the exponential phase of growth in the absence of stress (♦) or upon 30 min of exposure to 0.7 mg/L 5-flucytosine (■), or in the exponential phase of growth reached upon adaptation to 0.7 mg/L 5-flucytosine (▲). The different cell populations were washed with water and resuspended in 0.1M sodium phosphate buffer at pH 7.5. After addition of 20mg/L lyticase, the decrease in the OD_600nm of the cell suspension was measured periodically and indicated as a percentage of the initial OD_600nm. The indicated values are averages of at least three independent experiments. Error bars represent the corresponding standard deviation. * P<0.05; ***P<0.001. B) Comparison, by spot assays, of the susceptibility of <i>C</i>. <i>glabrata</i> KUE100 cells harvested in the exponential phase of growth in the absence of stress (control) or upon 30 min of exposure to 0.5 mg/L 5-flucytosine (30’ 5-FC), or in the exponential phase of growth reached upon adaptation to 0.5 mg/L 5-flucytosine (exp 5-FC) to Calcofluor White, in MMB agar plates. The inocula were prepared as described in Materials and Methods, and the cell suspensions used to prepare the spots 1:5 (b) and 1:25 (c) dilutions of the cell suspension used for column a. The images are representative of at least three independent experiments.</p

<i>CgFPS1</i> and <i>CgFPS2</i> expression decreases the accumulation of 5-flucytosine in <i>C</i>. <i>glabrata</i> cells.

<p>Time-course accumulation of [³H]-flucytosine in non-adapted KUE100 (♦), KUE100_<i>Δcgfps1</i> (■) and KUE100_<i>Δcgfps2</i> (▲) strains, during cultivation in BM liquid medium in the presence of 3mg/L of unlabelled 5-flucytosine. The indicated accumulation ratio values are averages of at least three independent experiments. Error bars represent the corresponding standard deviation. * P<0.05; **P<0.01.</p

New Mechanisms of Flucytosine Resistance in <i>C</i>. <i>glabrata</i> Unveiled by a Chemogenomics Analysis in <i>S</i>. <i>cerevisiae</i>

<div><p>5-Flucytosine is currently used as an antifungal drug in combination therapy, but fungal pathogens are rapidly able to develop resistance against this drug, compromising its therapeutic action. The understanding of the underlying resistance mechanisms is crucial to deal with this problem. In this work, the <i>S</i>. <i>cerevisiae</i> deletion mutant collection was screened for increased resistance to flucytosine. Through this chemogenomics analysis, 183 genes were found to confer resistance to this antifungal agent. Consistent with its known effect in DNA, RNA and protein synthesis, the most significant Gene Ontology terms over-represented in the list of 5-flucytosine resistance determinants are related to DNA repair, RNA and protein metabolism. Additional functional classes include carbohydrate and nitrogen—particularly arginine—metabolism, lipid metabolism and cell wall remodeling. Based on the results obtained for <i>S</i>. <i>cerevisiae</i> as a model system, further studies were conducted in the pathogenic yeast <i>Candida glabrata</i>. Arginine supplementation was found to relieve the inhibitory effect exerted by 5-flucytosine in <i>C</i>. <i>glabrata</i>. Lyticase susceptibility was found to increase within the first 30min of 5-flucytosine exposure, suggesting this antifungal drug to act as a cell wall damaging agent. Upon exponential growth resumption in the presence of 5-flucytosine, the cell wall exhibited higher resistance to lyticase, suggesting that cell wall remodeling occurs in response to 5-flucytosine. Additionally, the aquaglyceroporin encoding genes <i>CgFPS1</i> and <i>CgFPS2</i>, from <i>C</i>. <i>glabrata</i>, were identified as determinants of 5-flucytosine resistance. <i>CgFPS1</i> and <i>CgFPS2</i> were found to mediate 5-flucytosine resistance, by decreasing 5-flucytosine accumulation in <i>C</i>. <i>glabrata</i> cells.</p></div

The acquaglyceroporins CgFps1 and CgFps2 confer resistance to 5-flucytosine.

<p><b>A)</b> Comparison, by spot assays, of the susceptibility of the <i>C</i>. <i>glabrata</i> KUE100, KUE100_<i>Δcgfps1</i> and KUE100_<i>Δcgfps2</i> strains (B) to 5-flucytosine, in MMB agar plates. <b>B)</b> Fluorescence of exponential-phase BY4741 <i>S</i>. <i>cerevisiae</i> cells, harboring the cloning vector pGREG576 (control) or the pGREG576_<i>CgFPS1</i> plasmids (CgFps1_GFP), after 5h of galactose-induced recombinant protein expression. <b>C)</b> Comparison, by spot assays, of the susceptibility to 5-flucytosine of <i>S</i>. <i>cerevisiae</i> BY4741 and BY4741_<i>Δfps1</i> cells, harboring the cloning vector pGREG576 (v) or the same plasmid expressing the <i>CgFPS1</i> gene, in MMB agar plates. In A) and C) the inocula were prepared as described in Materials and Methods, and the cell suspensions used to prepare the spots 1:5 (b) and 1:25 (c) dilutions of the cell suspension used for column a. The images are representative of at least three independent experiments.</p

Arginine supplementation decreases the inhibitory effect exerted by 5-flucytosine in <i>C</i>. <i>glabrata</i>.

<p>The growth curves of parental <i>C</i>. <i>glabrata</i> KUE100 cells in the absence (□,◊) or presence (■,♦) of 0.5 mg/L 5-flucytosine are compared, upon supplementation (◊,♦) or not (□,■) with 60mg/L arginine. The displayed growth curves are the average of three independent experiments. Error bars represent the corresponding standard deviation. **indicates that there is a statistically significant (P<0.01) difference between the OD_600nm reached in the presence and absence of arginine, at the indicated times points.</p