05% Congo Red (w/v). SD1 in vitro samples were prepared by inoculating a single colony into Luria-Bertani (LB) medium grown to stationary phase at 37°C with agitation. The bacteria were harvested by centrifugation and washed twice with ice-cold PBS (6,000 × g, 15 min) at 4°C. The inoculum for in vivo experiments

was prepared by growing a typical SD1 colony selected from a TSA plate in LB medium overnight. Gnotobiotic piglets used for the animal experiments were delivered by Caesarian section at Tufts University Cummings School of Veterinary Medicine. Of several animals inoculated with SD1, three piglets were chosen for isolation of SD1 bacterial AZD8931 cells from the intestine in this comparative study. One of the piglets inoculated with 1 × 108 SD1 cells developed diarrhea 24 h later and was euthanized 4 d later when the gut contents GW3965 order were collected for bacterial purification. Another piglet inoculated with 5 × 108 SD1 cells developed diarrhea within 18 h and was euthanized 3 d post-inoculation. A third piglet inoculated with 5 × 109 SD1 cells developed diarrhea within 20 h and the

gut contents collected 2 d post-inoculation. SD1 bacterial cells were isolated from the gut contents as described previously [15]. Briefly, the gut contents from cecum and colon were pooled and transferred to sterile histological cups placed on ice, suspended in ice-cold PBS at 4°C and pelleted at 5,000 × g. After resuspension of the pellet in 65% isotonic Barasertib mouse Percoll solution and centrifugation at 14,500 × g, the bacterial layer near the bottom was collected using a 3-5 ml syringe with needle. The bacteria were washed twice with ice-cold PBS at 4°C and processed for proteomic analysis. Lysis of S. dysenteriae cells and trypsin digestion of extracted proteins After the PBS wash steps, bacterial cell pellets from in vitro or in vivo culture conditions were re-suspended in a hypotonic lysis buffer composed of 25 mM Tris-HCl (pH 7.8) with 150 μg/mL lysozyme, 0.05% Triton X-100, 5 mM EDTA, protease inhibitors (1 mM benzamidine and AEBSF) for 30 Morin Hydrate min at

room temperature (RT) with gentle agitation. The samples were then placed at -80°C until further processing. For nucleic acid digestion, bacterial samples suspended in the lysis buffer were thawed and gently agitated for 1 h at RT after the addition of DNase I, RNase and leupeptin (10 μg/mL each) and 20 mM MgCl2. Cell lysates were centrifuged at 16,000 × g for 30 min at 4°C, and the supernatants containing bacterial cell lysate proteins were recovered. Following cell lysis, the extracted bacterial proteins were precipitated in six volumes of ice-cold acetone at -20°C for at least 1 h. Acetone-precipitated proteins were recovered as a pellet after centrifugation at 5,000 × g for 10 min. The protein pellet was resuspended in 0.1 M TAB buffer, pH 8.5, and the total protein concentration measured using the BCA assay. Proteins were denatured in 0.