The Tripartite Type III Secreton of Shigella flexneri Inserts Ipab and Ipac into Host Membranes

Red Blood Cell Membrane Isolation

Bacteria and sheep RBCs were prepared as described above, except that they were resuspended in Tris-saline at 2.10¹¹ and 1.5.10¹⁰ cells/ml, respectively. Hemolytic reactions were prepared in 50-ml conical tubes with 600 μl RBCs, 120 μl bacteria, 860 μl Tris-saline, and a protease inhibitor cocktail (PIC¹; Complete™, Boehringer Mannheim). Samples were centrifuged at 2,000 g at 4°C for 10 min, incubated at 37 or 4°C for 1 h, resuspended by vortexing, and recentrifuged. Hemolysis was assessed spectrometrically as above. 800 μl of distilled water was added to each sample to lyse all RBCs nonspecifically (so that lysed membranes could be isolated even from those samples where no hemolysis had occurred), and these were vortexed and centrifuged again to remove bacteria. 2 ml of supernatant was collected, adjusted to 2.4 ml with Tris-saline and brought to 46% sucrose with 7 ml of 62% sucrose in Tris-saline containing the PIC. The mixtures were deposited at the bottom of SW41 centrifuge tubes (Beckman) and layered with 2 ml of 44% and then 25% sucrose in Tris-saline containing the PIC. Gradients were centrifuged at 15,000 g for 16 h at 4°C. The material at the 44/25% sucrose interface was collected, diluted in Tris-saline and concentrated by centrifugation at 450,000 g for 20 min 4°C in a TLA 100.3 rotor (Beckman). The pellets were resuspended in a minimal volume of buffer. Such samples were checked by transmission electron microscopy but no contaminating bacteria or bacterial ghosts were seen. When ³⁵S-labeled bacteria were used less than 0.01% of the initial radioactive input was recovered in RBC membranes. The protein content of the samples was estimated using Bradford's assay (Bio Rad) and an equivalent protein amount of each was separated by SDS-PAGE and Western blotted.

To assess the strength of association of Ipa proteins with RBC membranes, 100 μl of purified Shigella-lysed RBC membranes were incubated at 4°C for 1 h in Tris-saline or Tris-saline with 5 M NaCl, 8 M urea, or 0.2 M carbonate, pH 11. 300 μl of 62% sucrose in Tris-saline was then mixed with the membranes that were deposited at the bottom of 0.8-ml SW55 tubes (Beckman) and overlaid with 150 and 100 μl of 44 and 25% sucrose in Tris-saline. The gradients were spun overnight at 4°C at 15,000 g. The top 150 μl was collected from the gradients, diluted in Tris-saline, and concentrated by pelleting in a TLA 100.2 rotor for 20 min at 450,000 g at 4°C. The pellets were resuspended in a minimal volume of buffer.

Measurement of Ipa Protein Secretion

Antibodies

mAbs to IpaB and IpaC were a gift from Armelle Phalipon. mAbs against IpaA and IpgD were generated by immunizing female BALB/c mice with Ipa protein containing supernatants obtained after induction of secretion in Shigella flexneri with Congo red. In brief, 10–20 mg of protein was injected subcutaneously into both hind legs (5 injections at 3-d intervals) and the popliteal lymph nodes were fused with X63Ag8 lymphoma cells according to standard protocols. Hybridoma supernatants were screened by ELISA and immunoblotting and clones positive for IpaA and IpgD were subcloned by limiting dilution.

Immunoblotting

Conventional SDS-PAGE was performed and immunoblotting was performed using a semi-dry apparatus onto nitrocellulose or PVDF membranes and developed using the ECL or ECL Plus kits (Amersham) and X-OMAT film (Kodak).

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 gradient. The protein content of the membranes was separated 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 37°C, but not in fractions recovered after incubation with the mxiD mutant at 37°C or with the wild-type at 4°C (Fig. 2 A). By semiquantitative immunoblotting, the amount of IpaB, IpaC, IpgD, and IpaA recovered in the membrane fraction corresponded to ∼0.1% of the total amount of these proteins present initially 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).

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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 isolated 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 contact hemolysis with wild-type, ipaC, or mxiD strains at 37°C or 4°C. The blot probed with the anti-IpaB antibody was overexposed 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 37°C 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 isolated after contact with the wild-type at 37°C 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 measured after a second floatation in a sucrose density gradient. 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 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. Membranes 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, respectively. This indicated that IpaA and IpgD were not required for association of IpaB and IpaC with the membranes 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). Although this amount was vastly reduced as compared with that observed with the wild-type, it was significant as incubation of RBCs with the mxiD mutant at 37°C or with the wild-type at 4°C led to undetectable amounts of these proteins in the membranes. The small amount of IpaB associated 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 presence 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 relationship between hemolysis and Ipa protein secretion. We investigated the efficiency of hemolysis after incubation for 1 h between 4 and 42°C. No hemolysis was observed below 25°C and the reaction proceeded normally above 30°C (Fig. 3 A). The temperature dependence of lysis correlated with that of Ipa protein secretion after induction of the machinery with Congo red (Fig. 3 B).

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Figure 3

Parallel analysis of hemolysis and Ipa protein secretion. (A) The hemolytic assay was performed with bacteria 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 37°C. P stands for bacterial pellet and S for supernatant. (C) Wild-type bacteria were mixed with sheep RBCs in the presence 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 supernatant containing E. coli hemolysin A (HylA) or to the same supernatant to which proteinase K had been added (HylA+protease). (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 μM Congo red with or without centrifugation at 1,500 g for 10 min at 10°C, and incubated for 1 h at 37°C. (F) Wild-type (w.t.) or mxiD bacteria expressing AfaE were incubated with human RBCs, saline alone, saline with 30 μM Congo red (saline + CR), or RBCs with Congo red (RBCs + CR). They were treated as in the hemolytic assay, as was an identical volume of bacteria (bacterial extract) at the same concentration. Subsequently, 20 μl of the supernatants (for the bacterial extract, the bacterial pellet was resuspended) were separated on a 10% SDS-PAGE gel, blotted and probed with anti-IpaB and anti-IpaC mAbs, respectively.

The spa47 gene encodes a protein related to the β subunit of F1 mitochondrial ATPases which is essential for secretion (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. Addition 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 ∼20-fold but decreased hemolysis only 4-fold (Fig. 3C and Fig. D). This suggested that not all of the Ipa proteins that could be secreted were released during hemolysis. Indeed, 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 ∼50% of Ipa proteins (Fig. 3 F), had no effect on the efficiency of a subsequent 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 hemolysis 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 μg/ml), whether alone or in combination or even upon preincubation of bacteria (not shown). Next we added 1 mg/ml of proteinase K to the assay. This led to only a minor decrease in hemolytic efficiency (Fig. 3 C). This amount of protease instantly degraded E. coli hemolysin A, completely 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 pretreatment 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 surrounding medium.

To investigate the requirement for contact between bacteria and RBCs to obtain hemolysis, we used a derivative of S. flexneri carrying the AfaE afimbrial adhesin from enteropathogenic E. coli. This adhesin binds the decay accelerating factor, a ubiquitous surface glycoprotein, and confers 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 absence of centrifugation (Fig. 3 E). However, upon centrifugation, 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 37°C in the presence of human RBCs 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 hemolysis 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 centrifugation forces at 10°C and then either incubated at 37°C 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 ). 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). This indicated that centrifugation increased the surface of bacteria in contact with RBCs.

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Figure 4

Electron microscopy analysis of the interaction between bacteria and RBCs. Derivatives of wild-type bacteria expressing 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 10°C. 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 μm.

Although samples treated at 160 and 630 g looked similar, lysis occurred in the latter but not in the former (Table ), 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 (Shotton 1998). We compared the lysis efficiency of wild-type Shigella and that of an rfe mutant that expressed a complete LPS core but lacked the O-antigen (Sandlin et al. 1995; Rathman, M., P.J. Sansonetti, C. Parsot, and G. Tran Van Nhieu, submittedmanuscript 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 hemagglutinating RBCs (not shown), as the site on the DAF molecule 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 exposure to bacteria at 160 g (Fig. 4 C and Table ). 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 distance and the charge of the target membrane may be critical for induction of hemolysis.

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 assembled. This is consistent with evidence that MxiG is unstable in the mxiJ and mxiD mutants (Bahrani, F., and C. Parsot, unpublished observation). The secreton structure is composed of three different parts: (a) an external needle, (b) a neck, and (c) a large proximal bulb. In our images, it is difficult to discern the two bacterial membranes and thus to judge the localization of the bulb. Nevertheless the length of the neck domain is sufficient to traverse both membranes, implying that the bulb lies within bacterial cytosol. This putative cytoplasmic part was not recovered by Kubori et al. 1998 in secreton preparations, which were purified using a protocol derived from that for isolation of flagellar basal bodies (Aizawa et al. 1985). A cytoplasmic structure (other than the C-ring) is known to be associated with the bacterial flagellum and thought to contain the cytoplasmic F1-type ATPase FliI as well as several other components. This structure is not recovered in purified basal body preparations and has never been visualized (MacNab 1996; Minamino and MacNab 1999).

Inactivation of spa47, the homologue of the flagellar biosynthesis gene fliI, abolishes secretion. However, so far, it was unclear whether this ATPase was required for assembly and/or activity of the secretion machinery (Dreyfus et al. 1993). We showed that type III secretons were assembled by S. flexneri before host contact and that their structure did not grossly change when secretion was activated (in the presence of Congo red or in the ipaD or ipaB mutants). We also showed that secretion required energy and temperatures above 30°C (Fig. 3B and Fig. D). Low temperatures did not disassemble secretons (not shown). Thus ATP hydrolysis and conformational change(s) are likely to be required for Ipa secretion, as for flagellin export (Vogler et al. 1991). We propose that Spa47 is the azide-sensitive element of the secretion machine.

This work is dedicated to the memory of Thomas E. Kreis. We are grateful to Jacomine Krinjse-Locker for instructions on the use of neuraminidase and to Michel Chignard and Jean-Claude Manuguerra for human blood and HA assistance. A. Blocker wishes to thank in particular Philippe Clerc for his recollections of 15-yr-old unpublished data on the contact hemolytic activity of S. flexneri. She acknowledges Chantal LeBougenec, Tony Pugsley, and Elie Dassa for local advice and reagents and Lina Bernardini, Guy Cornelis, Mike Donnenberg, Bob MacNab, Ira Mellman, and Dennis Thomas for inspiring discussions, communication of results before publication, and/or critical reading of the manuscript. Finally, she is indebted to Gisou van der Goot, Gareth Griffiths, David Holden, and Paul Lazarow for their supportive friendship.

A. Blocker and K. Niebuhr were supported by National Institutes of Health and European Molecular Biology Organization postdoctoral fellowships, respectively. V. Cabiaux is a research associate from the Fonds National de la Recherche Scientifique, Belgium.