A family of secreted pathogenesis-related proteins in Candida albicans
Ontology highlight
ABSTRACT:
ORGANISM(S): Candida albicans
SUBMITTER: Steffen Rupp
PROVIDER: E-GEOD-28212 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community. Dual-species biofilms of S. mutans and C. albicans and single-species biofilms of S. mutans were cultivated in 24-well microtitre plates in YNBB medium. Transcriptional profiles of S. mutans in single- and dual-species biofilms were analysed at early (6 h) and late (10 h) logarithmic phase of the biofilm growth, as well as after 24 h when biofilms entered stationary phase. Transcriptional profiles of S. mutans grown in the dual-species biofilms were compared to profiles obtained for single-species biofilm from the same time point. Three biological and one to two technical replicas were used in the microarray study. RNA samples were labeled with Cy3 or Cy5 using the ULS fluorescent labeling kit (Kreatech, Germany). Seven hundred nanograms of Cy3 or Cy5 labeled RNA after fragmentation were hybridized to the microarray at 65M-BM-0C for 17 h using the Agilent hybridization chamber according to the manufacturer's instructions. The arrays were scanned using the Agilent DNA microarray scanner and the raw data were extracted using Agilent Feature Extraction software (v. 10.7).
2014-09-01 | E-GEOD-52543 | biostudies-arrayexpress
Project description:One of the first steps in bacterial cell division is the polymerization of the tubulin-like protein FtsZ at midcell. The dynamics of FtsZ polymerization is regulated by a set of proteins among which ZapA. A zapA mutation does not result in a clear phenotype in Bacillus subtilis. In this study we used a synthetic-lethal screen to find genes that become essential when ZapA is absent. Three transposon insertions were found in yvcL. Deletion of yvcL in a wild type background had only a mild effect on growth, but a yvcL zapA double mutant is very filamentous and sick. This filamentation is caused by a strong reduction in FtsZ polymerization, suggesting that YvcL is involved in an early stage of cell division. YvcL is 25 % identical and 50 % similar to the Streptomyces coelicolor transcription factor WhiA. WhiA is required for septation of aerial hyphae during sporulation. Using GFP fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP on chip assay did not provide clear evidence that YvcL functions as a transcription factor. To gain more insight into the function of YvcL, we searched for suppressors of the filamentous phenotype of a M-bM-^HM-^FyvcL M-bM-^HM-^FzapA mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implemented in the metabolic sensing of cell division. We conclude that YvcL (WhiA) is involved in cell division in B. subtilis through an as yet unknown mechanism. Comparing wild tpe Bascillus subtilus (n=3) with Bascillus Subtilis KS400 (n=2) and Bascillus subtilis KS696 (n=2)
2013-11-28 | E-GEOD-45824 | biostudies-arrayexpress
Project description:The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. Strains in which the rep1 gene is inactivated (M-bM-^HM-^Frep1 strain) are affected in aerial hyphae formation. This makes these strains instrumental to assess changes in global gene expression as a consequence of aerial growth. Microarray analysis revealed that only 31 genes in the M-bM-^HM-^Frep1 SG200 strain had a fold change in expression of >= 2. Twenty-two of these genes are up-regulated and half of them encode small secreted proteins (SSPM-bM-^@M-^Ys) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the M-bM-^HM-^Frep1 SG200 strain encode secreted cysteine-rich proteins (SCRPM-bM-^@M-^Ys). Interestingly, most of the SCRPM-bM-^@M-^Ys are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the M-bM-^HM-^Frep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that only minor changes occur in the expression profile when U. maydis forms aerial structures. Key words: aerial hypha, repellent, hydrophobin-like protein, Ustilago maydis, SSP, SCRP, fungal pathogenicity. To analyze expression changes in aerial hyphae upon deletion of the rep1 gene, strains SG200 and SG200M-bM-^HM-^Frep1 were grown on charcoal nitrate minimal array media supplemented with vitamins and 1% glucose at 22 C for 48 h. For each experiment three biological replicates were performed.
2011-05-01 | E-GEOD-21490 | biostudies-arrayexpress
Project description:Soybean toxin (SBTX) is an antifungal protein from soybeans with broad growth and filamentation inhibitory activity against many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina, and Cerospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute towards the design of novel antifungal drugs and/or for the development of transgenic plants resistant to pathogens. To gain new insights into the mode of action of SBTX, the polymorphic yeast C. albicans was chosen as a model organism, and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses, as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showd that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cells starving despite sufficient nutrient availability in the medium, and it can therefore be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold. For transcriptional profiling, the cells were growth in the presence or absence of SBTX (200 M-BM-5gM-bM-^HM-^YmL-1) in SDB/4.The cultures were grown until they reached an OD600 of approximately 0.5, after 16 and 18 h, for untreated and SBTX-treated cells. The cells were harvested by centrifugation (3000M-bM-^@M-"g at 25M-BM-0C), snap-frozen in liquid nitrogen and stored at -80M-BM-0C. Each pair of samples (untreated and SBTX-treated cells) constituted a single experiment, and two biologically independent experiments were carried out.
2013-12-31 | E-GEOD-47364 | biostudies-arrayexpress
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 ∆rbe1 or hyphae of ∆rbt4 deletion strains, respectively, indicating a compensatory function of both proteins. In a ∆rbe1/∆rbt4 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 ∆rbe1/∆rbt4 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.
2013-03-25 | GSE28212 | GEO
Project description:Biofilm formation is considered the most important factor involved in pathogenicity of Staphylococcus epidermidis.We investigated the role of two-component signal transduction system (TCS) srrAB, which was up-regulated under micro-aerobic condition, in the growth and biofilm formation of S. epidermidis.AsrrA-deficient mutant (M-bM-^HM-^FsrrA) derived from S. epidermidis1457 (SE1457), exhibited dramatic reduction in growth and biofilm formation underboth aerobic and micro-aerobic conditions, and more sensitive to several different types of antimicrobial agents, H2O2 and SDS. In New Zealand Rabbit model of S. epidermidis biofilm infection, M-bM-^HM-^FsrrA hardly formed biofilm compared to that of SE1457. Phenotypic alteration was restored to the wide-type levelwhen srrAB were complemented into M-bM-^HM-^FsrrA. Further study found that the initial adherence capacity and production of polysaccharide intercellular adhesion (PIA) in M-bM-^HM-^FsrrA were decreased, while extracellular DNA (eDNA) was increased. Transcriptional Analysisby qRT-PCR demonstrated that expression level of icaRin M-bM-^HM-^FsrrA was up-regulated compared to that of SE1457 under aerobic condition, while down-regulated under micro-aerobic condition;icaA and altE were down-regulated under both conditions. Expression of genes involved in respiratory metabolism, such as qoxB(quinol oxidase polypeptide II), ctaA(heme A synthase), and pfl(pyruvate formatelyase), etc. were down-regulated in M-bM-^HM-^FsrrAunder both conditions. Electrophoretic mobility shift assay (EMSA) revealed that phosphorylated SrrA bound to the promoter regions of icaR, icaA, atlE, qoxB,ctaA, andpflB just like binding its own promoter region srr. Taken together, our results demonstrate that srrAB may provide a mechanistic link between respiratory metabolism, environmental signals, and regulation of biofilm formation in S. epidermidis. Microarrays covering different S. epidermidis genomes were used to assess the impact of the two component system srrAB on growth and biofilm formation, by comparing WT with srrA mutant transcriptomes
2014-11-21 | E-GEOD-47101 | biostudies-arrayexpress
Project description:The strain bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H] improved salt resistance of bdf1M-bM-^HM-^F. To gain further insight into the mechanism of BDF1 in suppressing bdf1M-bM-^HM-^F salt sensitivity, DNA microarray analysis was performed to determine the reason for the salt sensitivity of bdf1M-bM-^HM-^F cells and the process of how coexpression of SIR2 and BDF2 improves salt resistance. Transcriptomic analysis under salt treatment (0.6 mol.L-1 NaCl for 45 min) was performed using three different strains: bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H], bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242] and bdf1M-bM-^HM-^F[pRS316][pYX242]. The transcription of 3244 genes were significantly changed( > 2-fold) in bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H] compared with bdf1M-bM-^HM-^F[pRS316][pYX242] upon NaCl stress (0.6 mol.L-1 NaCl for 45 min). Only 281 genes were significantly changed ( > 2-fold) in bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242] compared with bdf1M-bM-^HM-^F[pRS316][pYX242] upon NaCl stress (0.6 molM-bM-^HM-^YL-1 NaCl for 45 min). BF2:bdf1M-bM-^HM-^F[pRS316][pYX242]. BF2B: bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242]. BF2S:bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H]. BF2B vs BF2 under salt treatment (0.6 mol.L-1 NaCl for 45 min); BF2S vs BF2 under salt treatment (0.6 mol.L-1 NaCl for 45 min).
2014-05-26 | E-GEOD-47367 | biostudies-arrayexpress
Project description:Candida albicans is an opportunistic pathogenic yeast that is commensally found in variety of host niches. Rhb1 serves as an positive regulator of Tor1 kinase which is the central component of the TOR signaling in controlling several virulence factors. Here, we used microarray to determined changes in transcript profile while the cell lacks the RHB1 gene. 10 array generated by six batchs of individual experiment (from cell isolation to culture); each included one flask of wild type culture (SC5314) and one flask of rhb1M-bM-^HM-^F (CCT-D1)mutant culture.
2012-03-15 | E-GEOD-29128 | biostudies-arrayexpress
Project description:To investigate the regulatory mechanisms governing the malignant signature of different gliomas we analyzed microRNA expression profiles in human tumor samples of world health organization (WHO) grade I (benign tumors), II (low grade tumors) and IV (high grade tumors) and from primary cultures obtained from tumor samples of grade II and IV. Patients This study included tumor samples histologically verified as astrocytic gliomas obtained from patients who had undergone craniotomy for microsurgical tumor removal. According to the revised WHO classification, tumors were diagnosed as: grade I or pilocytic astrocytomas; grade II or diffuse fibrillary astrocytomas; grade IV or glioblastoma multiforme. Primary cell cultures from grade II and grade IV gliomas were also obtained and miRNA expression in these cultures were analyzed RNA extraction Total RNA, including small RNA, was isolated from tissue samples using the mirVanaTM miRNA Isolation Kit (Ambion) following the standard protocol. The quantity and quality of the purified RNA was evaluated by spectrophotometric analysis and electrophoresis on denaturing gel of acrylamide. Multiplex Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) The miRNAs were first converted to cDNA using Multiplex RT for TaqMan Array Human MicroRNA Panel. The RT Master mix included 100 mM each of dNTPs , 50 U/ml MultiScrabe reverse transcriptase (Applied Biosystems), 20 U/M-BM-5l RNase inhibitor (Applied Biosystems) and 10X RT Buffer. The 10 M-BM-5l reactions, including 7 M-BM-5l of RT master mix, 2 M-BM-5l of purified microRNA and 1 M-BM-5l of Multiplex RT Human primer pool (Applied Biosystem), were incubated in ice for 5 min and then in a thermal cycler for 30 min at 16M-BM-0C, 30 min at 42M-BM-0C, 5 min at 85M-BM-0C, and then hold at 4M-BM-0C. miRNA levels were normalized to the expression of small nucleolar RNAs, RNU44, RNU48 and RNU6B. All reverse transcriptase reactions, including no-template controls and RT controls, were run in duplicate. Real-time PCR was performed using a standard TaqMan PCR kit procedure on an M-bM-^@M-^\Real Time Fast 7900 HTM-bM-^@M-^] PCR System (Applied Biosystems). The 100 M-BM-5l PCR included 50 M-BM-5l RT product (before diluited 1:60) and 50 M-BM-5l TaqMan Universal PCR Master Mix (2X) (Applied Biosystems). The total volume were loaded into Card TaqMan Low Density Array Human MicroRNA Panel (Applied Biosystem) including a total of 384 human microRNAs publicated on databases www.sanger.ac.uk. The reaction cards was runned at 50M-BM-0C for 2 min and 95M-BM-0C for 10 min, followed by 40 cycles of 97M-BM-0C for 30s and 59,7M-BM-0C for 1 min. All reactions were run in triplicate. Analysis of data was performed using the SDS 2.3 software using the 2-M-bM-^HM-^FM-bM-^HM-^FCt (relative quantitative) method . The M-bM-^HM-^FCt of every miRNA was determined in relation to the endogenous control RNA U6 that was invariably expressed in all samples. The M-bM-^HM-^FM-bM-^HM-^FCt value was determined in relation to the calibrator, namely the normal brain tissue. Resulting data were grouped according to the tumor grading e selectioned using a cut-off value of 3. Results were expressed as M-bM-^@M-^\fold changeM-bM-^@M-^] over normal brain tissue. We analyzed two samples of grade I, two samples of grade II, two samples of grade IV gliomas. Four samples form norma brain were used as norma control. Primary cell cultures form grade II and grade IV samples were used for the analysis. All reverse transcriptase reactions, including no-template controls and RT controls, were run in triplicate.
2013-09-18 | E-GEOD-50924 | biostudies-arrayexpress
Project description:To gain insight into the targets of the PaMpk1 and PaMpk2 MAPK, we here determine the transcription profile of the mutants inactivated for PaMpk1, PaMpk2 and PaNox1 during vegetative growth at stages where they are required for proper development, in comparison with wild type. Microarrays representing 10556 coding sequences (CDS) predicted by the P. anserina genome project were used to estimate the transcription profiles (transcriptomes) during vegetative growth of three-day-old homokaryotic cultures of M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and the IDC343 PaNox1 mutants in comparison to wild type. Three zones corresponding to three developmental stages can be defined in such wild-type culture. From the growing edge the colony, the first five milimeters (zone 1) are constituted of hyphae lacking pigment and growing on the substrate. In this zone, very few hyphae are aerial and appressorium-like structures are not differentiated. In the second intermediate zone (zone 2), profuse aerial hyphae and appressorium-like structures are differentiated. This zone is about 7 mm wide and corresponds to the area where most perithecia are differentiated during sexual development of heterokaryotic mycelia. The zone that is most distal from the edge lack aerial hyphae (zone 3) accumulates a larger amount of pigment and differentiates few perithecia during sexual reproduction. It is about 1 cm wide. These three zones are easily visualized by the naked eye for sample recovery. The M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and the IDC343 PaNox1 mutants lack a clear zonation, since the three genes are involved in the signalling of stationary phase . Materials from these mutants were nonetheless recovered as three samples corresponding to the same zones, based mostly on the distance from the growing edge but also from subtle morphological differences. With GPL10116 : reference design, 4 strains (WT, M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and IDC343 PaNox1 with 3 growth conditions (zone1,2 and 3) each with four biological replicates, the common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5
2012-06-11 | E-GEOD-21331 | biostudies-arrayexpress