sonetti, 1987; Adam et al., 1995). This initiates a cycle of intra- and intercellular spreading (Niebuhr and Sansonetti, 1999). Only three Ipa proteins are essential for entry into HeLa cells and contact hemolysis (Clerc et al., 1986; San- sonetti et al., 1986): IpaB, IpaC, and IpaD (Sasakawa et al., 1989; Ménard et al., 1993). IpaB and IpaC have hydropho- bic regions that could form transmembrane helices. Both proteins form separate complexes with the same chaper- one, IpgC, within the bacterial cytoplasm (Ménard et al., 1994b). A small amount of IpaB is present in bacterial outer membrane fractions, from where it can be coprecipi- tated with IpaD (Ménard et al., 1994a). IpaD is a hydro- philic 35-kD protein that is also stored intracellularly and secreted upon host contact. ipaB and ipaD mutants consti- tutively secrete the remaining Ipa proteins suggesting that IpaB and IpaD play a role in secretion regulation (Ménard et al., 1994a; Parsot et al., 1995). IpaB and IpaC form a complex upon secretion from the bacterium (Ménard et al., 1994b). This complex is sufficient to induce membrane ruf- fling and entry into mammalian cells (High et al., 1992; Ménard et al., 1996; Tran Van Nhieu et al., 1999). As both proteins are hydrophobic, they could interact with the host plasma membrane to form a structure through which the remaining Ipa proteins become translocated. Indeed, IpaB and IpaC share structural similarities with YopB and YopD, respectively, from Yersinia species. These proteins are secreted by a type III secretion system and are re- quired for translocation of other Yops into host cells (Sory and Cornelis, 1994). Two other proteins, IpaA and IpgD, are secreted in re- sponse to Congo red and are likely to be secreted upon contact of bacteria with eukaryotic cells. Inactivation of ipaA and ipgD alters but does not abolish entry into HeLa cells (Allaoui et al., 1993b; Ménard et al., 1993). IpaA in- teracts with vinculin, an intracytoplasmic actin-associated host protein, during HeLa cell invasion (Tran Van Nhieu et al., 1997). IpgD shows homology to SopB, a protein found in Salmonella species. The latter is a phosphatidyl- inositol phosphatase which is translocated into eukaryotic cells and involved in tissue inflammation (Galyov et al., 1997; Norris et al., 1998). These data and the predicted hy- drophilic nature of IpaA and IpgD suggest that they are translocated into the host cell cytoplasm where they mod- ulate the invasiveness of S. flexneri. How do type III secretons mediate insertion and trans- location of proteins into the host cell? In flagellar biogene- sis, there is evidence for a canal within the structure (Mor- gan et al., 1995) through which flagellin may traffic until it reaches the tip of the structure and inserts into the grow- ing flagellum (Emerson et al., 1970). By analogy, the type III secreton may function by allowing protein traffic through a canal within its length, with insertion at the fla- gellum tip being replaced by insertion into the host mem- brane. The molecular mechanisms underlying these hy- pothetical functions and the subsequent translocation of substrate proteins remain to be established. Contact hemolysis is the ability of Shigella to lyse red blood cells with which they have been brought into contact by centrifugation. It occurs also with Yersinia species, re- quiring the yopB gene product for formation of 1.2–3.2- nm-pore within RBCs (Håkansson et al., 1996). To gain a mechanistic understanding of how the IpaB, IpaC, and IpaD proteins mediate the initial interaction of the bacte- rium with the host cell, we used contact hemolysis as a model of bacterial protein interaction with host mem- branes. In addition, we performed morphological analysis of the RBC/bacteria interaction and of type III secretons. By correlating biochemical and morphological data we propose a model of the earliest molecular events leading to S. flexneri entry into host cells. Materials and Methods Contact Hemolysis Assay Protocol. Sheep blood was obtained from BioMérieux (Marcy L’Etoile, France). Human blood was obtained from the Etablissement Français de Transfusion Sanguine (Paris, France). Bacteria were grown from overnight precultures diluted 1:100 in aerated flasks of Trypticase Soy Broth (TSB; Becton Dickinson) for 4 h at 378C to OD600 5 4. They were collected by centrifugation for 5,000 g for 7 min at 48C, washed in sa- line (150 mM NaCl) and resuspended at 1010 bacteria/ml at 48C. RBCs were washed 33 in saline by centrifugation at 2,000 g for 5 min at 48C and resuspended at 5.108/ml. The final resuspension buffer of both bacteria and RBCs contained 30 mM Tris at pH 7.5 (Tris-saline). 100 ml of bacteria were mixed with 100 ml of RBCs in round bottom 96-well plates, centri- fuged at 1,500 g for 10 min at 108C, and incubated at 378C for 1 h. The cells were resuspended and the samples recentrifuged. 100 ml of supernatant was transferred to a fresh plate where its optical density at 595 nm was measured. The baseline (B) of the assay was set with RBCs incubated with Tris-saline instead of bacteria and total hemolysis (T) was the value obtained when RBCs were incubated with saline containing 0.1% sodium dodecyl sulfate. The percentage of total hemolysis (P) was calculated us- ing the equation P 5 [(X 2 B)/(T 2 B)] 3 100, where X is the optical den- sity value of the sample analyzed. All data presented result from at least three independent experiments. Errors given are standard deviations. When AfaE-expressing Shigella (Clerc and Sansonetti, 1987) were used to lyse human RBCs, bacteria were resuspended at only 109 bacteria/ml. Avirulent S. flexneri expressing E. coli hemolysin A are described by Zychlinski et al. (1994). To obtain the hemolysin containing culture super- natants, bacteria were diluted 1:100 from overnight precultures into 8 ml of TSB and grown for 3 h at 378C. The bacteria were eliminated by centrif- ugation and the supernatant used immediately. The ipaA ipgD mutant strain was constructed by integration of pLAC-A carrying an ipaA::LacZ fusion (Ménard et al., 1993) into the large plasmid of the ipgD mutant strain. Osmoprotection Experiments. RBCs were resuspended in a 60-mM solu- tion of sucrose, raffinose (Sigma), or PEG1000 through 3000 (Fluka) made in Tris-saline. After incubation at 378C, the samples were cooled to stop the reaction, resuspended and immediately recentrifuged. Neuraminidase Treatment of RBC. 5 3 107 washed RBCs in 100 ml were treated with 0.5 U of neuraminidase, type V from Clostridium perfringens (Sigma) for 60 min on ice. The cells were washed 33 in Tris-saline and re- suspended at the same concentration for use in the hemolytic assay. Com- plete removal of sialic acid was verified by loss of the ability of HA virus, the cell receptor of which is sialic acid, to hemagglutinate the treated RBCs. Red Blood Cell Membrane Isolation Bacteria and sheep RBCs were prepared as described above, except that they were resuspended in Tris-saline at 2.1011 and 1.5.1010 cells/ml, respec- tively. Hemolytic reactions were prepared in 50-ml conical tubes with 600 ml RBCs, 120 ml bacteria, 860 ml Tris-saline, and a protease inhibitor cocktail (PIC1; Complete™, Boehringer Mannheim). Samples were centrifuged at 2,000 g at 48C for 10 min, incubated at 37 or 48C for 1 h, resuspended by vortexing, and recentrifuged. Hemolysis was assessed spectrometrically as above. 800 ml of distilled water was added to each sample to lyse all RBCs nonspecifically (so that lysed membranes could be isolated even from those samples where no hemolysis had occurred), and these were vor- 1. Abbreviations used in this paper: LPS, lipopolysaccharide; PIC, protease inhibitor cocktail. The Journal of Cell Biology, Volume 147, 1999 684