Equal volumes of the DL1 cultures and C. albicans hyphae, or final pH-adjusted fresh CSB medium as a control, were mixed, centrifuged (3,800 xg for 5 minutes) and the resulting loose pellets were incubated for 30 minutes at 36ºC aerobically in 5% CO2 without shaking to allow the S. gordonii cells to interact with the C. albicans hyphae in close proximity.
Growth protocol
Streptococcus gordonii strain Challis DL1 cells were grown to mid-log phase (approximately 108 cells/ml) in TY (5g/L Tryptone, 5 g/L yeast extract, 23 mM K2HPO4, pH 7.5) medium plus 5g/liter glucose (TY-G) under stationary conditions at 36ºC, aerobically with 5% CO2. Candida albicans strain UB1843 (SC5314) cells were grown overnight in YPD medium (1% w/v yeast extract, 2% w/v mycological peptone, 2% w/v glucose) shaking at 225 rpm, 37ºC aerobically for 16 hours to OD600 ~2.6. The resulting C. albicans cells were harvested by centrifugation (1,370 xg for 5 minutes), washed once in complete Salts-Biotin (CSB) nutrient-limited medium (7.5 mM Ammonium sulfate, 15 mM KH2PO4, 0.1 mM MgSO4•7H2O, 3.6µg/ml Biotin, 0.05 mg/ml CaCl2, 0.5% glucose, pH 6.5), resuspended in fresh CSB to OD600 ~ 1.0 (approximately 107 cells/ml) and incubated at 37oC aerobically with 225 rpm shaking for 16 hours to induce starvation. Candida hyphae formation was then induced by the addition of 1/10 volume 10% w/v aqueous glucose to the C. albicans culture. Incubation continued at 37ºC, 225 rpm for 3 hr and the final OD and pH were measured.
Extracted molecule
total RNA
Extraction protocol
RNA was prepared from the pelleted DL1-CSB and DL1-hyphae cell mixtures with a FastPrep120 Instrument (MP Biomedicals, Santa Ana, CA, USA) and RNAPro Blue RNA Extraction Kit (MP Biomedicals) for prokaryotic cells according to the manufacturer's directions. The extracted RNA was digested with DNase (Roche Applied Science, Indianapolis, IN, USA) and purified with QIAgen RNA mini spin columns (QIAgen, Germantown, MD, USA) according to the manufacturer's directions. RNA integrity was confirmed by comparison of the ratios of absorbance at 260/280 nm and 260/230 nm as quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, USA).
Label
Cy3,Cy5
Label protocol
cDNA was reverse transcribed using Superscript III (Life Technologies, Grand Island, NY, USA), end-labeled via amino-allyl-labeling (Life Technologies, Grand Island, NY, USA) and Cye-dye coupled (GE Healthcare/Amersham, Piscataway, NY, USA) to make microarray probes as previously described. Probes were purified over QIAgen PCR purification columns (QIAgen) to remove excess dye before use.
Channel 2
Source name
Streptococcus gordonii, alone, versus Streptococcus gordonii and hyphal Candida alicans
Equal volumes of the DL1 cultures and C. albicans hyphae, or final pH-adjusted fresh CSB medium as a control, were mixed, centrifuged (3,800 xg for 5 minutes) and the resulting loose pellets were incubated for 30 minutes at 36ºC aerobically in 5% CO2 without shaking to allow the S. gordonii cells to interact with the C. albicans hyphae in close proximity.
Growth protocol
Streptococcus gordonii strain Challis DL1 cells were grown to mid-log phase (approximately 108 cells/ml) in TY (5g/L Tryptone, 5 g/L yeast extract, 23 mM K2HPO4, pH 7.5) medium plus 5g/liter glucose (TY-G) under stationary conditions at 36ºC, aerobically with 5% CO2. Candida albicans strain UB1843 (SC5314) cells were grown overnight in YPD medium (1% w/v yeast extract, 2% w/v mycological peptone, 2% w/v glucose) shaking at 225 rpm, 37ºC aerobically for 16 hours to OD600 ~2.6. The resulting C. albicans cells were harvested by centrifugation (1,370 xg for 5 minutes), washed once in complete Salts-Biotin (CSB) nutrient-limited medium (7.5 mM Ammonium sulfate, 15 mM KH2PO4, 0.1 mM MgSO4•7H2O, 3.6µg/ml Biotin, 0.05 mg/ml CaCl2, 0.5% glucose, pH 6.5), resuspended in fresh CSB to OD600 ~ 1.0 (approximately 107 cells/ml) and incubated at 37oC aerobically with 225 rpm shaking for 16 hours to induce starvation. Candida hyphae formation was then induced by the addition of 1/10 volume 10% w/v aqueous glucose to the C. albicans culture. Incubation continued at 37ºC, 225 rpm for 3 hr and the final OD and pH were measured.
Extracted molecule
total RNA
Extraction protocol
RNA was prepared from the pelleted DL1-CSB and DL1-hyphae cell mixtures with a FastPrep120 Instrument (MP Biomedicals, Santa Ana, CA, USA) and RNAPro Blue RNA Extraction Kit (MP Biomedicals) for prokaryotic cells according to the manufacturer's directions. The extracted RNA was digested with DNase (Roche Applied Science, Indianapolis, IN, USA) and purified with QIAgen RNA mini spin columns (QIAgen, Germantown, MD, USA) according to the manufacturer's directions. RNA integrity was confirmed by comparison of the ratios of absorbance at 260/280 nm and 260/230 nm as quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, USA).
Label
Cy5,Cy3
Label protocol
cDNA was reverse transcribed using Superscript III (Life Technologies, Grand Island, NY, USA), end-labeled via amino-allyl-labeling (Life Technologies, Grand Island, NY, USA) and Cye-dye coupled (GE Healthcare/Amersham, Piscataway, NY, USA) to make microarray probes as previously described. Probes were purified over QIAgen PCR purification columns (QIAgen) to remove excess dye before use.
Hybridization protocol
cDNA probes were hybridized to aminosilane-coated glass slides spotted with unique 70-mers representing each open reading frame from the S. gordonii strain CH1 genome sequence (Genbank number CP000725.1) in replicates of six (obtained from the Pathogen Functional Genomics Resource Center; NIDCR Oral Microbe Microarray Initiative; GPL10584); each slide contained a total of 500 Arabidopsis thaliana 70-mers as controls. Slides were blocked during prehybridization at 42°C in blocking buffer (5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.4), 0.1% w/v SDS, 1% w/v bovine serum albumen (BSA), then washed successively with water and isopropanol before drying via centrifugation prior to hybridization. To accommodate potential increases in cDNA quantity based upon contributions from the C. albicans hyphae 2-fold more cDNA from the interaction samples was mixed with DL1-CSB samples for each interaction comparison. To account for biases during the labeling process, flip-dyes, in which the fluorescent labeling is reversed for each sample, were used. Cy3 labeled DL1-CSB cDNA was combined with Cy 5-labeled DL1-C. albicans cDNA in appropriate ratios; similarly Cy5-labled DL1-CSB and Cy3 labeled DL1-C. albicans cDNAs were combined. The dye-labeled cDNA mixtures were dried, resuspended in 50 µl of hybridization buffer (40% v/v formamide, 5x SSC, 0.1% SDS, 0.6 ug/uL Salmon Sperm DNA), heated at 95°C for 10 min, placed on ice, and individual mixtures added to each microarray slide under a Lifterslip (Erie Scientific Company, Portsmouth, NH, USA). Each slide was incubated at 42°C for 16 h in a sealed hybridization chamber (Corning Life Sciences, Tewksbury, MA, USA), then washed successively in 0.1% w/v SDS-2x SSC, 0.1% SDS-0.1x SSC, and 0.1x SSC and dried by centrifugation.
Scan protocol
The Axon 4200A (Axon Instruments, Foster City, CA, USA) was used to obtain digital images of the microarrays.
Description
Pellet of Streptococcus gordonii and CSB incubated for 30 minutes at 36ºC with 5% CO2 versus a mixed pellet of Streptococcus gordonii and hyphal Candida alicans, incubated together for 30 minutes at 36ºC with 5% CO2
Data processing
Data were analyzed using the TM4 Microarray Software Suite, consisting of Spotfinder, Midas, and MeV (http://www.tm4.org/). The fluorescence signals from the flip-dye hybridizations were measured using Spotfinder. Normalization and regularization of the data were performed using Midas (Lowess normalization, block mode, smooth parameter of 0.33, Cy3 reference; standard deviation regularization, block mode, Cy3 reference; flip-dye consistency checking and combination, using the data trim option of the standard deviation mode and keeping all data within 2.0 standard deviations; replicate spots were averaged to be used in the determination of the final ratio for each spot). Using MeV, with 100 permutations and a P value cutoff of 0.05 the final expression ratios were generated and used to determine the fold change for hybridization of each probe; fold changes were ranked from the greatest increase to the greatest decrease. Experimental cDNA probes from DL1 cells incubated with hyphae that hybridized to the oligonucleotide spots with average fold changes 1.6-times greater than or 1.6-times less than fold changes of DL1-CSB controls in two biologically independent experiments were considered to be potentially up- or down- regulated, respectively.
Transcriptome analysis of Streptococcus gordonii strain Challis DL1 indicates a role for the biofilm-associated fruRBA operon in response to Candida albicans hyphae formation
