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.

Project description:Oral streptococci, including Streptococcus gordonii, and Actinomyces naeslundii, are consistently found to be the most abundant bacteria in the early stages of dental plaque accumulation. These organisms interact physically (coaggregate) in vitro and in vivo. We hypothesized that coaggregation between S. gordonii and A. naeslundii leads to changes in gene expression in the partner organisms. Furthermore, we predicted that coaggregation-induced changes in phenotype contribute to the success of streptococci and actinomyces in dental plaque. To assess the responses of S. gordonii to coaggregation with A. naeslundii, RNA was extracted from S. gordonii cells 3 h after inducing coaggregation with A. naeslundii or from equivalent S. gordonii monocultures. The two RNA populations were reverse transcribed and compared by competitive hybridization with an S. gordonii genomic microarray. The most striking feature of the response to coaggregation was a profound change in expression of S. gordonii genes involved in arginine biosynthesis and transport. Subsequent experiments demonstrated that coaggregation with A. naeslundii stabilizes arginine biosynthesis in S. gordonii and enables growth under low-arginine conditions, such as those present in human saliva. Keywords: Cell-cell interaction The S. gordonii microarrays consist of 2195 70-mer oligonucleotides representing 2151 open reading frames, each repeated six times on the array. Chemically defined medium (CDM), was based in Tereleckyj’s FMC with minor modifications (Jakubovics et al., 2008). For coaggregate cultures, concentrated suspensions of S. gordonii DL1 (Challis) and A. naeslundii MG1 in CDM were mixed, vortexed and diluted to 1 x 108 cfu/ml. Monocultures were set up identically, except that A. naeslundii cells were omitted. Cultures were incubated at 37oC for 3 h prior to harvesting and extraction of total RNA. Purified RNA was reverse transcribed and cDNAs were labelled with Cy3 or Cy5 dye. cDNAs from coaggregate cultures and from S. gordonii monocultures were competitively hybridized with the S. gordonii microarray. Three independent sets of cultures were used, and flip dye pairs were included for two of the biological replicates (ie 5 hybridizations in total). In control experiments, cDNA derived from A.naeslundii monocultures did not hybridize with the S. gordonii microarrays. Data represent the ratios of gene expression in coaggregated S. gordonii compared with S. gordonii monocultured cells.

Project description:Biofilm formation on medically implanted devices by Candida albicans poses a significant clinical challenge. Here we compared biofilm-associated gene expression in two clinical C. albicans isolates, SC5314 and WO-1, to identify shared gene regulatory responses that may be functionally relevant. Among the 50 genes most highly expressed in biofilms relative to planktonic (suspension-grown) cells, we were able to recover insertion mutations in 25 genes. We observed that 20 of the 25 mutants have altered biofilm-related properties, including cell-substrate adherence, cell-cell signaling, and azole susceptibility. We focused on the most highly up-regulated gene in biofilms, RHR2, which specifies the glycerol biosynthetic enzyme glycerol-3-phosphate phosphatase. Glycerol is 5-fold more abundant in biofilm cells than planktonic cells, and an rhr2D/D strain accumulates 2-fold less biofilm glycerol than the wild type. Under in vitro growth conditions, the rhr2D/D mutant has reduced biofilm biomass and reduced adherence to silicone. The rhr2D/D mutant is severely defective in biofilm formation in vivo, in a rat catheter infection model. Expression profiling of the rhr2D/D mutant indicates that it has reduced expression of cell surface adhesin genes ALS1, ALS3, and HWP1, as well as a large fraction of all other biofilm up-regulated genes. Reduced adhesin expression is the cause of the rhr2D/D mutant biofilm defect, because overexpression of ALS1, ALS3, or HWP1 restores biofilm formation ability to the mutant in vitro and in vivo. Our findings indicate that internal glycerol has a regulatory role in biofilm gene expression, and that adhesin genes are among the main functional Rhr2-regulated genes. Gene expression profiles, in duplicate; (1) for biofilm vs. planktonic growth conditions for the two wild-type clinical isolates of Candida albicans (SC5314 and WO1-white/WO1-opaque), and (2) for rhr2M-NM-^T/M-NM-^T mutant and complemented strain, via RNA-deep sequencing using Illumina GA2 and HiSeq2000 platforms, respectively

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Streptococcus gordonii

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