The EMBO Journal vol. 1 1 no. 5 pp. 1 991 - 1999, 1992

IpaB of Shigella flexneri causes entry into epithelial cells and escape from the phagocytic vacuole

Nicola High, Joelle Mounier, Marie Christine Prevost' and Philippe J.Sansonetti2 Unite de Pathogenie Microbienne Moleculaire, Unite INSERM 199, and 'Station Centrale de Microscopie Electronique, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France 2Corresponding author Communicated by A.Danchin

By creating mutations within the Shigella flexneri ipaB gene, we have demonstrated that the invasion of epithelial cells is a three-step process encompassing adhesion on the cell surface, entry and lysis of the phagocytic vacuole allowing subsequent access to the cytoplasm. SC403, an insertion mutant which lacks expression of IpaB but still expresses downstream genes, has been particularly studied. It is non-invasive, does not elicit actin polymerization, but binds to HeLa cells indicating that an adhesion step occurs immediately prior to the entry process. The consequence of the inactivation of ipaB on the intracellular behaviour of S.flexneri was investigated using the macrophage cell line J774. SC403 was unable to lyse the phagocytic vacuole; moreover, this strain did not display the contact mediated haemolytic activity characteristics of Shigella. In addition to being a major component of the invasion complex, IpaB acts as a membrane-lysing toxin enabling escape to the cytoplasmic compartment. Key words: epithelial cells/hemolysin/invasion/Shigella

flexneri

Introduction Invasive bacteria have evolved specialized strategies allowing their entry and subsequent survival within non-phagocytic cells (Moulder, 1985). Bacterial determinants which mediate the entry process differ from species to species (Falkow, 1991). Subsequent steps of intracellular development are also variable. A major difference resides in the capacity of the internalized microorganism to lyse the membrane bound phagocytic vacuole in which it is contained. Whereas Salmonella and Yersinia are unable to escape the vacuole, Shigella and Listeria monocytogenes gain free access to the cytoplasm; this environment allows access to nutrients and interaction with cytoskeletal components promoting intracellular and cell to cell spread (Bernardini et al., 1989; Tilney and Portnoy, 1989; Mounier et al., 1990). For L. monocytogenes, invasion is at least a two-step process involving internalin, a surface protein which promotes entry (Gaillard et al., 1991) and listeriolysin-O which accounts for the subsequent lysis of the phagocytic vacuole (Gaillard et al., 1987; Portnoy et al., 1988). © Oxford University Press

Shigella provokes bacillary dysentery by a complex series of events which include entry into colonic epithelial cells (La Brec et al., 1964), intracellular multiplication and spread of bacteria to adjacent cells and to the lamina propria of intestinal villi. Entry into epithelial cells occurs in vitro through a process similar to phagocytosis since de novo polymerization of actin and accumulation of myosin are triggered by the bacterium (Clerc and Sansonetti, 1987). It has previously been demonstrated that S.flexneri elaborates a limited capacity to adhere to HeLa cells (Pal and Hale, 1989). Following entry, S.flexneri quickly lyses its phagocytic vacuole (Sansonetti et al., 1986), a property which has been correlated with the production of a contact haemolysin (Sansonetti et al., 1986; Clerc et al., 1987a) At least three major virulence phenotypes depend upon S.flexneri gaining free access to the cytoplasm: rapid intracellular growth (Sansonetti et al., 1986), intracellular and cell to cell spread (Bernardini et al., 1989; Vasselon et al., 1991) and rapid killing of macrophages (Clerc et al., 1987b). These various processes are essentially accomplished by proteins encoded by a 220 kb plasmid, pWR100 (Sansonetti et al., 1982, 1983). A 35 kb region of this plasmid has been cloned and shown to encode all the necessary functions required for entry into epithelial cells (Maurelli et al., 1985), although it is not in itself sufficient to confer full virulence. This invasion sequence can be divided into five genetic regions (Baudry et al., 1987, 1988; Sasakawa et al., 1986). One region specifies the biosynthesis of four polypeptides (Baudry et al., 1987; Buysse et al., 1987; Sasakawa et al., 1989) which are the dominant proteinaceous antigens eliciting a humoral immune response during infection by Shigella. These proteins have been termed 'invasion protein antigens' or IpA -D. Their respective molecular weights are 70 kDa, 62 kDa, 42 kDa and 38 kDa. IpaB and IpaC are consistently recognized by convalescent sera (Hale et al., 1985); they have also been found to be exported to the surface of the bacterium (Mills et al., 1988; Andrews et al., 1991). Particular interest is consequently paid to their possible role in the invasion process. The ipa gene cluster appears to be transcribed essentially as a single cistron with an accessory promoter upstream, ipaD (Sasakawa et al., 1989). Using transposon TnS mutagenesis, IpaA has been reported to have no obvious function (Sasakawa et al., 1986; Baudry et al., 1987). Investigations into the roles of IpaB-D have also relied on observing the phenotypes of transposon TnS mutants (Baudry et al., 1987; Sasakawa et al., 1988). Mainly due to the polarity of the mutations, no clear picture has emerged concerning the respective functions of these polypeptides in the invasion process. It was therefore decided to address first the function of IpaB by creating defined mutants directly in pWR 100 by a process of allellic exchange which would also allow reinitiation of downstream transcription. Using this general strategy it has been possible to 1991

N.High et al.

demonstrate that IpaB is a major invasin of S.flexneri. In addition to its role in mediating entry into epithelial cells it is also responsible for contact mediated haemolysis and as such is the key factor in the destruction of the phagocytic vacuole.

Results Construction of ipaB mutants and of plasmid pipaB which overexpresses IpaB The different steps of this construction are detailed in Materials and methods and summarized in Figure 1. M9OT, the invasive strain of S.flexneri used in this study belongs to serotype 5. Analysis of the proteins synthesized by the ipaB mutants Western blot analysis was carried out using serum obtained from a monkey convalescing from shigellosis as the first antibody probe (data not shown). This serum recognizes the four Ipa polypeptides. In addition to the absence of IpaB at the expected molecular weight in SC401, the Ipa polypeptides encoded by genes located downstream of the inserted Omega cassette also appeared not to be detected. These data confirmed that the genes constituting the ipa cluster were transcribed essentially as a polycistronic mRNA. In addition, the presence of a second transcript arising from a promoter located in front of ipaD was also identified (Sasakawa et al., 1989). This promoter allows the synthesis of small quantities of IpaD in ipaB mutants. The quantity produced was not sufficient to be detected by convalescent monkey serum. Significant amounts of this protein were however, visible in Western blots probed with an anti-IpaD

polyclonal antiserum kindly provided by Dr B.Adler (data not shown). Having confirmed the polar nature of the mutations in SC401, SC403 was constructed as described above with an independent constitutive promoter inserted in front of ipaC. To achieve better specificity, production of IpaB and IpaC was detected in M9OT and SC403 by using monoclonal antibodies H1O and J22-4 respectively. Results of the Western blots are shown in Figure 2. As compared with lane 2 showing expression of the 62 kDa IpaB polypeptide by M9OT, lane 1 shows that SC403 produced a truncate of 42 kDa corresponding to the gene product expected from the deletion. The faint band seen in both lanes at a higher molecular weight corresponds to a polypeptide recognized by natural antibodies contained in H1O mouse ascites fluid. As compared with lane 4 which demonstrates strong production of IpaC by M9OT, lane 3 shows that SC403, due to the presence of the inserted independent promoter, was still able to produce the same polypeptide but in lower quantity. Analysis of the virulence phenotype of mutants SC401 and SC403 The two mutants were non-invasive, however, they showed strong affinity for HeLa cell membranes. Following a 2 h infection period, bacteria could be seen to be bound to the HeLa cell monolayer. When the incubation period was extended for a further 2 h, HeLa cells became completely covered with bacteria as shown in Figure 3C and D respec-

tively for SC401 and SC403. Since this adherent phenotype may mask the presence of intracellular bacteria, following the 2 h incubation period, gentamicin was added in the medium at a concentration of 15 ytg/ml in order to kill

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Fig. 1. Organization of the ipa gene cluster in wild-type S.flexneri and in ipaB mutants SC401 and SC403. Map of plasmid plpaB which IpaB. Symbols: Bg, BglII; E, EcoRI; H, HindIII; P, PstI; S, Sall; Hc, HincII; B, BamHI. Promoters are indicated by arrows. Construction of SC401 consisted in the insertion of the Omega cassette within the ipaB gene sequence. This mutant produced a 17 kDa truncate of IpaB. Construction of SC403 encompassed partial deletion of the ipaB gene sequence and insertion of a promoter downstream of the Omega cassette in order to circumvent the polar effect observed on the transcription of ipaC in SC401. The arrow corresponds to the promoter of the pBR322 antisense RNA inserted downstream of the Omega cassette. pNH101 contains a 7.3 kb BamHI-HindIll fragment encompassing the entire ipa operon. overexpresses

1992

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extracellular microorganisms. Potentially intracellular bacteria were allowed to grow for a further 2 h period in the presence of the antibiotic. At the end of this period, bacteria were no longer binding on to the cell surface, and unlike the M9OT control, they could not be detected in the intracellular compartment, thus confirming that the ipaB mutations expressed a non-invasive phenotype. Since shigellae with rough lipopolysaccharide (LPS) often exhibit a more adhesive phenotype, subsequent analysis of the LPS of both M9OT and the ipaB mutants revealed no apparent difference in the length and number of 0-polysaccharide side chains (data not shown). It could therefore be concluded that the enhanced ability to adhere exhibited by the ipaB mutant was a direct result of the mutation. Transformation of SC403 by plpaB and induction of IpaB expression by incubation of the transformant for 1 h at 370C in the presence of IPTG restored the wild-type phenotype of HeLa cell invasion. In addition, if IpaC is a virulence product, these results confirm that its expression under the inserted promoter is nevertheless sufficient. Analysis of the adhesive capacity of M9OT and SC403, following growth at 30°C, a non-permissive temperature for the expression of virulence polypeptides, resulted in a total absence of binding similar to the non-invasive plasmidless derivative BS176. This observation indicated that the expression of this function, like the majority of Shigella virulence determinants is temperature regulated (Maurelli et al., 1984). Destruction of the ipaB gene therefore allowed identification of a temperature regulated adhesion step preceding the entry step. It is unlikely that a truncated

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and SC403 (D). Infections were carried out at

1993

N.High et al.

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Determination of the role of IpaB in triggering actin polymerization Penetration of HeLa cells by invasive shigellae has been shown to coincide with localized polymerization of actin and the accumulation of myosin at the site of bacterial entry (Clerc and Sansonetti, 1987). Since the adhesive mutants isolated in this study formed such an intimate contact with HeLa cell membranes, we investigated whether this apparent ability to adhere to HeLa cells was sufficient to trigger low levels of actin polymerization and therefore act as the initial signal for the induction of phagocytosis. Following a 120 min period of infection, the infected monolayers were fixed and processed. Despite the fact that all three adhesive mutants bound strongly to HeLa cell membranes, no evidence of aggregates of filamentous actin was observed at their site of binding as illustrated for SC403 in Figure 4C -D. Conversely, M9OT elicited large aggregates of F-actin at sites of bacterial interaction with the cytoplasmic membranes as shown in Figure 4A-B. This therefore indicated that IpaB is a major, if not exclusive, invasin involved in triggering entry through direct phagocytosis after initial adhesion to the target cell membrane has occurred. IpaB accounts for lysis of the membrane bound phagocytic vacuole and host cell killing It was not possible to analyse the consequences of the lack of expression of IpaB on the intracellular survival in HeLa 1994

cells, such mutants being unable to enter epithelial cells. The entry step could however, be bypassed by infecting J774 macrophages which phagocytose non-invasive microorganisms. The difference observed in the intracellular behaviour of SC403 and M9OT was striking. As illustrated in Figure 5, SC403 affected a phenotype similar to that of BS 176 after 3 h of intracellular life. As compared with M90T (Figure 5A), neither of these two strains caused significant destruction of the macrophages (Figure SB and C) and in both cases bacteria accumulated in a radial fashion around the nucleus (Figure SD). This pattern of distribution suggested that intracellular bacteria were unable to express the Ics phenotype which is characteristic of bacteria escaping the phagocytic vacuole and subsequently spreading intracellularly via interaction with the host cell microfilaments (Bernardini et al., 1989). Destruction of infected J774 macrophages by M9OT, BS176, SC403 and SC403/plpaB was then quantified as described in Materials and methods. Results are shown in Figure 6A. Height of the columns represents the percentage of residual macrophages present on dishes at various times following infection. The black portion of these columns represents the fraction of residual macrophages no longer able to exclude Trypan blue. SC403 and BS176 had not killed macrophages after 3 h of infection whereas M9OT and SC403/plpaB had achieved almost complete destruction of the monolayer during the same period. Demonstration that IpaB mediated very efficient killing of macrophages suggested that this protein, in addition to being an invasin, also acted as a cytotoxin in this particular cell assay system, thus suggesting that IpaB may be the contact haemolysin of

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Contrastingly membranes remained intact in 100% of cells infected by SC403 even after 3 h of intracellular life (Figure 7D and E) as already shown for BS176 (Clerc et al., 1987b). As expected from data presented in the previous section, cells infected by M9OT and SC403/plpaB presented symptoms of ongoing cytotoxicity with swelling of intracellular compartments as early as after 1 h of infection. These data indicate that IpaB accounts for the escape of intracellular bacteria from the phagocytic vacuole through its membranolytic (haemolytic) activity. In infected J774 macrophages which are sensitive to killing by shigellae (Clerc et al., 1987b), IpaB appears to be an active cytotoxin.

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Fig. 5. Destruction of J774 macrophages. Macrophages were infected, incubated at 37°C for 1 or 3 h, then fixed and observed after Giemsa staining. (A) M90T (1 h of infection); (B) BS176 (3 h); (C) and (E) SC403 (3 h). Bar, 10 niicrons.

IpaB is the contact haemolysin of S.flexneri As reported in Figure 6, the contact hemolytic activities of SC403 (Figure 6B-3), M9OT (Figure 6B-1) and BS176 (Figure 6B-2) were compared. Whereas M90T was strongly haemolytic, the degree of haemolysis mediated by SC403 was negligible, as observed for BS 176, and was comparable with the rate of spontaneous erythrocyte lysis (Figure 6B-6). The haemolytic phenotype of SC403 could be transcomplemented by plasmid plpaB in which expression of the ipaB gene was induced with IPTG. The amount of haemolysis increased as a function of the induction of the expression of IpaB for 1 h (Figure 6B-4) and 2 h (Figure 6B-5). In spite of only a partial restoration of the haemolytic phenotype, SC403/plpaB was restored to full invasive capacity in the HeLa cell assay.

IpaB accounts for lysis of the membrane bound phagocytic vacuole The striking differences in the intracellular behaviour of SC403 and M9OT were confirmed by observations made using transmission electron microscopy on infected J774 macrophages. As shown in Figure 7, phagosome membranes were lysed in 100% of the cells infected by M9OT or SC403/pIpaB within 1 h of infection (Figure 7A, B and C).

Discussion The ability of S.flexneri to induce its own phagocytosis by non-professional phagocytic cells such as human epithelial cells necessitates a close interaction between the bacterium and the target cell membrane. Since the kinetics of invasion are rapid (Clerc and Sansonetti, 1987), adhesion to epithelial cell membranes immediately prior to the initiation of the entry process is likely to be a transient step. Pal et al. (1989) demonstrated that S.flexneri elaborates limited adhesion at 4°C, a non-permissive temperature for invasion. No such data however, are available at 37°C, the physiological temperature for the development of infection. From the phenotype of the non-invasive ipaB mutants isolated in this study, it has become evident that an adhesion step precedes the entry process. As incubation proceeds the infected cells become progressively covered by the bacteria in a pattern which is reminiscent of the 'stacked-brick' phenotype of entero-aggregative Escherichia coli (Robins-Browne et al., 1988). The initial interaction between the adhesive mutants and HeLa cells occurs predominantly at the periphery of the cell in the region of the focal contacts (Burridge and Fath, 1989) which is particularly rich in actin and actin binding proteins, the essential motors of phagocytosis (Stendhal et al., 1980). Integrins which mediate the entry of Yersinia pseudotuberculosis into epithelial cells (Isberg and Leong, 1990) are also concentrated in this area. The initial adhesion step may be an important factor in targeting shigellae to their specific cell surface receptor. These mutants will therefore be convenient probes with which to identify this epithelial cell receptor. From the phenotype of SC401 and SC403, it is evident that just the ability to bind to epithelial cell membranes is not sufficient to trigger the cascade of events leading to phagocytosis since neither of these two mutants are capable of inducing even low levels of actin polymerization. This study has focused on the role of IpaB in cell invasion. Previous investigations had used random mutagenesis by TnS. Interpretation of these results was limited because these mutations had a polar effect on the transcription of the ipa operon. As a consequence, the phenotypes of the mutants obtained so far have always been interpreted as 'invasive' or 'non-invasive' with no further information on a particular step of the invasive process at which this mutant may be affected. By allelic exchange, we have constructed an ipaB mutant (SC403) in which a promoter was inserted downstream of the Omega cassette thereby directing expression of IpaC. When the invasive capacity of SC403 was examined, it was shown to express the same non-invasive, adhesive phenotype originally observed with the two other

1995

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Fig. 6. Destruction of infected J774 macrophages (A) and contact hemolytic activity (B). (A) The columns correspond to the percentage of initially seeded macrophages that remained bound to the tissue culture dish at various intervals. The black portion of these columns corresponds to dying cells which no longer excluded Trypan blue. (B) The amount of haemolysis is shown as the optical density measured at 540 nm, the band of absorption of haemoglobin. 1, M9OT; 2, BS176; 3, SC403; 4, SC401/plpaB, 1 h of IpaB expression, 5, SC401/pIpaB, 2 h of IpaB expression; 6, spontaneous haemolysis in reaction buffer.

mutants thus indicating that IpaB was essential for allowing shigellae to invade eukaryotic cells. This was confirmed by the demonstration that in trans expression of the ipaB gene alone from plasmid plpaB was sufficient to restore the invasive phenotype. Studies are in progress to understand

the molecular mechanisms by which IpaB elicits actin assembly and related events that characterize phagocytosis. In order to identify a further role for IpaB in the development of cell invasion, we bypassed the step of directed phagocytosis by using professional phagocytic cells such as J774 macrophages with SC403. SC403 affected a phenotype similar to BS176, a plasmidless, non-invasive derivative of M9OT. It invaded macrophages, grew slowly within the intracellular compartment, did not lyse host cells and affected an intracellular pattern suggestive of bacteria that were unable to lyse their membrane bound phagocytic vacuole. This hypothesis was confirmed by transmission electron microscopy of infected cells, showing that all intracellular mutants remained entrapped within a phagosome. Conversely, SC403/pIpaB had the intracellular behaviour of M9OT. It lysed macrophages within a very short period. In the few cells that supported intracellular growth, transmission electron mircoscopy confirmed the hypothesis, showing that 100% of the intracellular bacteria were free within the cytoplasm. These experiments demonstrated that entry and lysis of the membrane bound phagocytic vacuole are dependent on IpaB. Based on the original suggestion that the contact haemolysin of S.flexneri accounted for the membranolytic activity that carried out phagosome lysis (Sansonetti et al., 1986), SC403 was tested for haemolysis. IpaB is the contact haemolysin as confirmed by transcomplementation experiments in which the amount of haemolysis expressed by SC403 carrying pIpaB, a recombinant expression vector expressing IpaB under the control of 1996

the tac promoter, correlated with the degree of protein expression obtained in the presence of IPTG. These observations were further consolidated by the fact that purified IpaB is haemolytic (T.Vasselon, N.High and P.J.Sansonetti, manuscript in preparation). IpaB therefore appears to be a new member of the family of bacterial haemolysins (Welch, 1991). It has no sequence homology with any of the known structural genes of haemolysins (Baudry et al., 1988), but contains two highly hydrophobic domains which may ensure interaction between IpaB and eukaryotic membranes. Such a protein that can elicit entry of the microorganism when deposited on the surface of eukaryotic cells and subsequently become a membranolytic toxin when expressed within a closed phagosome is so far unique. It is concievable that constant accumulation of IpaB in the target cell membrane facing the bacterium as the phagocytic process progresses ultimately leads to the lysis of this membrane. In addition, we have previously demonstrated that the contact haemolytic activity was dependent on the pH of the reaction buffer (P.Clerc, unpublished data). Haemolysis increased by 100-fold as pH decreased from 7 to 5.5 which is the expected pH of a phagosome (Goren and Mor, 1988). It can be hypothesized that, like viral fusion proteins that become highly haemolytic at low pH (Helenius et al., 1983; Marsh, 1984), IpaB undergoes a conformational change in the phagosome which allows full expression of its membranolytic capacity.

Materials and methods Media Bacteria were grown routinely in Luria broth, Tryptic Soy broth (Diagnostics Pasteur, Marne-La-Coquette, France), or M9 minimal media supplemented with glucose (0.2%) and nicotinic acid (10 yog/ml). When required, solid

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Fig. 7. Expression of IpaB and lysis of the phagocytic vacuole within J774 macrophages. (A)-(C) Cells were infected for 1 h with M9OT.theLysis the phagosome membrane could be observed around all bacteria. (D) and (E) Cells were infected for 3 h with SC403. The membrane of phagosomes is intact and several bacteria may reside within a single vacuole as shown in (E). media were made by the addition of Bacto agar (1.5 %). Antibiotics were added where appropriate to the following final concentrations: ampicillin (100 Atg/ml); spectinomycin (50 Ag/ml). The ability to bind the dye Congo

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red (Sigma Chemicals, Poole, Dorset) was assessed by growth on Tryptic Soya agar to which Congo red had been added to a final concentration of 0.01%.

1997

N.High et a!. DNA methodology Restriction endonucleases and DNA modifying enzymes were used according to manufacturer's recommendations. Plasmid DNA from Shigella was purified essentially as described by Kado and Liu (1981). Plasmid DNA harboured by E.coli K-12 strains was isolated according to the method of Bimboim and Doly (1979). Invasion assay Bacterial invasion of HeLa cells was performed as previously described (Sansonetti et al., 1986). Infection of the monolayers was carried out for 3 h. To obtain a quantitative estimate of the number of internalized bacteria, HeLa cell monolayers were incubated for a further 3 h with gentamicin (20,ug/ml) added to the medium in order to eliminate extracellular bacteria. Double fluorescence staining of F-actin and invading bacteria Zones of F-actin accumulation within HeLa cells and the presence of invading bacteria were visualized by fluorescence microscopy essentially as described before (Clerc and Sansonetti, 1987). Bacteria were labelled by indirect immunofluorescence with anti-S.flexneri 5a LPS antiserum revealed by a goat anti-rabbit immunoglobulin antibody conjugated to rhodamine. Filamentous actin was visualized by staining the cells with NBD-Phalloidin for a 20 min period (Molecular Probes, Junction City, OR). Infection of J774 macrophages The mouse macrophage cell line J774 was propagated and infected as described in a previous paper (Clerc et al., 1987b). Assay for macrophage detachment and killing Macrophages were labelled for 18 h prior to infection in a culture medium containing 0.5 ACi/ml of [3H]uridine (Amersham Corp., Buckinghamshire, UK). Cells were then washed extensively and infection was carried out as described (Clerc et al., 1987b). After washing, the percentage of non-viable macrophages that still adhered to the dish was determined by Trypan blue (0.2% in PBS) staining (Phillips, 1973). The percentage of viable macrophages was determined by measuring the amount of radioactivity remaining in the dish. Adherent cells were lysed with 0.5% sodium deoxycholate in distilled water, 100 Al of the lysate were precipitated in 900 I1 of 5% trichloracetic acid, baked at 80°C for 30 min and soaked in NCS-OCS (Amersham) in a 1:9 ratio prior to counting. Assay for contact haemolysis Carried out according to the original description (Sansonetti et al., 1986).

Preparation of samples for electron microscopy Infected monolayers of J774 macrophages were fixed for 1 h at room temperature with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.1 M sucrose, 5 mM CaC12 and 5 mM MgCI2. The fixed monolayers were then washed and incubated overnight at 4°C in the same buffer. Further fixation was carried out in 1 % osmium tetroxide in the same buffer. After washing, cells were scraped off the surface of the dish, concentrated in agar and treated for 1 h in 1 % uranyl acetate. They were then dehydrated and embedded in Epon. Analysis of lipopolysaccharide side chains Lipopolysaccharide samples were prepared essentially as described by Westphal and Jann (1965). The component parts were separated as previously described (Laemmli, 1970) and transferred to a nitrocellulose membrane (Towbin et al., 1979). Detection of LPS side chains was achieved by solid phase immunosorbent assay.

Western blot analysis The presence of Ipa polypeptides was detected using essentially the same procedure as described for the LPS blot. Sera obtained from convalescent monkeys that had been experimentally infected with S.flexneri and mouse monoclonal antibodies (mAb) H 10 and J22-4, which respectively recognize IpaB and IpaC (A.Phalipon, J.Arondel, F.Nato, S.Rouyre, J.C.Mazie and P.J.Sansonetti, manuscript submitted), were used. The H1O mAb recognizes an epitope located between residues 194 and 202 on IpaB, and J22-4 mAb recognizes an epitope located between residues 24 and 33 on IpaC. Binding of the mAb was revealed using a goat anti-mouse immune serum labelled with alkaline phosphatase. The Protoblot System (Promega Corporation, Madison, WI) was used as the substrate for alkaline phosphatase. Conjugal mating experiments Overnight cultures of donor and recipient strains were mixed together in a ratio of 5:1 and grown overnight on TCS agar. Bacteria were then harvested and resuspended in PBS. The mating mixture was then plated out on to M9 minimal medium supplemented with nicotinic acid (10 jqg/ml) and

1998

spectinomycin (50 Ag/mil). Following incubation at 37°C for 48 h, individual colonies were screened for sensitivity to ampicillin. Spectinomycin resistant, ampicdillin sensitive colonies were selected for further analysis. In such clones it was assumed that a double recombination event had taken place.

General strategy of construction of ipaB mutants Construction involved direct insertion of a cassette within a selected restriction site or the creation of a deletion in the ipaB gene by removal of a specific restriction fragment. The cleaved fragment was then religated on the interposon Omega (Prentki and Kirsch, 1984), a 2 kb cassette which encodes resistance to spectinomycin and streptomycin. This region is flanked by T4 transcription and translation termination sequences. DNA fragments encompassing the mutated ipaB genes were then cloned into the suicide vector pJM703. 1 (Miller and Mekalanos, 1988), and the resulting plasmid was transferred into M9OT by conjugal mating. Transconjugants were then selected in which the wild-type gene has been replaced by the mutated ipaB gene via a double recombination event. Proper genetic rearrangement was checked by Southern hybridization. Construction of SC401 and SC403 mutants is summarized in Figure 1. Construction of the insertional mutant SC40 1 A 2 kb Pstl fragment comprising the ipaB gene was isolated from plasmid pNH101 shown in Figure 1 and cloned into pUC 18. The construction was partially digested with EcoRP, treatment with Klenow, and finally ligated to the Omega cassette contained on a 2 kb SmaI fragment. This final constructed was digested with PstI and the 4 kb fragment containing the Omega cassette flanked by the disrupted ipaB gene was cloned into PstI digested pJM703-1. Following conjugation into M90T and double crossing over, SC401 the final mutant, expressed a small truncated polypeptide of 17 kDa. Construction of SC403, a mutant with preserved transcription of ipaC Plasmid pNH1I1 was digested with Sall, releasing a 300 bp fragment. The larger fragment was treated with Klenow, then ligated to the Omega cassette contained on a 2 kb SniaI fragment. This construct, called pNH102, was partially digested with HindIll, treated with Klenow and religated in order to destroy the HindlIl site in the Omega cassette 300 bp upstream of the beginning of ipaC. The resulting plasmid was digested again with HindIII, treated with Klenow, then ligated to a 250 bp EcoRI fragment which had also been Klenow treated. The latter fragment was derived from pPIT7a2 and contains a constitutive promoter which directs the transcription of a small anti-sense RNA molecule involved in copy number control of pBR322 (Panayotatos et al., 1983). The selection of clones in which the promoter had been introduced in the correct orientation was achieved by restriction endonuclease analysis. The fragment was then ligated to EcoRI digested pJM703. 1. This mutation was introduced in plasmid pWR100 through a double recombination event, thus producing strain SC403 which was expected to produce a truncated polypeptide of 42 kDa. Construction of plasmid pipaB and overexpression of the IpaB polypeptide In order to express IpaB, a transcriptional fusion coupling the ipaB gene to the tac promoter harboured by vector pTTQ18 was created. The pTTQ series of vectors contain the laclq allele of the lac repressor gene ensuring maximal repression of the tac promoter in the absence of IPTG (Stark, 1987). Expression of gene fusions in these vectors is consequently very tightly controlled. This has the additional advantage of allowing host independence such that expression studies could be carried out directly in Shigella. The creation of this construct involved ligating a 2.14 kb EcoRV-PstI fragment derived from pNH101 into SnaI/PstI digested pTTQ18, thus creating p1paB. A map of this construct is also shown in Figure 1. Plasmid plpaB was introduced into either BS176, a non-invasive, plasmidless derivative of M9OT in order to study IpaB expression, or into SC403 in order to complement the deletion mutation. The resultant strains were grown to an OD650 of 0.5 in Luria broth. Cells were harvested and resuspended in an equal volume of prewarmed Luria broth containing 10 mM IPTG and appropriate antibiotics. The cells were then incubated for a further 1 or 2 h to allow induction of IpaB. Following this period, cells were harvested and strong expression of IpaB in BS176 was observed after separation of proteins by SDS-PAGE.

Acknowledgements We would like to thank Annick Fontaine and Maria-Lina Bernadini for their advice and stimulating discussions, and Claude Parsot for careful reading of this manuscript. N.J.H. was the recipient of an EMBO fellowship. This work was supported by the Thrasher Research Fund.

Invasion genes of S.flexneri

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Received on December 18, 1991; revised on January 30, 1992

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