Bacterial Strains and Culture Conditions
strain RIMD2210633 (KP-positive, serotype O3:K6) was used as the standard strain [11
]. The bacteria were cultured at 37°C with shaking in Luria-Bertani (LB) medium supplemented with 3% NaCl.
Deletion Mutants and Complementation of Deleted Genes in Mutant Strains of V. parahaemolyticus
Deletion mutant strains (ΔtdhAS, ΔT3SS1, ΔT3SS2, ΔVP1680, ΔVP1686, and ΔVPA0450) were previously described [9
]. Complementation of deleted genes was performed as previously described [20
]. Polymerase chain reaction (PCR) products were cloned into pSA19CP-MCS, and the plasmid construct was introduced into deletion mutant strains by electroporation.
Caco-2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich) containing 10% fetal bovine serum (FBS; Gibco BRL), and 100 μg/mL gentamicin (Sigma-Aldrich). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were seeded in 6-well culture dishes at a density of 2 × 105 cells/well. Caco-2 cells that had been cultured for 4 to 5 days were used for subsequent experiments.
At least 4 hours before infection, the culture medium was replaced with fresh DMEM (without supplements). After cultivation, bacteria were harvested by centrifugation and resuspended in phosphate buffered saline (PBS) (pH 7.4). The concentration was adjusted with PBS. Bacteria were added to each well (2 × 104 colony-forming units/dish). Infections were allowed to proceed at 37°C in 5% CO2.
Measurement of IL-8 Secretion Levels
The level of IL-8 released into the culture medium was assessed using an enzyme-linked immunosorbent assay (ELISA) kit (Pierce), in accordance with the manufacturer's instructions.
Preparation of Cell Lysates
Following the infection period, the medium was removed and the cells were washed once with cold PBS. The cells were solubilized using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 5 μg/mL aprotinin, 5 μg/mL leupeptin, 1% Triton X - 100, 1% sodium deoxycholate, and .1% sodium dodecyl sulfate [SDS]). Cell lysates were homogenized using a needle and syringe then centrifuged at 15,000 rpm for 10 minutes at 4°C. Total protein was determined using a BCA Protein Assay Kit (Pierce) and stored at −80°C.
Isolation of Soluble, Cytoplasmic, and Nuclear Extracts
Following bacterial infection, the cells were washed once with cold PBS and cells were lysed in Nuclei Lysis Buffer (10 mM Tris-HCl, pH 7.6, 10 mM NaCl, 3 mM MgCl2, .5% NP-40). Cell lysates were centrifuged at 2000 × g for 4 minutes at 4°C. Thereafter, the supernatants (cytoplasmic extracts) were transferred to a fresh tube, and the nuclear pellets were resuspended in RIPA buffer (nuclear extracts).
The cell lysates were mixed with sample buffer, boiled for 5 minutes, and separated through an SDS - polyacrylamide gel electrophoresis (PAGE). Following electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with primary antibody and incubated with horseradish peroxidase-conjugated secondary antibody. The blots were visualized with the enhanced chemiluminescence (ECL) Western Blotting Kit (GE Healthcare Bio-Sciences). Rabbit-anti NF-κBp65 and Lamin A antibodies were obtained from Santa Cruz Biotechnology. ERK1/2, p38 MAPK, ATF-2, CREB, c-Fos, c-Jun, B-actin, phospho-ERK1/2, phosphor-p38 MAPK, phosphor-ATF-2, and phosphor-CREB antibodies were purchased from Cell Signaling Technology.
Construction of IL-8 Luciferase Reporter Gene Constructs
The 5′-flanking region spanning from -133 to +44 bp of the IL-8 gene was created by PCR using the forward primer (5′-AGTGTGATGACTCAGGTTTGCC-3′), reverse primer (5′-AGCTTGTGTGCTCTGCTGTCTC-3′), and Caco-2 genomic DNA. PCR products were cloned into the pCR2.1-TOPO vector (Invitrogen). Each DNA insert was digested with KpnI and XhoI and cloned into pGL3 (digested with the same enzymes). This plasmid (pGL3 -133) was used as a standard construct.
Site-directed mutagenesis of the IL-8 promoter was performed with pGL3-133 plasmid and the PCR primers. The primers used for point mutation of the AP-1 site (TGACTCA to TATCTCA; mutation is underlined) were 5′-AGTGTGATATCTCAGGTTTGCC-3′ (forward) and 5′-GGCAAACCTGAGATATCACACT-3′ (reverse). For C/EBP (CAGTTGCAAATCGT to AGCTTGCAAATCGT), the point mutation primers were 5’-GGATGGGCCATAGCTTGCAAATCGTGG-3′ (forward) and 5′-CCACGATTTGCAAGCTATGGCCCATCC-3′ (reverse). For NF-κB (GGAATTTCCT to TAACTTTCCT), the point mutation primers were 5′-GTTGCAAATCGTTAACTTTCCTCTGACATAATG-3′ (forward) and 5′-CATTATGTCAGAGGAAAGTTAACGATTTGCAAC-3′ (reverse). These plasmid constructs were confirmed by sequencing.
Transfection and Luciferase Reporter Gene Assay
Caco-2 (1 × 105) cells transfected with the indicated constructs were seeded on 24-well plates and cultured for 36 hours in DMEM. Constructs (.4 μg) were mixed with .1 μg β-galactosidase (β-gal) expression vector, pCMV-β, in 25 μL of DMEM. The solution was mixed with .8 μL of Lipofectamine 2000 Reagent diluted in 25 μL of DMEM and incubated at room temperature for 20 minutes. After incubation, the cell culture medium was replaced with 200 μL of fresh DMEM (without supplements) and 50 μL of the constructs in DMEM solution were cotransfected into Caco-2 cells. After a 6-hour transfection, the medium was replaced with fresh DMEM. The next day, the cells were infected by bacteria for 6 hours, followed by the addition of gentamicin (100 μg/mL) to each well to avoid excess infection. After incubating for 12 hours, the cells were washed with 200 μL of ice-cold PBS and lysed by adding 100 μL of lysis buffer, supplied with the Luciferase Assay Kit (Promega). The lysates were assayed for luciferase activity and β-gal activity. Transfection efficiency for the luciferase activity was normalized against β-gal activity.
Caco-2 cells plated on glass cover slips in 6-well plates were infected by bacteria for 6 hours. After infection, the cells were fixed with 4% paraformaldehyde in PBS at room temperature for 10 minutes and washed 3 times with PBS. Cells permeabilized with PBS containing .1% Triton X - 100 for 7 minutes were treated with 3% BSA blocking buffer for 60 minutes. They were then incubated with primary antibody overnight at 4°C and washed 3 times with PBS. Secondary antibody conjugated with Alexa Fluor 568 (Molecular Probes) was incubated at room temperature for 60 minutes and stained with 500 nM DAPI (Molecular Probes) for 5 minutes at room temperature. NF-κBp65 (Santa Cruz Biotechnology) antibody was used at a dilution of 1/500, and secondary antibody at a dilution of 1/200.
RNA Extraction and Complementary DNA (cDNA) Synthesis
Total RNA was extracted from infected Caco-2 cells using TRIzol reagent (Invitrogen). In this process, RNA was treated with RNase-free DNase I (TaKaRa) to prevent carryover of genomic DNA. cDNA synthesis was prepared from 1 μg of the total RNA using the PrimeScript RT-Reagent Kit (TaKaRa), in accordance with the manufacturer's instructions.
Quantitative Real-Time Reverse Transcription PCR
Quantitative real-time reverse-transcription PCR (real-time RT-PCR) was performed in the LightCycler Real-Time PCR System (Roche Applied Science) with SYBER Premix Ex Taq (TaKaRa). Thermocycling was performed in a final volume of 20 μL containing 2 μL of cDNA, .2 μM of each primer, and 10 μL of SYBER Premix Ex Taq. After the reaction, the specificity of PCR products was confirmed with melt curve analysis and agarose gel electrophoresis.
The expression levels of IL-8 were normalized with the 18S ribosomal RNA housekeeping gene. Human-specific primers for IL-8 were 5′-CGGAAGGAACCATCTCACTG-3′ (forward) and 5′-AGCACTCCTTGGCAAAACTG-3′ (reverse). 18S primers were 5′-AAACGGCTACCACATCCAAG-3′ (forward) and 5′-GGCCTCGAAAGAGTCCTGTA-3′ (reverse).