time (h): 1 s/dmem(s) or s/d/dmem(d): D biological replicate: I strain: NCTC8239
Treatment protocol
Bacteria were co-cultured with human intestinal epithelial Caco-2 cells but with slight modifications. Briefly, Caco-2 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum. The cells were plated in a 24-well plate (1.3x105 cells per well) and incubated for 4 days. Just before the inoculation of bacteria, the cells were washed using phosphate buffered saline (PBS) three times and incubated in 1 ml glucose-negative DMEM (DMEM(-); Life Science Technologies, Carlsbad, CA) supplemented with 0.4% starch and 50 uM DCA (Wako, Osaka, Japan). Strain NCTC8239 was pre-cultured in FTG anaerobically for 18 h at 37ºC. The cultures were washed with PBS twice and 100 ul of bacterial culture (1-5 x 107 colony-forming units (CFU) per ml) were inoculated into Caco-2 cells and incubated in the CO2 incubator at 37ºC. The number of viable vegetative cells was determined by plating serially diluted samples onto Brain Heart Infusion (BHI) agar, incubating at 37°C for 24 h in anaerobic conditions, and calculating CFU. The number of heat-resistant spores was counted by plating heat-treated cultures onto BHI agar. The detection threshold was 200 CFU/ml.
Growth protocol
Bacterial strains and growth conditions. C. perfringens food poisoning (FP) type A strain NCTC8239 was purchased from the National Collection of Type Cultures. To achieve the sporulation, bacteria were inoculated into fluid thioglycollate (FTG; BD, Franklin Lakes, NJ) medium and incubated anaerobically for 18 h at 37ºC. One milliliter of the bacterial culture was passaged into 10 ml Duncan-Strong (DS) medium [31] and cultured for 24 h at 37ºC. One milliliter of the culture was then heated at 75ºC for 20 min and passaged into 10 ml of fresh DS medium. The heat treatment and passage were repeated until the amount of spores observed by phase contrast microscopy was greater than half of the total number of bacteria. These bacterial cells were heated and stored at -80°C with glycerol for future use.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with slight modifications. Bacterial cultures containing 2 × 107 CFU of C. perfringens were treated with RNAprotect Bacteria Reagent (Qiagen) according to the manufacturer’s instructions. After centrifugation at 5,000 × g for 10 min, the pellets were washed with SET buffer (25% sucrose, 50 mM EDTA (pH 8.0) and 50 mM Tris-HCl (pH 8.0)) at 5,000 × g for 10 min. The pellets, which were suspended in GTC buffer (4 M guanidine thiocyanate, 0.5% Na N-lauryl sarcosine, 25 mM sodium citrate (pH 7.0) and 0.1 M β-mercaptoethanol), were homogenized by passing 3 times through a 21-gauge needle to disrupt Caco-2 cell membranes. After being centrifuged at 5,000 × g for 10 min, the pellets were washed with SET buffer once. The bacterial cells were lysed by suspending in 100 µl SET buffer with 20 mg/ml lysozyme (Sigma) and 100 μg/ml proteinase K (Roche Applied Science) at 37ºC for 30 min. Following incubation, they were transferred to a tube containing zirconia beads (AMR, Gifu, Japan), vortexed for 5 min, and centrifuged at 21,130 × g for 5 min. Total RNA was extracted from the supernatants using TRI reagent LS (Sigma), and then purified with RNeasy MinElute Cleanup (Qiagen) according to the manufacturer’s instructions.
Label
Cy3
Label protocol
Total RNA prepared was labeled with the Agilent Low Input Quick-Amp WT Labeling Kit according to the manufacturer’s instructions.
Hybridization protocol
Before hybridization, 600 ng labeled cRNA of each product was fragmented and mixed with hybridization buffer according to the manufacturer's protocol (Agilent Technologies). Hybridizations were done for approximately 17h at 65°C. The slides were washed according to the manufacturer's manual, and scanning of microarrays was performed with 3µm resolution using a DNA microarray laser scanner (Agilent Technologies).
Scan protocol
Arrays were scanned using Agilent Scan software A8.3.1.
Description
C. perfringens Caco-2 in S/D/DMEM(-) 1 h
Data processing
Data were extracted using Feature Extraction 10.7.3.1. Microarray data were normalized with the bacterial control genes, log2 transformed.
