From bench to bedside: stealth of enteroinvasive pathogens. pathogenicity island 7 (SPI7), a large DNA region that is absent from the H 89 2HCl locus, composed of a regulatory gene, and genes. TviA is a positive regulator of the operon, which encodes functions for the biosynthesis and the export of the virulence-associated (Vi) capsular polysaccharide of strains used in this study are listed in Table 1. Unless noted otherwise, strains were routinely cultured at 37C in Luria-Bertani (LB) broth (10 g/liter tryptone, 5 g/liter yeast extract, 10 g/liter NaCl) or on LB agar plates (15 g/liter agar). For optimal expression of strains were cultured as described above in TYE broth for 3 h. A volume of 1 ml of bacterial culture was centrifuged for 2 min at 4C, and the supernatant was discarded. RNA was isolated by using the Aurum Total RNA minikit (Bio-Rad). The DNA-free kit (Life Technologies) was used to minimize DNA contamination. cDNA was generated from 0.1 g of RNA by using murine H 89 2HCl leukemia virus (MuLV) reverse transcriptase and reverse transcription-PCR (RT-PCR) reagents (Life Technologies) (22). As a control, a mock reaction with a mixture lacking the reverse transcriptase was performed to detect DNA contamination. SYBR green-based real-time PCR was performed (22) with the primers listed in Table 2. Data were analyzed by employing the comparative threshold cycle (and transcription to 16S rRNA levels. TABLE 2 Oligonucleotides used in this study for 5 min to synchronize infection. After 30 min, the supernatant was replaced with complete growth medium containing 0.1 mg/ml gentamicin. Cells were lysed with Tri reagent (Molecular Research Center) 3 h 30 min later, and total RNA was extracted according to the recommendations of the manufacturer. RNA was further purified by using the RNeasy MinElute Cleanup kit (Qiagen) and subjected H 89 2HCl to DNase treatment with the DNA-free kit (Life Technologies). Reverse transcription-PCR and real-time PCR were performed as outlined above. Analysis of FliC protein levels by Western blotting. The indicated strains were cultured in TYE broth for 3 h, as described above. Cell lysates corresponding to about 5 107 CFU were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore) (22). FliC and the housekeeping protein GroEL were analyzed by using rabbit H antiserum i (Difco), rabbit H antiserum d (Difco), and rabbit GroEL antiserum (Sigma), in conjunction with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Bio-Rad Laboratories). Chemiluminescence was detected by using a ChemiDoc-It system (UVP). Images were processed with Photoshop (Adobe) to adjust brightness and contrast uniformly to the entire image. Ethics statement. This H 89 2HCl study was performed in accordance with recommendations in the (25) All animal experimentation HGF was approved by the Institutional Animal Care and Use Committee of the University of California, Davis. Mouse strains and husbandry. C57BL/6 mice were purchased from Jackson Laboratory. throughout the experiment. Adoptive transfer and oral infection. Spleen and inguinal, brachial, cervical, and mesenteric lymph nodes were harvested from SM1 mice, and red blood cells were lysed by using ammonium-chloride-potassium (ACK) lysis buffer (Lonza) before labeling with carboxyfluorescein succinimidyl ester (CFSE) (29). The percentage of transgenic T cells was determined, and 800,000 H 89 2HCl to 1 1 106 cells were transferred intravenously into recipient mice. One day after the transfer, groups of mice were given 0.1 ml of a 5% sodium bicarbonate solution to neutralize the pH of the stomach and then intragastrically inoculated with 1 109 CFU of the indicated antigen presentation assay. Antigen presentation assays were performed as described previously (30). Briefly, spleens from C57BL/6 mice were digested by using collagenase D (Roche Diagnostics), and dendritic cells (DC) were enriched to a purity of >85 to 95% by using CD11c MicroBeads (Miltenyi Biotech). DCs, seeded at a density.
From bench to bedside: stealth of enteroinvasive pathogens