Figure 3. Parallel analysis of hemolysis and Ipa protein secretion. (A) The hemolytic assay was performed with bac- teria and sheep RBCs incubated for 1 h at the indicated temperatures. (B) Congo red–induced secretion in the absence of any RBCs was measured at 25, 30, and 378C. P stands for bacterial pellet and S for supernatant. (C) Wild-type bacteria were mixed with sheep RBCs in the pres- ence or in the absence of the indicated concentrations of sodium azide and/or 1 mg/ml of proteinase K. As a control for the activity of the protease, RBCs were also exposed to a bacterial culture super- natant containing E. coli hemolysin A (HylA) or to the same supernatant to which proteinase K had been added (HylA1protease). (D) Congo red–induced secretion was measured in the presence of increasing concentrations of azide. IpaB and IpaC content of the supernatants is shown. (E) Wild-type or mxiD bacteria expressing the afimbrial adhesin AfaE were mixed with human RBCs in the presence or in the absence of 30 mM Congo red with or without centrifugation at 1,500 g for 10 min at 108C, and incu- bated for 1 h at 378C. (F) Wild-type (w.t.) or mxiD bacteria expressing AfaE were incubated with human RBCs, saline alone, saline with 30 mM Congo red (sa- line 1 CR), or RBCs with Congo red (RBCs 1 CR). They were treated as in the hemolytic assay, as was an identical volume of bacteria (bacterial extract) at the same concentration. Subsequently, 20 ml of the supernatants (for the bacterial extract, the bacterial pellet was resus- pended) were separated on a 10% SDS- PAGE gel, blotted and probed with anti- IpaB and anti-IpaC mAbs, respectively. molysis ever became exposed to the external medium, we added antibodies to IpaB, IpaC, and/or IpaD to the hemolytic reaction. These reagents had no effect on hemolysis at any concentration tested (up to 100 mg/ml), whether alone or in combination or even upon preincuba- tion of bacteria (not shown). Next we added 1 mg/ml of proteinase K to the assay. This led to only a minor de- crease in hemolytic efficiency (Fig. 3 C). This amount of protease instantly degraded E. coli hemolysin A, com- pletely preventing hemolysis mediated by this protein (Fig. 3 C). Interestingly, the inhibitory effects of azide and proteinase K on hemolysis were additive (Fig. 3 C). This suggested that azide was acting by reducing secretion and proteinase K by degrading some of the secreted proteins as they were exiting the bacterium. Individual pretreat- ment of RBCs and bacteria with azide and proteinase K did not affect hemolysis (not shown), suggesting that any Ipa proteins at the bacterial surface before contact were not involved in hemolysis. These data confirmed that Ipa protein secretion and hemolysis were coupled in time and suggested that the process of Ipa protein transfer into the target membrane was poorly accessible from the sur- rounding medium. To investigate the requirement for contact between bac- teria and RBCs to obtain hemolysis, we used a derivative of S. flexneri carrying the AfaE afimbrial adhesin from en- teropathogenic E. coli. This adhesin binds the decay accel- erating factor, a ubiquitous surface glycoprotein, and con- fers upon bacteria the ability to hemagglutinate human RBCs (Pham et al., 1995). Despite their close binding to RBCs, these bacteria were unable to lyse RBCs in the ab- sence of centrifugation (Fig. 3 E). However, upon centrifu- gation, they lysed RBCs efficiently. Since the adhesion mediated by AfaE was not sufficient to induce extensive Ipa protein secretion (Fig. 3 F), AfaE-expressing bacteria were incubated at 378C in the presence of human RBCs The Journal of Cell Biology, Volume 147, 1999 688