Figure 2. Analysis of the Ipa proteins associated with RBCs membranes after contact hemolysis. RBCs membranes exposed to contact with various strains under different condition were iso- lated and examined for their Ipa protein content using SDS- PAGE and immunoblotting. Blots were revealed with the H16 anti-IpaB mAb, a mixture of K24, N9, H10, and J22 anti-IpaC mAbs or the 20G9 anti-IpgD and 322F7 anti-IpaA mAbs. The occurrence of contact hemolysis is indicated under each panel. (A) Ipa/Ipg protein associated with RBCs membranes after con- tact hemolysis with wild-type, ipaC, or mxiD strains at 378C or 48C. The blot probed with the anti-IpaB antibody was overex- posed to visualize the small amount of IpaB associated with RBC membranes lysed by the ipaC mutant. (B) Association of IpaB and IpaC with RBC membranes isolated after contact with wild- type bacteria at 378C and stripped, or not (mock), with 5 M NaCl (NaCl), 0.2 M carbonate, pH 11 (carbonate), and 8 M urea (urea). (C) Analysis of the association of IpaB, IpaC, IpgD, and IpaA with RBCs membranes brought into contact with various mutants. membranes, while IpgD and IpaA were located at the membrane periphery. To investigate further the mode of interaction of Ipa and Ipg proteins with RBCs membranes, we examined their presence in this fraction after contact of the ipaC, ipaA, ipgD, and ipaA ipgD mutants with RBCs. Mem- branes exposed to the ipaA, ipgD, or ipaA ipgD mutants contained relatively high amounts of IpaB and IpaC, and of IpgD or IpaA, or neither of the latter two proteins, re- spectively. This indicated that IpaA and IpgD were not re- quired for association of IpaB and IpaC with the mem- branes or for association of each other with membranes. In the ipaC mutant, reduced amounts of IpaB, IpaA and IpgD were associated with the membranes (see Fig. 2 A, in which the blots were overexposed to show more clearly the low levels of these proteins in these samples). Al- though this amount was vastly reduced as compared with that observed with the wild-type, it was significant as incu- bation of RBCs with the mxiD mutant at 378C or with the wild-type at 48C led to undetectable amounts of these pro- teins in the membranes. The small amount of IpaB associ- ated with membranes after incubation of RBCs with the ipaC mutant could account for the residual hemolysis of this strain. The low amount of IpaA and IpgD in these membranes suggested that these proteins require the pres- ence of IpaC and/or IpaB to associate with membranes. Ipa Protein Secretion Is Necessary but Not Sufficient for Contact Hemolysis To gain an understanding of how IpaB and IpaC were transferred into target membranes, we studied the rela- tionship between hemolysis and Ipa protein secretion. We investigated the efficiency of hemolysis after incubation for 1 h between 4 and 428C. No hemolysis was observed below 258C and the reaction proceeded normally above 308C (Fig. 3 A). The temperature dependence of lysis cor- related with that of Ipa protein secretion after induction of the machinery with Congo red (Fig. 3 B). The spa47 gene encodes a protein related to the b sub- unit of F1 mitochondrial ATPases which is essential for se- cretion (Venkatesan et al., 1992; Sasakawa et al., 1993). This led us to investigate the effect of sodium azide, an inhibitor of oxidative phosphorylation (to which RBCs should be insensitive), on secretion and hemolysis. Addi- tion of increasing amounts of azide progressively inhibited Ipa secretion induced by Congo red (Fig. 3 D), indicating that Ipa secretion required energy. However, the presence of 25 mM azide inhibited Congo red–induced secretion of IpaB z20-fold but decreased hemolysis only 4-fold (Fig. 3, C and D). This suggested that not all of the Ipa proteins that could be secreted were released during hemolysis. In- deed, during a hemolytic assay, only a small amount of Ipa proteins were recovered in the medium, the rest of these proteins remaining within the bacterium (inaccessible to a protease added to the medium; Fig. 3 F and not shown). In addition, not all the released Ipa proteins may be required to observe full hemolysis, for which a single pore per RBC is in theory sufficient. In support of this, pretreatment of bacteria with Congo red, which released z50% of Ipa pro- teins (Fig. 3 F), had no effect on the efficiency of a subse- quent hemolytic assay, even when novel protein synthesis was blocked (not shown). This may explain why lysed RBC membranes contained ,0.1% of the amount of Ipa proteins that were initially present in bacteria. In summary, Ipa secretion was an active process and secretion at the moment of contact was necessary for lysis. To determine whether the proteins responsible for he- Blocker et al. Type III Secreton-mediated Ipa Insertion into Membranes 687