(Fig. 1 D). This allowed estimation of the functional inner radius of the pore at 26 Å (60.4 Å, standard deviation, n 5 3). No osmoprotection was observed in lysis mediated by the ipaC strain (Fig. 1 B). This suggested that hemolysis induced by the ipaC mutant resulted either from forma- tion of a pore larger than 32 Å in diameter (the size of PEG 3000) or from destabilization of the membrane by another mechanism. IpaB and IpaC Are Tightly Associated with Lysed RBC Membranes To determine the components of the pore formed in the membrane during contact hemolysis, we isolated the lysed RBC membranes by floatation in a sucrose density gra- dient. The protein content of the membranes was sepa- rated by SDS-PAGE, blotted and probed with antibodies against IpaA-D and IpgD. IpaB, IpaC, IpgD, and IpaA were present in membranes recovered after incubation of RBCs with the wild-type at 378C, but not in fractions re- covered after incubation with the mxiD mutant at 378C or with the wild-type at 48C (Fig. 2 A). By semiquantitative immunoblotting, the amount of IpaB, IpaC, IpgD, and IpaA recovered in the membrane fraction corresponded to z0.1% of the total amount of these proteins present ini- tially in bacteria. IpaD was not detected in the membrane fraction, although the detection procedure was sensitive enough to reveal 0.1% of IpaD in bacterial extracts (not shown). Membranes isolated after contact with the wild-type at 378C were then incubated in the presence of agents known to release peripheral membrane proteins. The amount of Ipa and Ipg proteins associated with membranes was mea- sured after a second floatation in a sucrose density gradi- ent. After stripping with 5 M NaCl, 0.2 M carbonate, pH 11, or 8 M urea, the majority of IpaB and various amounts of IpaC remained associated with the membranes (Fig. 2 B). In contrast, IpgD and IpaA were lost even in the mock- treated sample (not shown). This indicated that IpaB, and to a lesser extent IpaC, were strongly associated with the Figure 1. Contact hemolytic activity of S. flexneri. Osmoprotec- tive analysis of the lytic activity. (A) The hemolytic assay was performed with bacteria and sheep RBCs incubated for 1 h at 378C; w.t. stands for strain M90T, for w.t. no spin the centrifuga- tion step before incubation at 378C was omitted. (B) Wild-type and ipaC or mxiD strains were incubated with sheep RBCs for 1 h at 378C in the presence of 30 mM of the different sugars indi- cated. (C) Wild-type bacteria were incubated with RBCs for var- ious times in the presence of 30 mM of the different sugars. (D) The lysis kinetics were evaluated from the data shown in B by the time necessary to obtain 50% of maximal lysis, t1/2. We used t1/2– t01/2 (t01/2 being the time without osmoprotectants) as an estimate of the time necessary for the osmoticant to diffuse inside the cell through the bacterially induced lesions. Accordingly 1/(t1/2-t01/2) is an estimate of the permeability of the sugar through the pore and was used to build a Renkin plot showing the relative perme- ability of the molecule versus its size. In this graph, the line was the best fitting curve determined by the computer program Cricket Graph and its intersection point with the x-axis puts the pore diameter at 2.25 nm. Consistently, PEG 3000 (estimated molecular diameter 3.2 nm) offers nearly complete osmoprotec- tion. The Journal of Cell Biology, Volume 147, 1999 686