Cm100

One, three and five-day-old cyprids (each samples, n = 4) were relaxed with 8% magnesium chloride51 (link)52 53 (link) for 1 h and washed thoroughly with 0.45 μm filtered 33 ppt sea water. The cyprids were then fixed with modified Karnovsky (2% paraformaldehyde and 2.5% double-distilled glutaraldehyde buffered with 0.1 M sodium cacodylate containing 3% sucrose, pH 7.4) overnight at 4 °C. The specimens were washed with cacodylate buffer, post-fixed with 1% aqueous osmium tetroxide, OsO₄, washed again with cacodylate buffer, dehydrated through a graded series of ethanol, infiltrated and embedded in Agar epoxy Resin 100 (Agar Scientific). The embedded specimens were polymerised for 48 h at 60 °C. Semithin sections were cut with glass knives on a Reichert Ultracut E ultramicrotome (Reichert-Jung) to locate the target structures. The semithin sections were stained with toluidine blue and examined under Leica DMRB microscope equipped with the Leica QWin Plus software (Leica, Wetzlar,Germany). Serial ultrathin sections (60–90 nm) were cut with diamond knives (Diatome, Switzerland). The ultrathin sections were collected on bare 75, 100 and 150-mesh copper grids, double stained with uranyl acetate (for 8 min) and lead citrate54 (link) (for 5 min) and examined with FEI CM100 and Hitachi ST7700 transmission electron microscopes operating at 80 and 100 kV.

S. Typhi containing the inducible vector pMMB207c with or without each fimbrial gene cluster was induced overnight at 37°C on LB plates with chloramphenicol and 50 μM IPTG. Bacteria were harvested in LB broth. For SDS-PAGE, bacteria were washed with a solution of 0.9% sodium chloride and then with a solution of 75 mM sodium chloride and 0.5 mM Tris pH 7.4. Extracellular proteins were extracted by heat treatment at 60°C during 15 min and were precipitated by addition of 10% trichloroacetic acid (Beloin et al., 2006 (link)). The concentration of proteins was normalized. The samples were loaded on SDS-PAGE 15% and stained with Coomassie R-250. The band of interest for Std and Stc (StdA and StcA) were cut from the gel, destained and digested by trypsin. Peptides were sequenced using LC-MS/MS at the Center for Advanced Proteomics Analyses (IRIC, Université de Montréal).
For electron microscopy, the 3–24 h-induced cultures were fixed with 2% glutaraldehyde for 30 min and adsorbed onto nickel formvar-carbon coated grids for 10 min and stained with 1% phosphotungstic acid. Bacteria were observed under Philips CM-100 or Hitachi H-7100 electron microscope.