structure-function analysis of the shigella virulence factor ipabinfections with enterobacteria of...

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.4.1269–1276.2001

Feb. 2001, p. 1269–1276 Vol. 183, No. 4

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Structure-Function Analysis of the Shigella VirulenceFactor IpaB

ANDREA GUICHON,1 DAVID HERSH,1 MARK R. SMITH,2 AND ARTURO ZYCHLINSKY1*

The Skirball Institute and Department of Microbiology, New York University Medical Center, New York, New York10016,1 and Intramural Research Support Program, SAIC Frederick, National Cancer Institute, Frederick Cancer

Research and Development Center, Frederick, Maryland 217022

Received 18 October 2000/Accepted 16 November 2000

Infection by the gram-negative bacterium Shigella flexneri results in dysentery, an acute inflammatory diseaseof the colon. Essential events in the pathogenesis of Shigella infections include bacterial invasion of epithelialcells, escape from the phagosome, and induction of apoptosis in macrophages. The Shigella virulence factorinvasion plasmid antigen B (IpaB) is required for all of these processes. Induction of apoptosis is dependenton IpaB binding to the cysteine protease caspase-1 (Casp-1). The activation of this enzyme triggers bothapoptosis and release of the proinflammatory cytokine interleukin-1b. Several IpaB mutants were generatedto correlate function with protein subdomains. We determined that the N-terminal portion of IpaB is necessaryfor stable expression of IpaB. A putative amphipathic a-helical domain preserves the structure of IpaB. Wefound 10 consecutive residues within the amino terminus of the hydrophobic region that play a critical role ininvasion, phagosomal escape, and cytotoxicity. An IpaB mutant carrying a mutation in this region binds toCasp-1 yet is not cytotoxic, even following direct delivery to the macrophage cytoplasm. These results indicatethat the association between IpaB and Casp-1 is only a step in the activation of macrophage apoptosis.

Infections with enterobacteria of the genus Shigella causedysentery, a severe bloody diarrhea. Dysentery is characterizedby an acute inflammation of the colon with mucosal erosion(11, 23). Development of shigellosis requires bacterial pene-tration across the intestinal epithelial barrier via M cells. Uponreaching the underlying lymphoid follicles, the bacteria areengulfed by resident macrophages (30, 38). Once inside a mac-rophage, Shigella escapes from the phagosome into the cyto-plasm and kills this cell by inducing apoptosis (42). The dyingmacrophage releases mature interleukin-1b (IL-1b) (40) andIL-18 (32), two cytokines important in the initiation of inflam-mation (34). Shigella also invades epithelial cells throughpathogen-directed endocytosis (23). Invasion of enterocytesand bacterial cell-to-cell spread enhance tissue damage (20,33).

The Shigella invasion plasmid antigens B (IpaB), IpaC, andIpaD are required for epithelial cell entry and phagosomeescape (14, 23). However, IpaB alone is sufficient to activatemacrophage apoptosis (5). The Ipa proteins interact with hostcells upon being secreted by a type III secretion apparatus(26). In the macrophage cytoplasm, IpaB binds to caspase-1(Casp-1; also called IL-1b converting enzyme [ICE]), a pro-apoptotic and proinflammatory cysteine protease that cleavesIL-1b and IL-18 to their biologically active forms (6, 37). Theactivation of Casp-1 leads to macrophage apoptosis by an asyet ill-defined pathway (15, 40). Casp-1-deficient macrophages’resistance to Shigella-induced cell death demonstrates thatCasp-1 is essential for this process (16).

IpaB contains a hydrophobic region (amino acids [aa] 310 to430) that contains two putative membrane-spanning domains

(aa 313 to 346 and 400 to 423) (3). IpaB is homologous to theSalmonella invasion protein B (SipB) and to the Yersinia outerprotein B (YopB). The similarity of IpaB to these proteins isparticularly high in the hydrophobic region (65 and 30% iden-tity to SipB and YopB, respectively) (9, 18). Interestingly, SipBis required for Salmonella invasion of epithelial cells (12, 17,18) and interacts with Casp-1 to trigger macrophage apoptosis(13). Unlike IpaB and SipB, YopB is not the effector moleculeof Yersinia-induced apoptosis (27, 28), and its function remainscontroversial (10, 21).

In this study, we investigate the regions of IpaB that arenecessary for invasion, phagosome escape, Casp-1 binding, andcytotoxicity. We generated ipaB mutants which were analyzedby functional complementation of a nonpolar ipaB deletionmutant strain (SF620) (25) or by testing purified recombinantIpaB mutant proteins in functional and binding assays. Wefound a region at the amino terminus of the hydrophobicdomain that is required for invasion of epithelial cells, escapefrom the phagosome, and induction of cell death. Unexpect-edly, this region is dispensable for Casp-1 binding.

MATERIALS AND METHODS

Bacterial strains and cell culture. The Shigella flexneri wild-type strain M90T(serotype 5A) was described previously (35). The ipaB deletion mutant strainSF620 (25) is an avirulent derivative of M90T that contains a nonpolar deletionof the ipaB gene. SF620 containing pGEX-KG-ipaB (gst-ipaB) or pGEX-KG(gst) was described before (5). SF620 was transformed with plasmid pUC19 (39)or with plasmids carrying either wild-type or mutant ipaB. Bacteria were grownat 37°C in tryptic soy broth supplemented with ampicillin (100 mg/ml) or kana-mycin (10 mg/ml) when necessary.

J774 and HeLa cells were grown at 37°C with 5% CO2 in RPMI 1640 mediumsupplemented with 10% decomplemented fetal calf serum (Gibco-BRL), 2 mMglutamine, and 50 mg each of penicillin and streptomycin per ml.

Cloning and mutagenesis of ipaB. Full-length ipaB was amplified by PCR fromp179 (22) using primers ipa-F (forward) and ipa-R (reverse) (Table 1) andcloned into pUC19 (pipaB) (39) by ligation to the HindIII and PstI sites of thepolylinker. Similarly, we constructed pipaBN75 and pipaBN146 by cloning a

* Corresponding author. Mailing address: Skirball Institute, NewYork University Medical Center, 540 First Avenue, New York, NY10016. Phone: (212) 263-7058. Fax: (212) 263-5711. E-mail: [email protected].

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fragment of ipaB (starting at positions corresponding to aa 75 and 146, respec-tively) into pUC19, using primers N75-F or N146-F, respectively, and ipa-R.Site-directed mutations and internal deletions in ipaB were created using pipaBor pGEX-KG-ipaB as templates. Mutations were produced using the Quick-Change site-directed mutagenesis kit (Stratagene) according to the manufactur-er’s protocol. This method uses complementary oligonucleotides encoding the

desired mutation. The sense strand oligonucleotides used in the mutagenesisreactions are listed in Table 1. All mutations were confirmed by restrictionanalysis and/or automated DNA sequencing.

Protein analysis. Cultures of exponentially growing bacteria were standardizedby measuring the optical density at 600 nm and harvested by centrifugation at10,000 3 g for 10 min. Crude bacterial extracts were obtained from the pellets,

TABLE 1. Oligonucleotides used in this studya

a Underlined letters represent non-ipaB sequences, such as restriction endonuclease sites generated to facilitate either cloningor screening of mutants and nucleotides introduced by site-directed mutagenesis.

b Mutagenic primers. Complementary oligonucleotides were also used but are not listed.

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and proteins of filtered (0.2-mm pore size) culture supernatants were precipitatedwith 10% trichloroacetic acid. Protein secretion was analyzed in basal conditionsfrom supernatants of cultures grown without specific inducers (24). Proteinsamples were analyzed by sodium dodecyl sulfate–10% polyacrylamide gel elec-trophoresis (SDS-PAGE). Immunoblotting procedures were carried out with themouse anti-IpaB monoclonal antibody (MAb) H16 and anti-IpaC MAb J22,kindly provided by Armelle Phalipon, Institut Pasteur (2, 31). Horseradish per-oxidase-labeled sheep anti-mouse immunoglobulin antibodies were used as sec-ondary antibodies and visualized by enhanced chemiluminescence.

For limited proteolysis of IpaB and IpaB mutants, supernatants from expo-nentially growing cultures were harvested and concentrated 50-fold by filtrationthrough a membrane with a cutoff value of 50 kDa. Samples were dilutedfourfold in 50 mM NaHCO3 and digested with 0.6 mg of trypsin (Sigma) per mlat 37°C. At different time intervals, aliquots (20 ml) were sampled and snap-frozen to stop the proteolysis. The protein samples were analyzed by immuno-blotting as described above.

Virulence assays. Infections of J774 and HeLa cells were performed as pre-viously described (35, 41) using a multiplicity of infection of 100. For macrophagecytotoxicity assays, J774 cells were grown in 96-well plates and infected in serum-free medium for 5 h. Cytotoxicity was quantified by measuring the release oflactate dehydrogenase enzyme from infected cells using the CytoTox 96 kit(Promega) following the manufacturer’s instructions.

Phagosomal escape was evaluated with a chloroquine resistance assay (7).Briefly, J774 cells infected for 1 h were incubated in the presence of gentamicin(50 mg/ml) with or without chloroquine (100 mg/ml) for an additional 1 h. Thecells were subsequently lysed and plated to determine the number of intracellularbacteria surviving the treatment. The percentage of bacteria that escaped fromthe phagosome was calculated as [(CFU from cells treated with gentamicinand chloroquine, corresponding to bacteria in the cytoplasm)/(CFU from cellstreated with gentamicin alone, corresponding to total intracellular bacteria)] 3

100. SF620 complemented with wild-type ipaB but not with ipaBC401 causedsignificant macrophage cytotoxicity in the time course of this experiment. Sincedying cells become permeable to gentamicin, the percentage of mutant bacteriaescaping the phagosome was evaluated relative to SF620 carrying vector alone.

To test for epithelial cell invasion, the number of intracellular bacteria ininfected HeLa cells was determined using a gentamicin protection assay asreported before (29). Briefly, HeLa cells infected for 1 h were incubated in thepresence of gentamicin (50 mg/ml) for an additional 3 h. Intracellular bacteriawere determined after lysing the infected cells, plating dilutions of the lysates,and counting the CFU. In the assays described above, the standard error wascalculated based on at least three independent determinations.

Purification of GST fusion proteins. Glutathione-S-transferase (GST), GST-IpaB, and GST-IpaB mutant proteins were produced as described before (5)from SF620 harboring plasmids encoding the corresponding genes. Bacterialcultures were induced with IPTG (isopropyl thiogalactopyronoside) for 3 h andpellets were subsequently lysed by French press. The lysates were incubated withglutathione-Sepharose beads (Pharmacia) for 4 h at 4°C, followed by threewashes of the beads with phosphate-buffered saline (PBS). The GST and GSTfusion proteins were used either coupled to the beads or after elution withglutathione, following the manufacturer’s protocol.

Microinjection. Microinjection experiments and the isolation of peritonealmacrophages were performed as previously described (5). Briefly, a monolayer ofcells was microinjected (0.3 3 10211 to 0.7 3 10211 ml/cell) with coded samples(750 mg of protein per ml, 2.25 to 5.25 fg of protein per cell) using an Eppendorfmicroinjection system. After microinjection, cells were incubated for 4 to 6 h at37°C and then stained with 1 mM propidium iodide in PBS. Injected cells wereidentified and scored for propidium iodide uptake into the nucleus, which allowsvisualization of dead cells. The results are the averages of at least four experi-ments with a minimum of 600 cells microinjected per sample.

Casp-1 binding assay. J774 cells were radiolabeled with [35S]methionine,lysed, and centrifuged to obtain a nucleus-free supernatant. GST or GST fusionproteins coupled to beads were incubated with the cell lysate for 3 h at 4°C andthen washed five times with RIPA buffer (1% Triton X-100, 0.5% deoxycholicacid, 0.1% SDS, 50 mM Tris-HCl [pH 7.5], 0.15 M NaCl). The proteins bound tothe beads were resolved in a 5 to 15% gradient SDS-PAGE gel and exposed toa PhosphorImager. As described before (5), the only radioactive protein bindingto IpaB was identified as Casp-1 by immunoblotting. Since the Casp-1 antibodiescross-react with IpaB mutant degradation products, the detection of boundradiolabeled Casp-1 is more sensitive and reproducible than the immunoblot inthese cases.

RESULTS AND DISCUSSION

ipaB truncation mutants. The S. flexneri nonpolar ipaB de-letion mutant strain SF620 is unable to invade epithelial cellsor cause macrophage apoptosis. Introduction of wild-type ipaBin trans (SF620/pipaB) fully complements SF620 for these twophenotypes (see Fig. 4) (25, 36, 41). The levels of cytoplasmicand secreted IpaB in SF620/pipaB were slightly higher but notsignificantly different from those in wild-type Shigella by im-munoblot analysis (see Fig. 2A), even though ipaB is a high-copy-number plasmid (pUC19) and under a heterologous pro-moter. Menard et al. (25) originally described comparableresults obtained using the same ipaB mutant strain (SF620)complemented with a similar plasmid.

To determine the regions necessary for the different func-tions of IpaB, we designed truncations of either the amino-terminal (IpaBN75 and IpaBN146) or the carboxy-terminal(IpaBC311 and IpaBC401) segments of the protein (Fig. 1A).We tested constructs bearing these deletions for their ability tocomplement SF620 in assays for expression, secretion, macro-phage cytotoxicity, phagosome escape, and epithelial cell inva-sion.

To examine whether these IpaB truncations were expressedand secreted, we analyzed the bacterial extracts and culturesupernatants by immunoblotting. IpaB lacking the N terminus(IpaBN75) was detected, albeit in low amounts, in the bacteria,but was not secreted (Fig. 2B and C). We could not detectsecreted IpaBN75 even after overexposure of the film (datanot shown). Further truncation of the N terminus (IpaBN146)appears to make expression of the protein even lower, since itwas not detected in bacterial extracts.

We could detect the translated product of ipaBN146 inhighly concentrated (at least 10-fold) samples of bacterial ex-tracts, confirming that this truncated product was synthesized(data not shown). In contrast, loss of the C-terminal region inIpaB (IpaBC311 and IpaBC401) did not abrogate the expres-sion or the secretion of the protein compared to wild-type IpaBlevels (Fig. 2B and C). In the immunoblot analysis, we detectedseveral IpaBC401 degradation products that are not present inthe other truncates or in wild-type IpaB. The significance ofthis degradation remains to be determined.

We measured the capacity of the ipaB truncates to comple-ment SF620 for killing of the macrophage-like cell line J774.The level of cytotoxicity observed for SF620 with wild-typeipaB expressed in trans was used as a reference (100%) tocalculate the killing potential of SF620 carrying the truncatesor vector alone. Strains encoding ipaBN75 and ipaBN146 wereseverely attenuated for macrophage cytotoxicity (Fig. 3A).Among the C-terminal truncation mutants, IpaBC311 was notcytotoxic, while IpaBC401 retained an intermediate ability tokill macrophages.

Using a gentamicin protection assay, we evaluated the ca-pacity of the ipaB truncates to complement SF620 for invasionof HeLa cells, an epithelial cell line. The number of intracel-lular bacteria recovered after infection with SF620 comple-mented with wild-type ipaB was used as a reference (100%).Similar to the results obtained for cytotoxicity, ipaBN75 andipaBN146 did not complement SF620 for HeLa cell invasion(Fig. 3B). ipaBC311 was also unable to restore invasion, while

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SF620 containing ipaBC401 was 10-fold more invasive thanSF620 with vector alone.

Escape from the phagosome is a prerequisite for Shigella toinduce macrophage apoptosis (41). We tested whether SF620complemented with either ipaBC311 or ipaBC401 escapesfrom the phagosome using a chloroquine resistance assay (7).This assay was done in J774 macrophages, which are phago-cytic and therefore internalize both invasive and noninvasivebacteria. The percentage of cytoplasmic bacteria was calcu-lated relative to the total number of intracellular bacteria asdescribed in Materials and Methods. Both the control straincarrying the vector alone and the one with ipaBC311 wereunable to lyse the phagosomal vacuole, displaying 7% 6 1%and 6% 6 1% escape, respectively. Conversely, a high percent-age of bacteria expressing IpaBC401 (62% 6 6%) reached thecytoplasm of the macrophage.

The N-terminal region of IpaB appears to be relevant formaintaining normal levels of cytoplasmic protein. Not surpris-ingly, mutants IpaBN75 and IpaBN146 were impaired in cyto-toxicity and invasion. The region truncated in IpaBN146 maps,in part, to a coiled-coil structure between aa 120 and 180, aspredicted by the algorithm of Berger et al. (4). The low proteinlevels detected for the amino-terminal IpaB truncation mu-tants may be due to lower transcription or translation. Both ofthese mutants use a different translation initiation site, whichcould modify the translational level. In the bacterial cytoplasm,the chaperone IpgC prevents IpaB degradation prior to itssecretion (26). This interaction could also be altered in mu-tants IpaBN75 and IpaBN146 and cause protein instability.Further tests are necessary to determine why the N-terminalregion of IpaB affects protein levels.

We determined that the C-terminal 179 aa in IpaB are, tosome degree, dispensable for function, since a mutant with adeletion in this region (ipaBC401) partially complementsSF620. In contrast, truncation of the entire hydrophobic region(ipaBC311) causes a complete loss of activity. These data implythat the hydrophobic domain is either necessary for function orimportant to maintain the structure of another critical domain.HeLa cell invasion was complemented to a lesser extent thancytotoxicity by ipaBC401. This discrepancy may reflect differ-ences in the sensitivity of each method in measuring IpaBactivity. Alternatively, the C terminus of IpaB may play a moreimportant role in epithelial cell invasion than in macrophagekilling.

Alanine-scanning mutagenesis of ipaB. The results de-scribed above indicate that the hydrophobic core of IpaB isessential for activity. Since charged or polar residues in hydro-phobic domains may play a role in protein function (19), wemutated each of these residues. Our mutations resulted inalanine substitution of 3 polar and 13 charged aa betweenresidues 306 and 400 in IpaB (Fig. 1B). Each mutant was testedfor its capacity to complement SF620 for macrophage cytotox-icity and epithelial cell invasion. All of these ipaB mutantscomplemented SF620 for killing and invasion similar to wild-type levels (data not shown). These data suggest that thecharged and polar residues in the hydrophobic domain do notplay a crucial role. Alternatively, single substitutions of theseaa may be insufficient to cause a significant alteration in pro-tein function.

Internal-deletion ipaB mutants. We constructed indepen-dent and consecutive internal deletions of 8 or 10 aa in theregion between aa 167 and 352 and a deletion between aa 410

FIG. 1. Schematic representation of mutations generated in IpaB. (A) We generated two C-terminal (IpaBC401 and IpaBC311) and twoN-terminal (IpaBN75 and IpaBN146) constructs. The positions of the corresponding first and last aa in each truncate are indicated. (B)Alanine-scanning. The indicated amino acids, either charged or polar, were substituted by alanine. (C) Internal, nonoverlapping deletions. Twentydeletions of either 8 or 10 residues were generated. Only the starting positions of the deletions are shown.



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and 417 (Fig. 1C). These deletions covered part of the coiled-coil region and the two putative transmembrane domains inthe hydrophobic region. The phenotypes of these deletionswere assayed as described above. Six of these deletion mu-tants, encoding ipaBD247–256, ipaBD257–266, ipaBD267–276,ipaBD277–286, ipaBD307–316, and ipaBD410–417, killed mac-rophages very inefficiently (Fig. 4A). Furthermore, these mu-tants could not invade HeLa cells (Fig. 4B). The rest of thedeletion mutants fully complemented SF620 for both cytotox-icity and HeLa cell invasion.

Of the mutants impaired in function, only IpaBD410–417was expressed at very low levels, while IpaBD247–256 (data notshown), IpaBD257–266, IpaBD267–276, IpaBD277–286, andIpaBD307–316 were expressed (Fig. 4C) and secreted (Fig. 5,lane 09) at levels similar to wild-type IpaB. To investigatewhether these IpaB mutants had conformational alterations,we tested their susceptibility to limited proteolysis by trypsin(1) and analyzed them by immunoblotting. IpaBD307–316(Fig. 5) and the fully functional deletion mutant IpaBD237–246(data not shown) had a proteolytic pattern similar to that ofwild-type IpaB, suggesting that IpaBD307–316 folds correctly.

In contrast, the proteolysis of IpaBD247– 256, IpaBD257–266,IpaBD267–276, and IpaBD277–286 yielded products of 29 to34 kDa which are not observed in wild-type IpaB. Also, thefull-length products of these mutants seem to be more resistantto trypsin digestion. Taken together, these results suggest thatthese IpaB mutants do not fold correctly. The loss of functionobserved in strains expressing IpaBD247–256, IpaBD257–266,and IpaBD277–286 is probably due to their structural alter-ations. According to our secondary-structure analysis, thesemutations map to an amphipathic a-helix between aa 240 and280 that is predicted by the algorithms of Frishman and Argas(Fig. 6) (8). Our data suggest that this structure may be nec-essary to maintain IpaB in its native conformation.

There are two putative transmembrane regions in IpaB (Fig.6) (3). The mutations in IpaBD307–316, IpaBD317–324,IpaBD325–334, and IpaBD335–342 all map within the first ofthese regions (aa 313 to 346). Interestingly, among these mu-tants only IpaBD307–316, in which only 3 aa of the putativetransmembrane region are deleted, was impaired in function,as tested by complementation of SF620. The second putativemembrane-spanning region in IpaB (aa 400 to 423) maps be-yond the IpaBC401 truncate, which conserves function. A de-letion mutation in the second putative membrane-spanningregion, IpaBD410– 417, was poorly expressed and thereforewas inactive in our functional assays. It remains to be deter-mined whether the inefficiency of this mutant in complement-ing for invasion and cytotoxicity is intrinsic to the mutant’sfunction or a reflection of the low level of expression.

Analysis of IpaBD307 and Casp-1 binding domain in IpaB.The partial tryptic digestion of IpaBD307–316 suggests that

FIG. 2. Expression and secretion of IpaB truncates. (A) Whole-celllysates (lys) and culture supernatants (sup) of SF620 carrying pipaB,wild-type strain M90T, and SF620 carrying vector alone were analyzedby immunoblotting with an anti-IpaB MAb. The expression of IpaB inSF620/pipaB is slightly higher but not significantly different from thatin the wild-type Shigella strain. Whole-cell extracts (B) and culturesupernatants (C) of SF620 carrying vector alone or SF620 expressingIpaB or the indicated truncates were analyzed by immunoblotting withan anti-IpaB MAb. IpaBC311 and IpaBC401 are expressed and se-creted at detectable levels. In contrast, IpaBN75 is expressed but notsecreted, and IpaB146 is neither expressed nor secreted. The molec-ular sizes are indicated (in kilodaltons).

FIG. 3. Cytotoxicity and invasiveness of ipaB truncation mutants.Macrophage cytotoxicity (A) and epithelial cell invasion (B) of SF620complemented with plasmids encoding ipaB truncates. Cells were in-fected with SF620 carrying vector alone or SF620 expressing IpaB orthe indicated truncates. Cytotoxicity and invasiveness were assayed asdescribed in Materials and Methods.



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this mutant has a normal structure. Nevertheless, IpaBD307–316 is unable to mediate macrophage cytotoxicity and HeLacell invasion. To further investigate ipaBD307–316, we testedits ability to complement SF620 for phagosome escape. Thepercentage of SF620 complemented with ipaBD307–316 thatescapes to the cytoplasm (10% 6 1%) is similar to that ofSF620 carrying vector alone (7% 6 1%). This result suggeststhat aa 307 to 316 are also critical for lysing the phagosome.

Since escape from the phagosomal vacuole is a prerequisitefor cytotoxicity and IpaBD307–316 is impaired in this function,we microinjected purified protein into the macrophage cyto-plasm to test the cytotoxicity of this mutant. Microinjection ofGST-IpaB fusion protein but not of GST into macrophagescauses cell death (5). After microinjection of purified GST

fusion proteins, macrophage cytotoxicity was evaluated by pro-pidium iodide exclusion (5). The number of dead macrophageswas scored by fluorescence microscopy. Our positive control,GST-IpaB, and our negative control (GST) caused 85% 6 5%and 24% 6 4% cytotoxicity, respectively. GST-IpaBD307–316was poorly cytotoxic (30% 6 5%). Thus, residues 307 to 316 inIpaB are crucial for the induction of macrophage death.

The interaction of IpaB with Casp-1 triggers the cell deathprogram in Shigella-infected macrophages (5, 16). To map theregion of IpaB involved in this interaction, we purified the GSTfusion proteins GST-IpaB, GST-IpaBC311, GST-IpaBD307–316, and GST-IpaBC401 and tested their ability to associatewith Casp-1. The recombinant proteins were incubated withradiolabeled J774 lysates as previously described (5). Casp-1

FIG. 4. Cytotoxicity, invasiveness, and expression of ipaB internal deletion mutants. Macrophage cytotoxicity (A) and epithelial cell invasion(B) of SF620 complemented with plasmids encoding ipaB internal deletion mutants. Cells were infected with SF620 carrying vector alone or SF620expressing IpaB or the indicated IpaB deletion mutants. Mutants are labeled with the corresponding starting position. The wild-type strain M90Twas also included as a control. Cytotoxicity and invasiveness were assayed as described in Materials and Methods. (C) Whole-cell extracts of SF620carrying vector alone or SF620 expressing IpaB or the indicated IpaB deletion mutants were analyzed by immunoblotting. IpaBD257–266,IpaBD267–276, IpaBD277–286, and IpaBD307–316 are expressed at levels comparable to wild-type IpaB. In contrast, IpaBD410–417 is expressedat significantly lower levels.



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precursor and mature forms bound to GST-IpaB but not to thenegative control GST (Fig. 7). Casp-1 interacted with GST-IpaBC401 and GST-IpaBD307–316 but did not associate withGST-IpaBC311. These results indicate that the Casp-1 bindingdomain is located in the hydrophobic region of IpaB that isamino terminal to aa 401. In addition, since GST-IpaBD307–316 binds to Casp-1 but is not cytotoxic, it is possible thatresidues between 307 and 316 in IpaB are involved in regulat-ing the activation of Casp-1. The precise mechanism of thisprocess is still under study.

Between residues 307 and 316 there is only one polar (C309)and one charged (K312) aa. As described in the section on

alanine-scanning mutagenesis, the replacement of these resi-dues with alanine did not affect IpaB function, suggesting thatthe activity resides in the hydrophobic residues of this se-quence. These residues are necessary to maintain a functionalIpaB either by having intrinsic activity or by indirectly affectinga different functional region. Interestingly, the sequence ofIpaB between aa 307 and 316 is identical, except for a singleconservative substitution, to the corresponding segment in theIpaB homologue SipB. In contrast, YopB, which is not in-volved in the induction of apoptosis (27, 28), does not containthis aa sequence. Since the hydrophobic region in IpaB ishomologous to SipB, and SipB can complement SF620 forHeLa cell invasion (12) and macrophage cytotoxicity (our un-published observation), our results provide a structure-func-tion link between these two proteins. We speculate that the

FIG. 5. Proteolytic profile of IpaB internal deletion mutants. Cul-ture supernatants from SF620 expressing IpaB or the indicated IpaBdeletion mutants were subjected to limited proteolysis by trypsin andanalyzed by immunoblot. The time course of trypsin digestion is indi-cated (in minutes), and molecular sizes are shown in kilodaltons. Thepattern observed for IpaBD307–316 but not for IpaBD247–256,IpaBD257–266, IpaBD267–276 or IpaBD277–286 is similar to that forwild-type IpaB.

FIG. 6. Summary of IpaB mutant results. IpaB and IpaB mutants are schematically represented by solid bars with the corresponding results ofthe assays performed. The predicted domains in IpaB, coiled-coil, amphipathic a-helix, hydrophobic, and putative transmembrane (PTM), areindicated. Wild-type IpaB, truncation mutants, alanine mutants, and internal deletion mutants are shown. Internal deletion mutants with aphenotype similar to the wild type are grouped together and independently from those with a different phenotype. The results described in thiswork are summarized as positive (1), intermediate (1/2), and negative (2); refer to the text for quantitative details. For the proteolytic pattern,wt indicates a wild-type pattern, 5wt indicates similar to wild-type pattern, and Þwt indicates different from wild-type pattern.

FIG. 7. Binding of IpaB mutants to Casp-1. GST-IpaB mutantscoupled to beads were incubated with lysates from metabolically la-beled J774 macrophages. Proteins eluted from the beads were resolvedin a 5 to 15% gradient SDS-PAGE. GST-IpaB and GST are thepositive and negative controls, respectively. The molecular sizes areindicated (in kilodaltons). The bands observed in the top and bottompanels correspond to the Casp-1 precursor and mature forms, respec-tively. GST-IpaBC401 and GST-IpaBD307–316 but not GST-IpaBC311 bind to Casp-1.



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hydrophobic domain of SipB also constitutes the functionalcore. Future research on the interaction between IpaB andCasp-1 as well as on the role of IpaB in invasion will allow usto better understand how IpaB and its homologues work.

ACKNOWLEDGMENTS

This work was supported by grants from the NIH (AI 42780–01) andthe WHO (V27/181/108).

We thank A. B. Hittelman for his help with the pipaBN75 construct.We acknowledge Rashmi Hegde and Tim Cardozo for their valuableadvice. We thank A. Aliprantis, H. Hilbi, R. Menard, J. Moss, W.Navare, R. Puro, and Y. Weinrauch for careful revision of the manu-script.

REFERENCES

1. Barzu, S., J. Arondel, S. Guillot, P. J. Sansonetti, and A. Phalipon. 1998.Immunogenicity of IpaC hybrid proteins expressed in the Shigella flexneri 2avaccine candidate SC602. Infect. Immun. 66:77–82.

2. Barzu, S., F. Nato, S. Rouyre, J.-C. Mazie, P. J. Sansonetti, and A. Phalipon.1993. Characterization of B-cell epitopes on IpaB, an invasion-associatedantigen of Shigella flexneri: identification of an immunodominant domainrecognized during natural infection. Infect. Immun. 61:3825–3831.

3. Baudry, B., M. Kaczorek, and P. J. Sansonetti. 1988. Nucleotide sequence ofthe invasion plasmid antigen B and C genes (ipaB and ipaC) of Shigellaflexneri. Microb. Pathog. 4:345–357.

4. Berger, B., D. B. Wilson, E. Wolf, M. M. Tonchev, and P. S. Kim. 1995.Predicting coiled coils by use of pairwise residue correlations. Proc. Natl.Acad. Sci. USA 92:8359–8263.

5. Chen, Y., M. R. Smith, K. Thirumalai, and A. Zychlinsky. 1996. A bacterialinvasin induces macrophage apoptosis by directly binding ICE. EMBO J.15:3853–3860.

6. Dinarello, C. 1998. Interleukin-1b, interleukin-18, and the interleukin-1 betaconverting enzyme. Ann. N.Y. Acad. Sci. 856:1–11.

7. Finlay, B. B., and S. Falkow. 1988. Comparison of the invasion strategiesused by Salmonella cholera-suis, Shigella flexneri and Yersinia enterocolitica toenter cultured animal cells: endosome acidification is not required for bac-terial invasion or intracellular replication. Biochimie 70:1089–1099.

8. Frishman, D., and P. Argos. 1996. Incorporation of non-local interactions inprotein secondary structure prediction from the amino acid sequence. Pro-tein Eng. 9:133–142.

9. Hakansson, S., T. Bergman, J. C. Vanooteghem, G. Cornelis, and H. Wolf-Watz. 1993. YopB and YopD constitute a novel class of Yersinia Yop pro-teins. Infect. Immun. 61:71–80.

10. Hakansson, S., K. Schesser, C. Persson, E. E. Galyov, R. Rosqvist, F.Homble, and H. Wolf-Watz. 1996. The YopB protein of Yersinia pseudotu-berculosis is essential for the translocation of Yop effector proteins across thetarget cell plasma membrane and displays a contact-dependent membranedisrupting activity. EMBO J. 15:5812–5823.

11. Hale, T. L. 1991. Genetic basis of virulence in Shigella species. Microbiol.Rev. 55:206–224.

12. Hermant, D., R. Menard, N. Arricau, C. Parsot, and M. Y. Popoff. 1995.Functional conservation of the Salmonella and Shigella effectors of entry intoepithelial cells. Mol. Microbiol. 17:781–789.

13. Hersh, D., D. Monack, M. Smith, N. Ghori, S. Falkow, and A. Zychlinsky.1999. The Salmonella invasin SipB induces macrophage apoptosis by bindingto caspase-1. Proc. Natl. Acad. Sci. USA 96:2396–2401.

14. High, N., Mounier J., M. C. Prevost, and P. J. Sansonetti. 1992. IpaB ofShigella flexneri causes entry into epithelial cells and escape from the phago-cytic vacuole. EMBO J. 11:1991–1999.

15. Hilbi, H., Y. Chen, K. Thirumalai, and A. Zychlinsky. 1997. The interleukin1b-converting enzyme, caspase 1, is activated during Shigella flexneri-inducedapoptosis in human monocyte-derived macrophages. Infect. Immun.65:5165–5170.

16. Hilbi, H., J. E. Moss, D. Hersh, Y. Chen, J. Arondel, S. Banerjee, R. A.Flavell, J. Yuan, P. J. Sansonetti, and A. Zychlinsky. 1998. Shigella-inducedapoptosis is dependent on caspase-1 which binds to IpaB. J. Biol. Chem.273:32895–32900.

17. Hueck, C., M. Hantman, V. Bajaj, C. Johnston, C. Lee, and S. Miller. 1995.Salmonella typhimurium secreted invasion determinants are homologous toShigella Ipa proteins. Mol. Microbiol. 18:479–490.

18. Kaniga, K., S. Tucker, D. Trollinger, and J. E. Galan. 1995. Homologs of the

Shigella IpaB and IpaC invasins are required for Salmonella typhimuriumentry into cultured epithelial cells. J. Bacteriol. 177:3965–3971.

19. Klann, A. G., R. A. Hull, T. Palzkill, and S. I. Hull. 1994. Alanine-scanningmutagenesis reveals residues involved in binding of pap-3-encoded pili. JBacteriol. 176:2312–2317.

20. LaBrec, E. H., H. Schneider, T. J. Magnani, and S. B. Formal. 1964. Epi-thelial cell pentetration as an essential step in the pathogenesis of bacillarydysentery. J. Bacteriol. 88:1503–1518.

21. Lee, V. T., and O. Schneewind. 1999. Type III machines of pathogenicyersiniae secrete virulence factors into the extracellular milieu. Mol. Micro-biol. 31:1619–1629.

22. Maurelli, A. T., B. Baudry, H. d’Hauteville, T. L. Hale, and P. J. Sansonetti.1985. Cloning of plasmid DNA sequences involved in invasion of HeLa cellsby Shigella flexneri. Infect. Immun. 49:164–171.

23. Menard, R., C. Dehio, and P. J. Sansonetti. 1996. Bacterial entry intoepithelial cells: the paradigm of Shigella. Trends Microbiol. 4:220–226.

24. Menard, R., P. Sansonetti, and C. Parsot. 1994. The secretion of the Shigellaflexneri Ipa invasins is activated by epithelial cells and controlled by IpaB andIpaD. EMBO J. 13:5293–5302.

25. Menard, R., P. J. Sansonetti, and C. Parsot. 1993. Nonpolar mutagenesis ofthe ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexnerientry into epithelial cells. J. Bacteriol. 175:5899–5906.

26. Menard, R., P. J. Sansonetti, C. Parsot, and T. Vasselon. 1994. Extracellularassociation and cytoplasmic partitioning of the IpaB and IpaC invasins of S.flexneri. Cell 79:515–525.

27. Mills, S. D., A. Boland, M.-P. Sory, P. van der Smissen, C. Kerbourch, B. B.Finlay, and G. R. Cornelis. 1997. Yersinia enterocolitica induces apoptosis inmacrophages by a process requiring functional type III secretion and trans-location mechanisms and involving YopP, presumably acting as an effectorprotein. Proc. Natl. Acad. Sci. USA 94:12638–12643.

28. Monack, D. M., J. Mecsas, N. Ghori, and S. Falkow. 1997. Yersinia signalsmacrophages to undergo apoptosis and YopJ is necessary for this cell death.Proc. Natl. Acad. Sci. USA 94:10385–10390.

29. Mounier, J., T. Vasselon, R. Hellio, M. Lesourd, and P. J. Sansonetti. 1992.Shigella flexneri enters human colonic Caco-2 epithelial cells through thebasolateral pole. Infect. Immun. 60:237–248.

30. Perdomo, O. J., J. M. Cavaillon, M. Huerre, H. Ohayon, P. Gounon, andP. J. Sansonetti. 1994. Acute inflammation causes epithelial invasion andmucosal destruction in experimental shigellosis. J. Exp. Med. 180:1307–1319.

31. Phalipon, A., J. Arondel, F. Nato, S. Rouyre, J. C. Mazie, and P. J. San-sonetti. 1992. Identification and characterization of B-cell epitopes of IpaC,an invasion-associated protein of Shigella flexneri. Infect. Immun. 60:1919–1926.

32. Sansonetti, P., A. Phalipon, J. Arondel, K. Thirumalai, S. Banerjee, S. Akira,K. Takeda, and A. Zychlinsky. 2000. Caspase-1 activation of IL-1b and IL-18are essential for Shigella flexneri induced inflammation. Immunity 12:581–590.

33. Sansonetti, P. J., and J. Arondel. 1989. Construction and evaluation of adouble mutant of Shigella flexneri as a candidate for oral vaccination againstshigellosis. Vaccine 7:443–450.

34. Sansonetti, P. J., J. Arondel, J.-M. Cavaillon, and M. Huerre. 1995. Role ofIL-1 in the pathogenesis of experimental shigellosis. J. Clin. Investig. 96:884–892.

35. Sansonetti, P. J., D. J. Kopecko, and S. B. Formal. 1982. Involvement of aplasmid in the invasive ability of Shigella flexneri. Infect. Immun. 35:852–860.

36. Thirumalai, K., K. Kim, and A. Zychlinsky. 1997. IpaB, a Shigella flexneriinvasin, colocalizes with interleukin-1b converting enzyme in the cytoplasmof macrophages. Infect. Immun. 65:787–793.

37. Thornberry, N. A. 1994. Interleukin-1b converting enzyme. Methods Enzy-mol. 244:615–631.

38. Wassef, J. S., D. F. Keren, and J. L. Mailloux. 1989. Role of M cells in initialantigen uptake and in ulcer formation in rabbit intestinal loop model ofshigellosis. Infect. Immun. 57:858–863.

39. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phagecloning vectors and host strains: nucleotide sequences of the M13mp18 andpUC19 vectors. Gene 33:103–119.

40. Zychlinsky, A., C. Fitting, J. M. Cavaillon, and P. J. Sansonetti. 1994.Interleukin-1 is released by murine macrophages during apoptosis inducedby Shigella flexneri. J. Clin. Investig. 94:1328–1332.

41. Zychlinsky, A., B. Kenny, R. Menard, M. C. Prevost, I. B. Holland, and P. J.Sansonetti. 1994. IpaB mediates macrophage apoptosis induced by Shigellaflexneri. Mol. Microbiol. 11:619–627.

42. Zychlinsky, A., M. C. Prevost, and P. J. Sansonetti. 1992. Shigella flexneriinduces apoptosis in infected macrophages. Nature 358:167–168.



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