texed and centrifuged again to remove bacteria. 2 ml of supernatant was collected, adjusted to 2.4 ml with Tris-saline and brought to 46% sucrose with 7 ml of 62% sucrose in Tris-saline containing the PIC. The mixtures were deposited at the bottom of SW41 centrifuge tubes (Beckman) and layered with 2 ml of 44% and then 25% sucrose in Tris-saline containing the PIC. Gradients were centrifuged at 15,000 g for 16 h at 48C. The mate- rial at the 44/25% sucrose interface was collected, diluted in Tris-saline and concentrated by centrifugation at 450,000 g for 20 min 48C in a TLA 100.3 rotor (Beckman). The pellets were resuspended in a minimal vol- ume of buffer. Such samples were checked by transmission electron mi- croscopy but no contaminating bacteria or bacterial ghosts were seen. When 35S-labeled bacteria were used less than 0.01% of the initial radio- active input was recovered in RBC membranes. The protein content of the samples was estimated using Bradford’s assay (Bio Rad) and an equiv- alent protein amount of each was separated by SDS-PAGE and Western blotted. To assess the strength of association of Ipa proteins with RBC mem- branes, 100 ml of purified Shigella-lysed RBC membranes were incubated at 48C for 1 h in Tris-saline or Tris-saline with 5 M NaCl, 8 M urea, or 0.2 M carbonate, pH 11. 300 ml of 62% sucrose in Tris-saline was then mixed with the membranes that were deposited at the bottom of 0.8-ml SW55 tubes (Beckman) and overlaid with 150 and 100 ml of 44 and 25% sucrose in Tris-saline. The gradients were spun overnight at 48C at 15,000 g. The top 150 ml was collected from the gradients, diluted in Tris-saline, and concentrated by pelleting in a TLA 100.2 rotor for 20 min at 450,000 g at 48C. The pellets were resuspended in a minimal volume of buffer. Measurement of Ipa Protein Secretion With Congo Red. 1-ml aliquots of Shigella cultures, grown as above were washed in Tris-saline and resuspended in 500 ml of the same buffer. 200 mM of Congo red was added to the bacteria and the cells were incubated for 10 min at 378C. The bacteria were pelleted at 14,000 g at 48C for 15 min. 10 ml of supernatant and of the pellet resuspended in 500 ml of Tris-saline were separated by SDS-PAGE and Western blotted. In Hemolysis. After the final centrifugation 20 ml of hemolytic assay su- pernatant and 20 ml of a resuspended reaction containing an equivalent number of bacteria incubated in the absence of any inducer of secretion were separated by SDS-PAGE and Western blotted. Antibodies mAbs to IpaB and IpaC were a gift from Armelle Phalipon. mAbs against IpaA and IpgD were generated by immunizing female BALB/c mice with Ipa protein containing supernatants obtained after induction of secretion in Shigella flexneri with Congo red. In brief, 10–20 mg of protein was in- jected subcutaneously into both hind legs (5 injections at 3-d intervals) and the popliteal lymph nodes were fused with X63Ag8 lymphoma cells according to standard protocols. Hybridoma supernatants were screened by ELISA and immunoblotting and clones positive for IpaA and IpgD were subcloned by limiting dilution. Immunoblotting Conventional SDS-PAGE was performed and immunoblotting was per- formed using a semi-dry apparatus onto nitrocellulose or PVDF mem- branes and developed using the ECL or ECL Plus kits (Amersham) and X-OMAT film (Kodak). Electron Microscopy Whole Mount Negative Stain. Bacteria were grown for 2 h at 378C from 1:50 dilutions of overnight precultures in 8 ml of TSB. 2 ml of bacteria were collected, washed in filter sterilized PBS, and resuspended in 20 ml of the same buffer. The bacteria were osmotically and mechanically shocked by a 1:4 dilution into a 60-ml drop of distilled water deposited on parafilm. Often lysis of dividing bacteria was favored by incubation of the samples on ice for a few hours before dilution in water. After a few minutes, 200 mesh Formvar-coated copper microscopy grids were placed on top of the drops. After a brief incubation at room temperature, the grids were rinsed with a flow of drops of 2% phosphotungstic acid at pH 7. After drying, the samples were observed in a Philips CM12 electron microscope working in standard conditions. Image Processing. Negative micrographs were digitized on a rotary drum scanner using a 10-mm/pixel square scanning aperture. Windowing and processing of the particle images was performed on digitized micro- graphs, using SPIDER software and WEB interactive selection program (Franck et al., 1996) on a Unix Digital Workstation. After contrast nor- malization, the whole set of selected image was subjected to a reference free alignment process (Penczek et al., 1992) to generate an average pro- jection image of the secreton. The resolution limit estimation was per- formed using both differential phase-residual (Franck et al., 1981) and Fourier ring correlation criterion (Saxton and Baumeister, 1982). Resin Embedding of RBC and Bacteria. AfaE-expressing bacteria and human red blood cells were mixed as in for the hemolytic assay but in 500-ml tubes. Hemagglutination occurred and samples were either centrifuged at various g forces for 10 min or left to sediment at 1 g at 108C. Samples were fixed for 1 h at room temperature with 1.2% glutaraldehyde, and 0.05% ruthenium red (RR) in 0.1 M cacodylate buffer, pH 7.4, to better visualize the carbohydrate surrounding cell membranes (Handley, 1991). Samples were rinsed with 0.05% RR in cacodylate buffer and post-fixed with 1% OsO4, 0.05% RR in cacodylate buffer for 1 h at room temperature. Cells were rinsed with H2O and embedded in 2% agarose (Type VII; Sigma). After gelling on ice samples were dehydrated with an increasing acetone series and embedded in Epon. Thin sections were conventionally stained. Results The Hemolytic Activity of S. flexneri Is Due to Formation of a 25-Å Pore within RBC Membranes To study the interaction of S. flexneri with a simple cell membrane, we reinvestigated its contact hemolytic activ- ity. Bacteria were mixed with RBCs, centrifuged at 1,500 g for 10 min at 108C and incubated at 378C for 1 h. Release of haemoglobin was monitored as described in Materials and Methods. As previously reported, contact hemolysis did not occur without centrifugation (Fig. 1 A), and strains carrying mutations in mxiD, ipaB, or ipaD were non- hemolytic, whereas strains carrying mutations in ipaA, ipgD, or in both ipaA and ipgD displayed normal hemoly- sis (Allaoui et al., 1993a,b; Ménard et al., 1993). The ipaC mutant had z10% hemolytic activity. This low activity had not been detected before because previously the wild-type strain induced only 40% hemolysis (Barzû et al., 1997), whereas, in the current assay, it caused 75–100% hemoly- sis. These results confirmed that integrity of the secretion machinery and IpaB, IpaC, and IpaD, but neither IpaA nor IpgD, were required for hemolysis. The residual activ- ity of the ipaC mutant suggested that, in the absence of IpaC, another factor could still destabilize the RBC mem- brane, albeit inefficiently. To determine whether hemolysis was due to the forma- tion of a pore within the RBC membrane, we performed osmoprotection experiments. Lysis of RBCs by bacterial toxins forming hydrophilic pores in membranes proceeds through osmotic shock (Menestrina et al., 1994). This can be prevented by addition of osmotic protectants at 30 mM to the medium. If the molecule is too large to pass through the pores, it counterbalances the increased intracellular pressure. Osmoprotectants of intermediate sizes induce a size-dependent increase of the half time of hemolysis that can be used to estimate pore size. Molecules larger than PEG1000 allowed significant protection against lysis gen- erated by wild-type bacteria and protection increased with the size of the molecule (Fig. 1 B). A time course analysis of hemolysis in the presence of different osmoprotectants (for example, Fig. 1 C) was used to derive a Renkin plot (Renkin, 1954; Ginsburg and Stein, 1987) showing the rel- ative permeability of the osmoprotectants versus their size Blocker et al. Type III Secreton-mediated Ipa Insertion into Membranes 685