RCF g 1 160 350 630 1400 0 6 0 1.1 6 1.6 33.2 6 5.4 55.7 6 13.5 69.7 6 9.4 Table I. Effect of Centrifugation Force on Hemolytic Efficiency Untreated Sialidase treated Mock treated RBCs 0 6 0 23.3 6 11.1 35 6 10.1 36.5 6 9.4 ND 0 6 0 3.9 6 1.9 45.4 6 2.6 45.0 6 10.4 ND Figure 4. Electron microscopy analysis of the interaction be- tween bacteria and RBCs. Derivatives of wild-type bacteria ex- pressing the afimbrial adhesin AfaE were mixed with human RBCs and either left to sediment at (A) 1 g, or centrifuged at (B) 1,400 g for 10 min at 108C. In C and D RBCs were mock treated or treated with sialidase, respectively, before mixing with the bacteria, and the samples were centrifuged at 160 g. The samples were fixed and prepared for transmission electron microscopy. Bar, 5 mm. and Congo red. Induction of Ipa protein secretion in the immediate vicinity of RBCs did not lead to lysis but the same procedure in the presence of centrifugation did (Fig. 3 E). This indicated that Congo red did not inhibit hemoly- sis and suggested that Ipa proteins could not insert into the RBC membrane after release from the bacterium. These data indicated that Ipa secretion was necessary but not sufficient for hemolysis. Hemolysis Requires Extensive Surface Apposition and Reduced Distance between Bacteria and RBCs Given that bacteria adhering to cells via the AfaE adhesin were unable to cause hemolysis without centrifugation at 1,500 g, we examined the consequence of this physical treatment on the cells. Mixtures of RBCs and AfaE- expressing bacteria were exposed to different centrifuga- tion forces at 108C and then either incubated at 378C to assay hemolysis or prepared for examination by transmission electron microscopy. Hemolysis did not occur at 160 g but was complete at 630 g (Table I). In samples centrifuged at 160–630 g, the RBC membrane was deformed to line that of bacteria (Fig. 4 B). In the sample which had not been centrifuged, bacteria associated with RBCs adhered at a tangent rather than over their entire surface (Fig. 4 A). The values presented in this table are percentages of total hemolysis. All reactions were performed with wild-type bacteria expressing the AfaE adhesin and human RBCs untreated, sialidase treated, or mock treated. This indicated that centrifugation increased the surface of bacteria in contact with RBCs. Although samples treated at 160 and 630 g looked simi- lar, lysis occurred in the latter but not in the former (Table I), suggesting that the distance between bacterial and RBC membranes might also be critical for hemolysis. To test this possibility, we shortened this distance artificially, by removing either bacterial lipopolysaccharide (LPS) or sialic acid, a major constituent of the RBC surface (Shot- ton, 1998). We compared the lysis efficiency of wild-type Shigella and that of an rfe mutant that expressed a com- plete LPS core but lacked the O-antigen (Sandlin et al., 1995; Rathman, M., P.J. Sansonetti, C. Parsot, and G. Tran Van Nhieu, manuscript submitted for publication). The rfe mutant was as efficient as the wild-type at lysing RBCs at any g force applied (not shown). Then, we treated RBCs with neuraminidase to remove sialic acid. This treatment did not prevent AfaE carrying Shigella from hemaggluti- nating RBCs (not shown), as the site on the DAF mole- cule which is sialylated is distant from the SCR-3 domain to which AfaE binds (Pham et al., 1995). This treatment allowed lysis to occur efficiently at 160 g (Fig. 4 D) whereas mock-treated RBCs remained intact after expo- sure to bacteria at 160 g (Fig. 4 C and Table I). Surface charge is known to affect the ability of proteins to insert into artificial membranes (van der Goot et al., 1991) and the absence of LPS or the removal of sialic acid affect the surface changes of bacteria and RBCs. Thus, both the dis- tance and the charge of the target membrane may be criti- cal for induction of hemolysis. The Type III Secreton Is a Multicopy Tripartite Structure Constitutively Assembled at 378C To understand why contact was critical for hemolysis, we searched by electron microscopy for the structure encoded by the mxi and spa genes in osmotically shocked and nega- tively stained bacteria. Most bacteria were unaffected by osmotic shocking, while some, mostly those undergoing bi- nary fission, were partially lysed and a few appeared as complete membrane ghosts. No structures were visible on intact bacteria. Some structures, resembling the type III secretion apparatus of Salmonella, were visible at the pe- riphery of partially lysed bacteria (Fig. 5 A, inset). In the ghosts, many structures were visible dispersed over the whole bacterial surface (50–100/cell) and they were not identical to that seen in partially lysed bacteria (Fig. 5 A). Blocker et al. Type III Secreton-mediated Ipa Insertion into Membranes 689