Liposomes Recruit IpaC to the Shigella flexneri Type III Secretion Apparatus Needle as a Final Step in Secretion Induction

Materials.The S. flexneri ipaC null mutant (strain SF621) and ipaA-ipgD null mutant were from P. J. Sansonetti (Institut Pasteur, Paris, France) (18). The S. flexneri mxiH null strain was generated by A. Allaoui (Brussels, Belgium) (3). Antibodies were from E. V. Oaks, Walter Reed Army Institute for Research (Silver Springs, MD). Sheep and goat red blood cells were from the Colorado Serum Company (Denver, CO). Alexa Fluor-labeled secondary antibodies were from Molecular Probes (Eugene, OR). DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPG {1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]}, and SM were from Avanti Polar-Lipids, Inc. (Alabaster, AL). Hank's balanced salt solution (HBSS) and Chol were from the Sigma Chemical Co. (St. Louis, MO). Minimum essential medium with Earle's salts (MEME) was from Gibco-BRL. All other chemicals were reagent grade.

Preparation of liposomes from sheep erythrocytes.Liposomes were prepared from sheep erythrocytes as described previously (2, 12). Briefly, 15 ml of erythrocyte suspension was collected by centrifugation, washed three times with 10 mM sodium phosphate (pH 7.0)-150 mM NaCl (phosphate-buffered saline [PBS]), and subjected to hypotonic lysis with deionized water. The resulting erythrocyte ghosts were collected by centrifugation and washed four times with PBS. The ghosts were used directly to assay for IpaC mobilization to the S. flexneri surface, or the lipids were extracted with chloroform and methanol (2). The extracted lipid fraction was then dried under vacuum conditions and resuspended in 2 ml of PBS. To ensure that the procedure resulted in liposome preparations of uniform size, the suspension was pulse sonicated, centrifuged, and extruded 10 times at 45°C through a 100-nm-pore-size membrane (1).

Preparation of liposomes having a defined composition.Commercial lipids were resuspended in chloroform and mixed in the following ratios to achieve 1 mg/ml: (i) DOPC; (ii) DOPG:DOPC (1:3); (iii) SM:DOPC (1:3); (iv) Chol:DOPC (1:3); and (v) SM:Chol:DOPC (1:1:2). Lipid mixtures were dried under vacuum conditions. The resulting lipid films were resuspended in 1 ml of PBS, sonicated to form liposomes, and extruded through a 100-nm-pore-size membrane as described above (1).

Incubation of S. flexneri with liposomes.To determine the ability of liposomes to promote the mobilization of IpaC to the S. flexneri TTSA needle tip, a modification of the standard contact-mediated hemolysis assay was used (21, 22). Briefly, S. flexneri were grown to the log phase at 37°C in tryptic soy broth (TSB) containing 0.1% DOC and 50 μg/ml kanamycin (100 μg/ml ampicillin was included when appropriate). The bacteria were collected, resuspended in 200 μl of PBS, and combined with either 200 μl of erythrocyte-derived liposomes or defined liposomes. After 90 min at 37°C, the bacteria and associated proteins were fixed with formaldehyde and the reaction was quenched with glycine. The presence of IpaC on the S. flexneri surface was determined by immunofluorescence microscopy. To reduce exposure risks, an ipaA-ipgD null strain was initially used for many of these experiments. This strain has a wild-type phenotype for secretion control, invasion, and hemolysis (18). To determine the region of IpaC required for its recruitment to and maintenance at the S. flexneri TTSA needle tip, previously characterized ipaC mutants of S. flexneri ipaC null strain SF621 were used (21, 26).

Fluorescence microscopy.Bacteria were dried on a coverslip, blocked with 1% bovine serum albumin in PBS, and immunolabeled with rabbit polyclonal anti-MxiH, anti-IpaB, and/or mouse monoclonal anti-IpaC antibodies. Alexa Fluor 488 goat anti-rabbit and/or Alexa Fluor 488 or 568 anti-mouse immunoglobulin G (IgG) was utilized for fluorescence detection. Imaging was performed using an Olympus IX-81 microscope equipped with 488- and 568-nm-excitation laser lines, a 60× objective (numerical aperture, 1.45), and a Hamamatsu electronic microscopy charge-coupled device. Phase-contrast images were also obtained. Student's t tests were performed using the resulting data, as needed.

Electron microscopy.When mxiH is subcloned behind the inducible lac promoter of pRK2 and protein expression is induced in mxiH null mutant SH116 (giving mxiH pRK2/SH116), long needles (some more than 300 nm in length) are generated, making them more readily visible on the bacterial surface (11, 25). This strain was grown at 37°C in TSB containing 100 μg/ml ampicillin, 50 μg/ml kanamycin, 0.1% DOC, and 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside). Cells were harvested by centrifugation, resuspended in the appropriate liposome preparation, and incubated for 90 min at 37°C. Glutaraldehyde was added to achieve a final concentration of 4%, and the samples were placed on either carbon-Formvar or holey-carbon electronic microscopy grids. The samples were treated with rabbit polyclonal anti-IpaB antibodies followed by 5-nm-diameter gold-labeled goat anti-rabbit IgG F(ab′)₂ and with mouse monoclonal anti-IpaC antibodies followed by 10-nm-diameter gold-labeled anti-mouse IgG F(ab′)₂. Negative staining with 2% uranyl acetate permitted visualization of the samples. Samples were imaged using an FEI Tecnai G² field emission transmission electron microscope at an electron acceleration voltage of 200 KeV.

Induced secretion of TTSS proteins.Induction of type III secretion in S. flexneri can be examined by measuring protein secretion following the addition of Congo red (CR). CR stimulates a rapid burst of protein secretion by the S. flexneri TTSS, thus mimicking the response seen upon host cell contact (11). Bacteria were grown in a single broth culture, and then the culture was divided equally for incubation with liposomes or other treatments to ensure that identical numbers of bacteria were represented in the Western blot analysis. After a 20-min incubation with various liposome preparations, bacteria were removed by centrifugation. The resulting supernatant fraction from identical numbers of cells was assessed for IpaB, IpaC, and IpaD secretion by Western blot analysis using rabbit anti-IpaC antibodies and mouse monoclonal anti-IpaB and -IpaD antibodies and the corresponding Alexa Fluor 680 goat anti-mouse IgG and Alexa Fluor 800 goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Images were obtained and relative protein ratios quantified using an Odyssey Infrared Imager (LI-COR, Lincoln, NE).

Invasion of cultured HeLa cells.The ability of S. flexneri to invade cultured cells was measured by a modified gentamicin protection method (11). To assess the impact of IpaB localization on invasion, these assays were performed using HBSS to avoid the effects that MEME has on tip complex composition (data not shown). Bacteria for these assays were grown under several sets of conditions, including those that promote localization of IpaB or of IpaB plus IpaC on the bacterial surface. Student's t tests were performed using the resulting data as needed.

IpaC can be induced to localize to the S. flexneri surface as part of the TTSA needle tip complex.We have previously shown that IpaD alone localizes to the TTSA needle tip of S. flexneri in the log phase in TSB. IpaB colocalizes with IpaD only when S. flexneri bacteria are incubated in the presence of DOC during the log phase; however, IpaC has not previously been observed on the S. flexneri surface (7, 19, 24). Because IpaC is required for translocon pore formation and contact-mediated hemolysis, it is likely that it must become a part of the tip complex upon contact with erythrocytes. Therefore, the contact-mediated hemolysis assay was modified to visualize IpaC on the bacterial surface. Bacteria were grown in the presence of DOC to the log phase and then incubated with the erythrocytes. Indeed, mouse anti-IpaC antibodies and Alexa Fluor 488 goat anti-mouse IgG detected IpaC on the S. flexneri surface; however, the released hemoglobin interfered with microscopy (data not shown). Confirmation of this event was achieved when erythrocyte ghosts were used. Under these conditions, IpaC could be clearly visualized on the surface of 25% of the observed S. flexneri bacteria (Fig. 1B). Furthermore, IpaC was visible only in areas where IpaB was also detected (Fig. 1D), indicating colocalization.

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FIG. 1.

Immunofluorescence microscopy was used to demonstrate the presence of IpaC on the S. flexneri surface following incubation with sheep erythrocyte ghosts. Panel A shows an immunofluorescence micrograph of IpaB localization. IpaC localization is seen in panel B, and a merged image composed of the images presented in panels A and B is seen in panel D. Panel C shows a differential interference contrast micrograph.

The central hydrophilic region of IpaC may be important for its maintenance at the S. flexneri surface.We have previously shown that IpaC possesses distinct functional domains (9, 21, 26). Therefore, we sought to use these constructs to determine the regions within IpaC that are required for its stable maintenance at the S. flexneri surface. Erythrocyte liposomes were used in these experiments and induced IpaC surface localization in 20% of the observed bacteria. Because the TTSS signal is located at the IpaC N terminus, an IpaC^Δ1-62 mutant lacking residues 1 to 62 (IpaC^ΔI; 21) is not secreted. Thus, as expected, IpaC^Δ1-62 failed to be detected on the S. flexneri surface after addition of the liposomes (Fig. 2). Although an IpaC^Δ176-260 mutant lacking central hydrophilic residues 176 to 260 (IpaC^ΔII; 21) exhibits residual invasiveness (10%) in S. flexneri SF621 (21), the mutant protein did not associate with the S. flexneri surface (Fig. 2). In contrast, truncations of the C-terminal portion of IpaC, including IpaC^Δ260-363 (IpaC^ΔIII; 21) and IpaC^Δ344-363 (an effector region deletion referred to as 1035; 26), did not prevent maintenance of IpaC on the S. flexneri surface after the addition of erythrocyte-derived liposomes. Similarly, an IpaC^Δ62-170 mutant with its hydrophobic region removed (IpaC^ΔH; 21) still associated with the S. flexneri surface (Fig. 2). This mutant is noninvasive and has greatly reduced stability and secondary structure content as a purified recombinant protein (13). However, if the secondary structure of residues 176 to 260 remains intact, stability may be increased in vivo by its inclusion as a part of the tip complex. Based on these findings, it is possible that the central hydrophilic portion of IpaC is involved in forming a stable interaction with IpaB on the surface of S. flexneri following contact with host cells.

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FIG. 2.

Immunofluorescence microscopy (IFM) was used to determine whether different IpaC deletion mutants could be retained at the S. flexneri surface following incubation with sheep erythrocyte-derived liposomes. No IpaC was observed on the surface of S. flexneri ipaC null strain SF621 bacteria or of bacteria that synthesized IpaC with either an IpaC^Δ1-62 or IpaC^Δ176-260 deletion mutant. IpaC^Δ260-363, IpaC^Δ344-363, and IpaC^Δ62-170 deletion mutants all stained positively for IpaC on the S. flexneri surface after incubation with liposomes. WT, wild type.

The importance of liposome composition in IpaC surface localization.The sheep erythrocyte-derived liposomes that mobilized IpaC to the S. flexneri surface are reported to have a high content of SM and Chol (12). With this in mind and to better define the requirements for IpaC recruitment to the S. flexneri TTSA surface, defined liposomes composed of DOPC, SM, and Chol were generated. Indeed, the SM/DOPC/Chol (1:2:1) liposomes were efficient at promoting the stable recruitment of IpaC to the S. flexneri surface (Fig. 3). This is consistent with work of van der Goot et al. that showed that liposomes rich in SM and Chol induced TTSS-dependent secretion of IpaB (27).

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FIG. 3.

Immunofluorescence microscopy (IFM) was used to demonstrate the recruitment of IpaC to the S. flexneri surface in the presence or absence of liposomes composed of DOPC, SM, and Chol. The negative control was formed by incubation of bacteria with PBS plus DOC.

To further investigate the impact that defined liposomes have on IpaC surface localization in S. flexneri, a variety of liposomes prepared using DOPC, DOPG, Chol, or SM or combinations of these were examined. A summary of those results is given in Table 1. Although all of the lipids tested were found to increase IpaC recruitment to the S. flexneri surface, the results with respect to efficiency of recruitment differed depending upon the liposome composition (Table 1). Whereas 10% of the bacteria exposed to DOPC or Chol alone exhibited surface-localized IpaC, nearly 30% of the bacteria incubated with either SM or mixed liposomes composed of SM/DOPC/Chol (1:2:1) exhibited IpaC surface localization. These data suggest an important role for SM in promoting the recruitment of IpaC to the S. flexneri surface. The particulate nature of the liposomes is most likely responsible for the relatively small percentage of bacteria with IpaC detected on their surfaces.

IpaC recruitment to the S. flexneri surface occurs concomitantly with induction of type III secretion.Because SM and SM/DOPC/Chol liposomes increase the surface localization of IpaC and because liposomes containing SM and Chol were previously implicated in type III secretion induction (27), we examined the effects of the presence of erythrocyte and defined liposomes on the induced secretion of IpaB and IpaC by identical numbers of S. flexneri bacteria. As shown in Fig. 4, erythrocyte liposomes induced S. flexneri Ipa protein secretion, whereas a synthetic liposome mixture of SM/DOPC/Chol induced even greater secretion of Ipa proteins (Fig. 4). Neutral liposomes consisting only of DOPC had little or no effect on IpaB or IpaC secretion, but the addition of the acidic lipid DOPG also increased type III secretion (Fig. 4). When the data from this representative immunoblot were quantified (Fig. 4B and C), IpaB and IpaC levels reflected the qualitative trend represented in Fig. 4A, with the results for IpaC being the more striking (Fig. 4C). It thus appears that once IpaC is recruited to the S. flexneri surface, type III secretion is induced for all the Ipa proteins and, presumably, for the subsequently secreted TTSS effectors.

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FIG. 4.

Immunoblot analysis of IpaB and IpaC secreted by S. flexneri in the presence of different liposome types. (A) Results for PBS plus DOC (the control for uninduced secretion), liposomes derived from erythrocytes (red blood cells [RBC]), or defined liposomes composed of DOPC, DOPG, SM, and/or Chol. (B and C) Densitometry scans of IpaB (B) and IpaC (C) present in the immunoblot represented in panel A provide a quantitative measure of Ipa protein secretion by S. flexneri in the presence of different lipid preparations. The intensity of the signal at 680 nm was used to quantify the Alexa Fluor 680-labeled goat anti-rabbit IgG used to detect rabbit anti-IpaB antibodies. The intensity at 800 nm was used to quantify the Alexa Fluor 800-labeled goat anti-mouse IgG used to detect mouse anti-IpaC antibodies.

IpaC is located at the TTSA needle tip after liposome addition.We previously used immuno-gold labeling and electron microscopy to demonstrate that both IpaD and IpaB are associated with the TTSA needle tip (7, 19). To confirm that IpaC localizes to the TTSA needle tip complex upon lipid-induced type III secretion, this methodology was employed again (Fig. 5). In order to reduce the nonspecific background observed during the uranyl acetate staining of bacteria after erythrocyte ghosts or lipids were added, defined liposomes were used to promote IpaC mobilization. The liposome preparation used was DOPC/SM/Chol (1:2:1) based on the data presented in Table 1. Following incubation of bacteria with liposomes, they were doubly immuno-gold labeled using rabbit anti-IpaB antibodies with 5-nm-diameter gold-labeled goat anti-rabbit IgG F(ab′)₂ for IpaB and mouse monoclonal anti-IpaC antibodies with 10-nm-diameter gold-labeled goat anti-mouse IgG F(ab′)₂ for IpaC (Fig. 5). The resulting micrographs show that IpaB and IpaC are present at the tip of the needles, which confirms the colocalization shown by immunofluorescence (Fig. 1). Immuno-gold labeling appears only at the distal end of the needle structures, suggesting a specific association of these proteins following secretion induction. It thus appears that IpaC, together with MxiH, IpaD, and IpaB, contributes to the formation of a quaternary complex at the needle tip of the S. flexneri TTSA following incubation of the bacteria with DOC and liposomes.

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FIG. 5.

Transmission electron microscopy of TTSA needles protruding from the S. flexneri surface (see white arrowheads) after incubation with liposomes composed of SM, DOPC, and Chol (1:2:1) was used with immuno-gold particle labeling. The presence of 5-nm-diameter gold particles indicates labeling for IpaB (*) and that of 10-nm-diameter gold particles indicates IpaC labeling (**). No IpaB or IpaC labeling was observed for an mxiH null mutant (not shown). IpaB labeling but no IpaC labeling was seen for the ipaC null SF621 prior to the addition of liposomes (not shown).

The status of the S. flexneri TTSA needle tip influences the efficiency of bacterial invasion of HeLa cells.We have now shown that the maturation process of the TTSA tip complex occurs in three steps. For step 1, IpaD alone is present at the needle tip of S. flexneri bacteria grown to the log phase in TSB. Addition of DOC induces step 2 by mobilizing IpaB to create the MxiH-IpaD-IpaB ternary tip complex. Here we show that step 3 is the mobilization of IpaC to the needle tip complex after incubation of bacteria with the ternary complex on their surfaces with specific liposomes. To understand the importance of these needle tip complexes with respect to S. flexneri invasion of cultured cells, bacteria were grown to the mid-log phase in TSB, exposed to conditions known to promote either IpaB or IpaB-IpaC recruitment to the S. flexneri surface, and then tested for their relative levels of invasion of HeLa cell monolayers. Since the presence of MEME, like that of DOC, results in the recruitment of IpaB to the bacterial surface without inducing type III secretion (unpublished observations), the gentamicin-protection assay was performed with HBSS to avoid possible bias in determining the importance of DOC-induced IpaB surface presentation for S. flexneri invasiveness. When the bacteria were incubated for 30 min in the absence or presence of DOC after growth in TSB, clear differences in invasion efficiency were observed (Table 2). The addition of DOC enhanced S. flexneri invasion more than threefold, indicating that the DOC-mediated recruitment of IpaB to the TTSA needle tip resulted in the formation of a productive complex at the tip of the TTSA needle and that the ternary complex is important in host cell invasion (Table 2). This finding supports the proposal that the ternary complex present on the S. flexneri surface is physiologically relevant. Thus, the DOC-mediated events represent the second step in TTSA complex maturation and may be a prerequisite step for type III secretion induction.

The MxiH-IpaD-IpaB complex appears to represent the mature tip complex that is required for optimally productive host cell contact. Thus, the subsequent membrane-IpaB interaction would logically represent the type III secretion trigger that promotes IpaC recruitment to the TTSA needle tip and the simultaneous injection of IpaB and IpaC into the host cell membrane and cytoplasm. If this is the case, when IpaC localizes prematurely to the tip, interaction with the host cell would no longer be optimal and there would be a reduction in invasion. In line with this, when the bacteria were incubated in DOC combined with the DOPC/SM/Chol liposomes, invasion levels still increased relative to those seen with S. flexneri incubated in the absence of any supplement; however, the levels were reduced relative to those seen with bacteria incubated with DOC alone (Table 2). When known artificial inducers of S. flexneri type III secretion, namely, CR and serum, were incubated with the bacteria (with no DOC present), invasion levels also decreased relative to the levels seen with the DOC control (Table 2). In fact, incubation with serum decreased invasion levels to below those seen without DOC. Based on these findings, it is possible to suggest that the optimal TTSA tip complex for S. flexneri invasion of cultured cells is the ternary complex that includes MxiH, IpaD, and IpaB. The subsequent induction and secretion of IpaC comprise the final step in triggering the induction of full type III secretion.