These structures appeared as composed of three parts: (a) a needle 11 nm in diameter, (b) a neck 10 nm high and 21 nm wide, and (c) a bulb 44 nm wide and 27 nm high (Fig. 5 B). This bulb did not appear to have cylindrical symmetry since we saw several different views of it (Fig. 5 A). Upon alignment and averaging of a gallery of the 22 best pre- served structures on the micrograph displayed in Fig. 5 A, we obtained a two-dimensional density map of the bulb at 2.8 nm resolution (Fig. 5 B). This tripartite structure is not identical to that seen by Kubori et al. (1998). They pre- sented partially lysed bacteria in which secretons were vi- sualized at the cell periphery and lacked the bulb. When examined by this method, intact bacteria and, to a lesser extent, partially lysed ones have a thickness which could explain why secreton structures seemed absent or partial, respectively, at their surface. No structures were found in mxiD, mxiJ, and mxiG mu- tants (not shown). As these three strains carry mutations within genes coding for components of the type III secre- tion system, we concluded that the structures observed in the wild-type corresponded to the type III secretons. No obvious differences were detected in the morphology of secretons in ipaB, ipaC, and ipaD mutants relative to the wild-type (Fig. 5 C), nor in their distribution or number (not shown). Likewise, we saw no differences in secreton morphology or distribution of the wild-type which had been induced to secrete with Congo red (Fig. 5, A and C). This indicated that activation of secretion, as seen in ipaB and ipaD mutants or upon incubation of the wild-type with Congo red, did not result in or from gross alterations of secreton structure or number. We also concluded that the nonhemolytic phenotypes of the ipaB, ipaC, and ipaD mutants did not result from major anomalies in the struc- ture of their secretons. Discussion Nature and Composition of the Translocator Osmoprotection experiments showed that S. flexneri in- serts a hydrophilic pore into RBCs. Amongst the five pro- teins secreted upon contact with eukaryotic cells, only IpaB and IpaC were tightly associated with RBC mem- branes isolated after contact-hemolysis. IpaA and IpgD were peripherally associated with RBC membranes while IpaD was not found in the membrane fraction. Since ipaA and ipgD mutants are not impaired in hemolysis, associa- tion of IpaA and IpgD with membranes is unrelated to pore formation. As IpaB and IpaC are the only secreted proteins with predicted a-helical hydrophobic regions ca- pable of spanning membrane bilayers, it is likely that they are the sole bacterial components inserted into the host membrane during cell entry. Whether the IpaB/IpaC pore structure contains host components also can not be as- sessed with our experimental setup. However, studies of the association of Ipa proteins secreted in the presence of Congo red with artificial membranes have yielded only the same four proteins associated with liposomes and these proteins form a nonselective, gated channel of 91 pS in conductance within lipid bilayers (De Geyter, C., F. Hom- blé, R. Wattiez, P.J. Sansonetti, P. Falmagne, J.M. Ruyss- chaert, C. Parsot, and V. Cabiaux, manuscript submitted for publication). We also showed that IpaB and IpaC are both required to form the pore. Indeed IpaB alone is un- able to form the pore structure since lysis mediated by the ipaC strain was insensitive to osmoprotection yet led to a small amount of IpaB protein becoming associated with lysed RBC membranes (Figs. 2 B and 3 A). When se- cretion occurs in the absence of any membrane, IpaB and IpaC are found solely as a complex in the medium (Blocker, A., unpublished observation), suggesting that they associate as they exit the secreton. Hence, to be in- serted efficiently into the target membrane IpaB and IpaC must form a complex which may remain associated within lipid bilayers. Although the ipaD mutant was nonhemolytic, we did not recover IpaD in lysed RBC membranes. Given the constitutive secretion phenotype of the ipaD mutant, the re- quirement of IpaD for hemolysis probably reflects the role of this protein in controlling secretion. IpaA and IpgD were associated peripherally with lysed RBC membranes, yet they were dispensable for hemolysis. As these proteins were found associated with RBC membranes lysed by the wild-type but not the ipaC mutant, they probably associate with the membranes via IpaB and/or IpaC. Evidence is mounting that both IpaA and IpgD proteins are translo- cated within the host cell at the time of entry when they modulate the actin rearrangements induced by IpaC (Tran Van Nhieu et al., 1997, 1999; Bourdet-Sicard, 1999; Nie- buhr, K., N. Jourhi, A. Allaoui, P. Sansonetti, and C. Par- sot, manuscript in preparation). This would suggest that IpaA and IpgD associate with the cytoplasmic face of the host membrane. Further work is required to establish the domains in each protein involved in these interactions, their topology relative to the membrane as well as the role of these interactions in cell entry. Structure of the Type III Secreton Electron microscopy studies showed that the type III secretons are assembled before any contact with target cells. In the mxiD, mxiG, and mxiJ mutants, no secretons were detected, which suggests that components of the secreton are degraded when the machinery can not be as- sembled. This is consistent with evidence that MxiG is un- stable in the mxiJ and mxiD mutants (Bahrani, F., and C. Figure 5. Morphological identification and analysis of the type III secretons of S. flexneri. (A) Osmotically shocked and negatively stained wild-type Shigella which had been induced to secrete with Congo red were visualized by electron microscopy. Arrows show the position of the secreton at the margin of bacteria. The neck and bulb are inside the body of the bacterium while the needle is protruding outside the outer membrane. Inset shows the periphery of partially lysed bacteria. Only the protruding needle is clearly visible. The neck is poorly distinguished and the bulb is masked inside the body of the bacterium. With the method used here one can not assume that the two lines at the margin of the bacterium are its inner and outer membranes. (B) Image of the secreton bulb resulting from align- ment and averaging of the 22 best preserved secretons found in A and deduced projection density map of the averaged image at 2.8 nm resolution. (C) Typical secretons found in wild-type, ipaB, ipaC and ipaD strains. Bars: (A) 200 nm; (B) 50 nm. Blocker et al. Type III Secreton-mediated Ipa Insertion into Membranes 691