Supporting Materials and Methods
MS and Peptide Sequencing Analyses of Citrobacter rodentium Secreted Proteins
. Secreted proteins were precipitated with 10% trichloroacetic acid and the pellets were solubilized in 2D sample buffer (8 M urea/2 M thiourea/4% CHAPS/20 mM Tris/0.002% bromophenol blue). Proteins were analyzed by 2D gels using 18-cm Immobiline Dry Strips (pH 3-10 or 6-11) according to the manufacturer’s instructions (Amersham Pharmacia). Gels were stained with Sypro Ruby (Bio-Rad) and visualized on a UV lightbox. Spots of interest were excised manually and analyzed by MS and/or peptide sequencing. In-gel digestion of proteins was performed on the Investigator ProGest Robot (Genomic Solutions, Ann Arbor, MI) as described (1) . Samples of high protein abundance were analyzed by a liquid chromatography (LC)-MS system consisting of a Nanoflow Liquid Chromatography system equipped with FAMOS Autosampler (LC Packings–Dionex, San Francisco, CA), and an LCQ Deca Ion Trap Mass Spectrometer (Thermo Finnigan, San Jose, CA). Peptides were separated by reversed phase HPLC on a PicoFrit Column PFC7515-PP18-5 (New Objective, Woburn, MA). A flow rate of 200 nl/min was used to separate the peptides. The column effluent was sprayed directly into the mass spectrometer. Low-protein abundance samples were analyzed on an API QSTAR Pulsar Hybrid MS/MS Mass Spectrometer (Applied Biosystems/MDS SCIEX, Concord, Canada) equipped with a Nanospray Ion Source (Proxeon, Odense, Denmark). Before the analysis, samples were purified and concentrated on ZipTips (Millipore, Billerica, MA). The API QSTAR Pulsar was also used for de novo peptide sequencing. Spectra were searched against National Center for Biotechnology Information (NCBI, Bethesda, MD) DataBase with the help of MASCOT (Matrix Science, London) or SONAR (Proteometrics, Winnipeg, Canada) search engines.Virulence Assays of C. rodentium Locus of Enterocyte Effacement (LEE) Mutants in Mice.
Three different strains of mice, inbred C57BL/6 and C3H/HeJ from The Jackson Laboratory, as well as outbred NIH Swiss from Harlan Sprague-Dawley (Indianapolis), were used in this study. The mice were housed in the animal facility at the University of British Columbia in direct accordance with guidelines drafted by the University of British Columbia’s Animal Care Committee and the Canadian Council on the Use of Laboratory Animals. WT C. rodentium and its LEE deletion mutants were grown in LB broth overnight in a shaker at 200 rpm, and 100 m l of the cultures was used to infect mice by oral gavage. To assess the ability to colonize the intestines and cause colonic hyperplasia of the LEE gene deletion mutants, groups of five NIH Swiss or C57BL/6 mice were infected. The infection was repeated at least once for all of the mutants. For infection of the highly susceptible C3H/HeJ mice by C. rodentium, the survival of infected mice were assessed daily for signs of morbidity over the course of the infection. When any mouse became moribund, it was immediately killed. For bacterial virulence assays using NIH Swiss and C57BL/6 mice, the mice were killed at day 6 or 10 postinfection. To check for bacterial colonization, colonic tissues plus fecal pellets were homogenized in PBS, using a Polytron Tissue Homogenizer, and serially diluted before being plated on MacConkey agar (Difco) or LB agar supplemented with appropriate antibiotics. We combined the colonic tissue and the fecal pellets to determine the total bacterial burden in the mouse colon at the time of death. MacConkey agar is selective for Gram-negative bacteria, on which C. rodentium forms colonies with a highly distinctive and identifiable morphology not typical of Escherichia coli. When necessary, the identity of C. rodentium was verified by PCR analysis using primers annealing to Citrobacter eae (intimin) gene. To score colonic hyperplasia, the first 4 cm of the distal colon starting from the anal verge was collected and weighed after fecal pellets were removed. Colon from mice infected by WT C. rodentium is significantly heavier than that of control, uninfected mice, and colonic epithelial hyperplasia is further assessed by microscopic histological analysis. For histological analysis, the last 0.5 cm of the colon from mice infected by C. rodentium was fixed in 10% neutral buffered formalin, processed, cut into 3-m m sections, and stained with hematoxylin and eosin (H&E). Histological staining (H&E) was done by the Morphological Services Laboratory at the Department of Pathology and Laboratory Medicine of the University of British Columbia.1. Houthaeve, T., Gausepohl, H., Mann, M. & Ashman, K. (1995) FEBS Lett. 376, 91-94.