Proteomic analysis of Candida albicans after serial systemic infection in murine model
ABSTRACT: This study assessed proteomic profile of Candida albicans after serial systemic infection in a murine model. The animals were infected initially by wild-type C. albicans SC5314 (WT) with an inoculum of of 3.5x105 cells via lateral tail vein. Then, five days post-infection, the animals were euthanized and their kidneys were removed, homogenized in lysis buffer, plated on SDA and incubated for 24 h at 35 °C. Colonies recovered from infected kidney were used to prepare inoculum for the subsequent infections as described for WT, totalizing five serial passages (P1-P5) and they were also used to protein extraction. By LC-MS/MS, 479 proteins were identified, with 56 proteins statistically significant in abundance in P1, 29 proteins in P3 and 97 proteins in P4. Regarding biological processes, the majority of proteins were related to carbohydrate metabolism, stress response and amino acid metabolism. The proteins were also categorized according to their potential role in virulence factors such as biofilm production, yeast-to-hyphae transition, phenotypic switching, proteins related to stress response and uncharacterized proteins. Therefore, serial infection associated with proteomic approach enabled to deepen the knowledge about host-pathogen interaction.
Project description:The dataset contains ChIP-Seq data of the Set3 and Hos2 proteins in Candida albicans, assayed in two morphological phases (yeast and hypha). The Set3 and Hos2 proteins in the respective strains carry 9myc epitopes and ChIP was performed with an anti-myc antibody. Included samples are the following: 1 input and 1 ChIP sample of an untagged wild type strain as negative control assayed in the yeast phase, 1 input and 3 ChIP biological replicates of the Set3-9myc strain in the yeast phase, 1 input and 2 ChIP biological replicates of the Set3-9myc strain in the hypha phase, 1 input and 2 ChIP biological replicates of the Hos2-9myc strain in the yeast phase, 1 input and 2 ChIP biological replicates of the Hos2-9myc strain in the hypha phase, 1 input and 3 ChIP biological replicates of Set3-9myc in a set1delta/delta background in the yeast phase. ChIP-Seq was performed of Candida albicans strains in two morphological phases (yeast and hypha). Yeast-phase cells were grown to the exponential phase in YPD at 30C. Hyphal differentiation was induced by resuspending the cells in YPD+20% Fetal Calf Serum and a shift of the growth temperature to 37C. Induction was performed for 30 minutes. Cells were crosslinked with 1% formaldehyde for 15 minutes at room temperature.
Project description:Sfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1,YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. Indeed, we show that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central “switch on/off” proteins to coordinate the regulation of C. albicans morphogenesis. ChIP was performed in 2 independently grown C. albicans sfl1 or sfl2 homozygous mutant strains expressing (sfl1-CaEXP-SFL1-HA or sfl2-CaEXP-SFL2-HA, respectively) or not (sfl1-CaEXP or sfl2-CaEXP, respectively) SFL1-HA or SFL2-HA (-HA, 3'-triple-HA-tagged alleles of SFL1 or SFL2) under the control of a methionine-repressible promoter (Total samples = 8; 2xCaEXP-SFL1-HA, 2xCaEXP-SFL2-HA, 2xCaEXP control for SFL1-HA ChIP and 2xCaEXP control for SFL2-HA ChIP).
Project description:We report that a transcriptional regulator that originated in the lineage that gave rise to multiple host-associated Candida species is a key component of the circuitry that governs the C. albicans cell surface composition. Specifically, we show that the transcription regulator ZCF21 controls the expression of genes encoding multiple cell surface proteins and cell wall modifying enzymes. Transcriptome (RNA-seq) analysis of 2 Candida albicans strains (reference strain and zcf21 deletion mutant) grown under 4 culture conditions (YPD broth at 30°C for 24h; YPD broth at 30°C in the presence of 15 mM caffeine for 24h; Todd-Hewitt agar at 37°C for 24h; Todd-Hewitt agar at 37°C for 24h in the presence of 5 mM caffeine).
Project description:Candida albicans, a major opportunistic fungal pathogen is frequently found together with Streptococcus mutans in dental biofilms associated with severe childhood tooth-decay, a prevalent pediatric oral disease. Previous studies have demonstrated that S. mutans and C. albicans synergizes virulence of plaque-biofilms in vivo. However, the nature of this bacterial-fungal relationship in this cross-kingdom biofilm remains largely uncharacterized. Using iTRAQ based quantitative proteomics, we found that proteins associated with carbohydrate metabolism such as alpha-1,4 glucan phosphorylase, Hexokinase-2, Isocitrate lyase and malate synthase were significantly upregulated in C. albicans in the mixed-species biofilms (P<0.05). C. albicans proteins associated with growth/morphogenesis such as pH-responsive protein-2, Fma1p and Hsp21 were also induced. Conversely, S. mutans proteins in the tricarboxylic acid cycle such as citrate synthase and in the pentose phosphate pathway such as Ribose-5-phosphate isomerase A as well as proteins associated with sugar transport systems were upregulated indicating enhanced carbohydrate metabolism. Interestingly mixed-species biofilm microenvironment had a lower pH than S. mutans single-species biofilms. This observation was supported by proteomics, wherein proteins associated with lactate and formate assimilation such as Glyoxalase and putative NADPH-dependent methylglyoxal reductase proteins were significantly upregulated in the mixed-species biofilms (P<0.05). Furthermore, we unexpectedly found that S. mutans derived glucosyltransferase B (GtfB), responsible for co-adhesion via glucans, can also contribute to C. albicans growth and carbohydrate metabolism by providing glucose and fructose from sucrose breakdown. These findings demonstrate synergistic bacterial-fungal interactions within mixed-species biofilms and a novel GtfB cross-feeding role. Taken together, quantitative proteomics provides new insights into this virulent cross-kingdom oral biofilm.
Project description:The diploid fungal pathogen Candida albicans is a highly heterozygous organism, with numerous non-synonymous substitutions often seen within two alleles. RNA-sequencing of the wild-type strain SC5314 has revealed 233 genes with significant levels of allelic expression imbalance. Overall percentage protein identity comparisons were significantly lower in these differentially expressed alleles. This suggests that two different, perhaps functionally divergent, proteins are being expressed at significantly different quantities by the two alleles of a single gene. Previously, gene expression levels have been correlated with structural factors such as GC content, ORF length and codon usage. Here, these factors were first correlated with overall gene expression data to decipher the relationship they have with gene expression in Candida albicans. These relationships were then used to assess the contribution of these factors to allelic expression imbalance. GC content and codon usage did not differ significantly in differentially expressed alleles whereas ORF length was found to be significantly lower in the allele with lowest expression. This surprising result goes against the overall trend observed between length and gene expression. Differences in GC content and ORF length between alleles correlated strongly with percentage protein identity, suggesting an indirect link between these factors and allelic expression imbalance. One sample (SC5314: wild-type strain) assessed in triplicate and compared to the reference diploid genome
Project description:Although Candida albicans and Candida dubliniensis are most closely related, both species significantly behave differently with respect to morphogenesis and virulence. In order to gain further insight into the divergent routes for morphogenetic adaptation in both species, we investigated qualitative along with quantitative differences in the transcriptomes of both organisms by cDNA deep sequencing. Following genome-associated assembly of sequence reads we were able to generate experimentally verified databases containing 6016 and 5972 genes for C. albicans and C. dubliniensis, respectively. About 95% of the transcriptionally active regions (TARs) contain open reading frames while the remaining TARs most likely represent non-coding RNAs. Comparison of our annotations with publically available gene models for C. albicans and C. dubliniensis confirmed approximately 95% of already predicted genes, but also revealed so far unknown novel TARs in both species. Qualitative cross-species analysis of these databases revealed in addition to 5802 orthologs also 399 and 49 species-specific protein coding genes for C. albicans and C. dubliniensis, respectively. Furthermore, quantitative transcriptional profiling using RNA-Seq revealed significant differences in the expression of orthologs across both species. We defined a core subset of 84 hyphal-specific genes required for both species, as well as a set of 42 genes that seem to be specifically induced during hyphal morphogenesis in C. albicans. Species specific adaptation in C. albicans and C. dubliniensis is governed by individual genetic repertoires but also by altered regulation of conserved orthologs on the transcriptional level. We investigated qualitative along with quantitative differences in the transcriptomes of both organisms by cDNA deep sequencing. In a first step, we reevaluated the in silico predicted gene models by collecting experimental data using FLX - technology for sequencing strand-specific and normalized cDNA libraries derived from blastospores and hyphae. In the second step, quantitative RNA-Seq (GAIIX) was applied to C. albicans hyphal cells and C. dubliniensis blastospore and hyphal cells to complement reevaluation of the gene models with FLX data as well as to measure differential gene expression across the species with two biological replicates.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare transcriptome profiling of control of C. albicans ATCC 10231 (RNA-seq) to transcriptome profiling of garlic oil-treated C. albicans ATCC 10231 and to evaluate protocols for optimal high-throughput data analysis Methods:SDB medium, C. albicans cells and garlic oil were added to two separate 50 mL conical flasks. The initial cell concentrations in culture were both 105 CFU/mL. The cultures were incubated in a water bath shaker at 28 ºC with shaking at 150 rpm for 12 h. Garlic oil or PBS buffer were then added to the cultures to make the concentrations of garlic oil arrived at 0 (control) and 1.25 μl/mL, respectively. The cultures were continuously incubated at the same conditions for 5 h. The cells were then sampled and centrifuged. The cell precipitates in the control and 1.25 μl/mL garlic oil groups were quickly separately frozen at -80 ºC. Then total RNA were repared from the cell pellets. Results:The RNA sequencing results showed that many genes in C. albicans exposed to garlic oil were differentially expressed. Nearly three thousand genes were differentially expressed, with either an increase or decrease of more than twofold. Most of them were down regulated while a small number were upregulated. Conclusions: Our study indicated that garlic oil induced differential expression of some critical genes, such as genes for cellular response to drugs, oxidation-reduction processes, pathogenesis, and the cellular response to starvation. Moreover, the differentially expressed genes were mainly clustered in 19 KEGG pathways, such as oxidative phosphorylation, spliceosome, cell cycle, protein processing in endoplasmic reticulum, pyrimidine metabolism, meiosis, RNA transport, ribosome biogenesis, and RNA degradation. The mRNA profiles of C. albicans ATCC 10231 (ck) and garlic oil-treated C. albicans ATCC 10231 (sample1) were generated by deep sequencing, in one time, using Illumina Higseq 2500.
Project description:Candida albicans demonstrates three main growth morphologies yeast, pseudohyphal and true hyphal forms. Cell separation is distinct in these morphological forms and the process of separation is closely linked to the completion of mitosis and cytokinesis. In Saccharomyces cerevisiae the small GTPase Tem1 is known to initiate the mitotic exit network, a signalling pathway involved in signalling the end of mitosis and initiating cytokinesis and cell separation. We have characterised the role of Tem1 in C. albicans, and demonstrate that it is essential for mitotic exit and cytokinesis, and that this essential function is signalled through the kinase Cdc15. Consistent with its role in activating the mitotic exit network Tem1 localised to spindle pole bodies in a cell cycle dependent manner. Cells depleted of Tem1 displayed highly polarised growth but ultimately fail to complete cytokinesis and re-enter the cell cycle following nuclear division. At the transcriptional level genes downregulated following the depletion of Tem1 where significantly enriched for genes whose expression peaks early in the cell cycle and for those associated with glycolysis. For expression analysis triplicate RNA samples were generated from exponentially growing C. albicans yeast cells and from cells following 8 h inhibition of TEM1 expression. Gene expression was determined using RNA-seq on the Illumina HiSeq 2000 platform.
Project description:Recent studies have shown that the transcriptional landscape of the pleiomorphic fungus Candida albicans is highly dependent upon growth conditions. Here using a dual RNA-seq approach we identified 299 C. albicans and 72 Streptococcus gordonii genes that were either up- or down-regulated specifically as a result of co-culturing these human oral cavity microorganisms. Seventy five C. albicans genes involved in responses to chemical stimuli, regulation, homeostasis, protein modification and cell cycle were statistically (P ≤0.05) upregulated, while 36 genes mainly involved in transport and translation were down-regulated. Upregulation of filamentation-associated TEC1 and FGR42 genes, and of ALS1 adhesin gene, concurred with previous evidence that the C. albicans yeast to hypha transition is promoted by S. gordonii. Increased expression of genes required for arginine biosynthesis in C. albicans was potentially indicative of a novel oxidative stress response. The transcriptional response of S. gordonii to C. albicans was less dramatic, with only eight S. gordonii genes significantly (P ≤0.05) up-regulated ≥ twofold (glpK, rplO, celB, rplN, rplB, rpsE, ciaR, and gat). The expression patterns suggest that signals from S. gordonii cause a positive filamentation response in C. albicans, while S. gordonii appears to be transcriptionally less influenced by C. albicans. Five Samples; Sample 1 - Candida albicans cells grown in hypha inducing conditions for two hours; Sample 2 - Candida albicans cells grown in hypha-inducing conditions for two hours before co-culture with Streptococcus gordonii cells for one hour in a 2:1 rato; Sample 3 - Candida albicans cells grown in hypha-inducing conditions for two hours before culture in Streptococcus gordonii media for one hour; Sample 4 - Candida albicans cells grown in hypha inducing conditions for two hours, filtered to remove Candida albicans cells and media added to Streptococcus gordonii cells for one hour; Sample 5 - Streptococcus gordonii cells alone for one hour. All samples extracted and sequenced in biological triplicate using Illumina HiSeq2500. Samples 1, 2 and 3 aligned to the reference genome for Candida albicans and Samples 2, 4 and 5 aligned to the reference genome for Streptococcus gordonii.