The actin-based motility defect of a Shigella flexneri rmlD rough LPS mutant is not due to loss of IcsA polarity

Microbial Pathogenesis 35 (2003) 11–18 www.elsevier.com/locate/micpath

The actin-based motility defect of a Shigella flexneri rmlD rough LPS mutant is not due to loss of IcsA polarity Luisa Van Den Bosch, Renato Morona* School of Molecular and Biomedical Science, University of Adelaide, North Terrace, Adelaide, SA 5005, Australia Received 20 January 2003; received in revised form 12 March 2003; accepted 16 March 2003

Abstract Shigella flexneri requires the outer membrane protein IcsA(VirG) and lipopolysaccharide (LPS) for efficient actin-based motility (ABM) within mammalian cells which is essential for virulence. Wild type strains of S. flexneri 2a such as 2457T have smooth LPS whose O antigen (Oag) chains have two modal lengths and IcsA predominantly located at one pole on their cell surface. In contrast, rough LPS mutants lack Oag chains, have IcsA on lateral and polar regions of the cell surface, and are defective for ABM. In this study we directly compared the phenotype of a S. flexneri producing non-IcsP/SopA cleavable IcsA (IcsAp) with that of a rough LPS mutant. IcsAp was located on lateral and polar regions of smooth LPS bacteria, and was fully functional in ABM assays (HeLa cell monolayer plaque and F-actin comet tail formation) which contrasts with the R-LPS phenotype. This indicates that loss of polar IcsA localisation in R-LPS mutants is unrelated to their ABM defect, and suggests that Oag may directly contribute to IcsA-mediated ABM. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Shigella flexneri; Lipopolysaccharide; rmlD; Actin-based motility; F-actin comet tail; IcsA

1. Introduction Shigella flexneri bacteria cause bacillary dysentery in humans by invading and replicating inside intestinal epithelial cells and triggering apoptosis in local macrophages which results in an acute inflammatory response [1,2]. Virulence is the result of the coordinated and interdependent activity of many virulence determinants that are encoded on the chromosome and on the virulence plasmid (VP). The outer membrane of S. flexneri plays a key role in virulence as it contains a number of critical virulence factors, including the outer membrane protein IcsA (also called VirG) [3,4] and lipopolysaccharide (LPS). The IcsA protein is essential for nucleating actin-based motility (ABM), a process which is used by S. flexneri to spread and move inside the cytoplasm of infected cells, and to spread to adjacent cells by means of bacteria-containing extrusions of the plasma membrane (or filopods) which penetrate into these cells [2]. The 116 kDa IcsA protein is anchored into the outer membrane by its carboxy-terminal end (called beta domain), and in wild type cells the protein * Corresponding author. Tel: þ 61-8-8303-4151; fax: þ61-8-8303-7532. E-mail address: [email protected] (R. Morona).

is predominantly located at one of the cell poles [5 – 7]. Sequences within the 85 – 95 kDa amino-terminal alpha domain (IcsA’) target synthesis and export of IcsA to the old cell pole [8 – 10]. IcsA’ is secreted into the culture medium due to the low level activity of an outer membrane protease termed either IcsP [11] or SopA [12] that cleaves IcsA between amino acids 758 and 759 [13,14]. The IcsP/SopA protease contributes to the polarised distribution of IcsA [15,16]. LPS has three distinct regions: lipid A, core sugars and O antigen (Oag) polysaccharide chains, and these intact LPS molecules are termed smooth LPS (S-LPS). The lipid A region anchors the molecule into the outer membrane, and the Oag chains which are linked to the lipid A via the core sugar region extend into the external milieu. The basic Oag repeat unit (RU) of almost all S. flexneri serotypes is composed of three rhamnose and one N-acetyl glucosamine sugar units. LPS molecules lacking Oag chains as a result of mutations affecting synthesis and assembly of the Oag RU, and also their linkage to the core sugar region, are termed rough LPS (R-LPS). These mutations affect virulence and the cell surface expression, localisation and function of IcsA [17 –25].

0882-4010/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0882-4010(03)00064-0

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A S. flexneri 2457T rmlD (rfbD) < Km R mutant (RMA723, 2457T‘rough’) is unable to synthesize Oag due to a block in dTDP-rhamnose synthesis and has R-LPS [25]. Like similar rfb mutants [22], it has high levels of IcsA on both the lateral and polar regions of its cell surface. 2457T‘rough’, while still able to invade mammalian cells in tissue culture, has an ABM defect as it is unable to plaque on HeLa cells and has a defect in F-actin comet tail formation. The infrequent F-actin comet tails have an altered morphology; they are short and distorted compared to those seen for 2457T [25]. Previous studies have characterised S. flexneri strains with mutations that inactivate either the IcsP/SopA cleavage site in IcsA (resulting in an altered protein herein termed IcsAp) or the icsP gene that encodes the IcsP protease [11 –14,26]. These mutations have little effect on virulence, and actually enhanced intracellular motility and caused an earlier onset of virulence in Sereny and plaque assays. Intriguingly, in these mutants IcsA/IcsAp was localised on both the lateral and polar regions of the cell surface, suggesting that strict polar localisation of IcsA in S. flexneri is not essential for its function in ABM and virulence. In comparison, as described above, S. flexneri R-LPS mutants also have IcsA on lateral and polar regions of the cell surface but are avirulent and have an ABM defect. Despite these findings, several studies have implied that loss of polar IcsA localisation due to R-LPS mutations was responsible for loss of ABM [23,24,27]. We have recently shown that Oag chains can mask the presence of IcsA on the cell surface of S. flexneri [28]. This suggested to us the absence of Oag chains in the R-LPS strains permits detection of IcsA on lateral cell surfaces with antibodies. In this study, we directly compare for the first time the phenotype of a S. flexneri R-LPS strain with that of a smooth LPS S. flexneri strain producing IcsAp. This comparison supports our hypothesis that IcsA localisation is unrelated to its loss of function in S. flexneri R-LPS mutants.

2. Results and discussion 2.1. Construction of strains producing IcsA and IcsAp To allow direct comparison of relevant phenotypes, isogenic wild type and R-LPS strains producing either wild type IcsA or IcsAp were constructed. The icsA gene was deleted in strains 2457T and 2457T rough [RMA723] by allelic exchange mutagenesis using pRMA920 as described in the Methods and Methods, resulting in 2457T DicsA < TcR [RMA2041] and 2457T rmlD < KmR DicsA < TcR [RMA2043]. The pBR322-based plasmids pIcsA (encoding wild type IcsA) and pIcsAp (encoding IcsAp) expressing icsAþ and icsAp, respectively, from the native promoter were then electroporated into the DicsA < TcR strains resulting in 2457T DicsA < TcR (pIcsA) [RMA2090, 2457T‘smooth þ

IcsA WT’], 2457T DicsA < Tc R (pIcsA p) [RMA2092, 2457T‘smooth þ IcsAp’], 2457T rmlD < KmR DicsA < TcR (pIcsA) [RMA2107, 2457T‘rough þ IcsAWT’] and 2457T rmlD < KmR DicsA < TcR (pIcsAp) [RMA2108, 2457T‘rough þ IcsAp’]. Western immunoblotting with anti-IcsA showed that these strains produced similar amounts of IcsA (data not shown). In addition we confirmed by Western immunoblotting of culture supernatants that IcsAp was non-cleavable since 2457T‘smooth þ IcsAp’ and 2457T‘rough þ IcsAp’ did not secrete the 85 – 95 kDa IcsA’ into the culture media (data not shown). 2.2. Comparison of virulence related properties The S-LPS strains 2457T‘smooth IcsA WT ’ and 2457T‘smooth þ IcsAp’ were fully virulent as determined by their ability to form plaques on HeLa cell monolayers. No differences in either plaque size or plaquing efficiency were observed between these strains and 2457T (data not shown). These results also show that the non-IcsP cleavable IcsAp is fully functional, a result which is consistent with previous reports [14,26] and with the phenotype of a 2457T icsP mutant [11]. Like 2457T‘rough’ [25], the R-LPS strains 2457T‘rough þ IcsAWT’ and 2457T‘rough þ IcsAp’ were unable to plaque on HeLa cell monolayers (data not shown). We then determined if pIcsA and pIcsAp had any effect on the F-actin comet tail formation defect associated with the rmlD mutation [25]. HeLa cells infected with the S-LPS strains 2457T‘smooth þ IcsAWT’ and 2457T‘smooth þ IcsAp’ and the R-LPS strains 2457T‘rough’, 2457T‘rough þ IcsAWT’, and 2457T‘rough þ IcsAp’, were stained with FITC-phalloidin to detect F-actin comet tails. While approximately 25 – 50% of S-LPS 2457T‘smooth þ IcsAWT’ (27%, n ¼ 134) and 2457T‘smooth þ IcsAp’ (54%, n ¼ 110) bacteria were associated with F-actin and Factin comet tails (Fig. 1, panels b, c), this was less frequently observed for the R-LPS strains (2457T‘rough’ (4%, n ¼ 271; P , 0:05 when compared to 2457T‘smooth þ IcsAWT’); 2457T‘rough þ IcsA’ (3%, n ¼ 201; P , 0:05 when compared to 2457T‘smooth þ IcsAWT’); 2457T‘rough þ IcsAp’ (2%, n ¼ 292; P , 0:005 when compared to 2457T‘smooth þ IcsAp’)) (Fig. 1, panels d, e, and f). The F-actin comet tails formed by the R-LPS strains had altered an morphology compared to those of the S-LPS strains, being both thinner and distorted, as previously described [25]. The F-actin comet tails formed by 2457T‘smooth þ IcsAp’ had a similar morphology to those formed by 2457T‘smooth þ IcsAWT’, in agreement with the results of Fukuda et al. [14]. This contrasts with the results of d’Hauteville et al. [13] who reported that a S-LPS S. flexneri M90T derived strain producing IcsAp formed F-actin comet tails with altered morphology. The phenotype of 2457T‘rough þ IcsAp’ shows that IcsAp was unable to suppress the plaque and F-actin comet tail formation defects caused by rmlD < KmR mutation.

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Fig. 1. Detection of F-actin comet tails produced by S. flexneri growing within HeLa cells. HeLa cell monolayers were infected with S. flexneri strains and stained to detect F-actin comet tails. S. flexneri bacteria were detected by indirect immunofluorescence staining with rabbit anti-Oag antibody and a Texas Redconjugated secondary antibody (seen here as Red), and F-actin was detected by staining with FITC-phalloidin (seen here as Green). The strains in each panel are: a, 2457T DicsA < TcR [RMA2041]; b, 2457T DicsA < TcR (pIcsA) [RMA2090]; c, 2457T DicsA < TcR (pIcsAp) [RMA2092]; d, 2457T rmlD < KmR [RMA723]; e, 2457T rmlD < KmR DicsA < TcR (pIcsA) [RMA2107]; f, 2457T rmlD < KmR DicsA < TcR (pIcsAp) [RMA2108]. In each panel, the arrowheads indicate the location of an F-actin comet tail. The experiment was repeated on three occasions and typical results are shown.

As IcsAp is fully functional in 2457T‘smooth þ IcsAp,, IcsAp is still dependent on S-LPS for activity in ABM. Strains with the rmlD < KmR mutation (Fig. 1, panels d, e, f) did not have an absolute defect in ABM; they exhibited some intracellular spreading since they were localised throughout the cytoplasm of infected HeLa cells when compared with S-LPS 2457T DicsA < TcR [RMA2041] bacteria which were located in a cluster close to the nucleus (Fig. 1, panel a). 2.3. Cell surface localisation of IcsAp We used indirect immunofluorescence microscopy to compare the cell surface localisation of IcsAp in a smooth LPS background with that of IcsA in the R-LPS. S. flexneri strains grown in LB to early logarithmic grow phase were immuno-stained to detect IcsA. 2457T‘smooth þ IcsAWT’ had polarly localised IcsA (Fig. 2, panel a) and was identical to 2457T (data not shown) whereas 2457T‘smooth þ IcsAp’ (Fig. 2, panel b) had IcsA on lateral and polar regions of its cell surface. This results shows that the non-cleavable form of IcsA is not strictly polarly localised which supports the findings of d’Hauteville et al. [13] but not that of Fukuda et al. [14]. Additionally, the polar localisation of IcsA on 2457T‘smooth þ IcsAWT’ shows that localisation of IcsA was not affected by icsA

being expressed from a plasmid. Strains 2457T‘rough þ IcsAWT’ and 2457T‘rough þ IcsAp’ (Fig. 2, panels d,e) were identical to 2457T‘rough’ (Fig. 2, panel c) and had IcsA/IcsAp on both lateral and polar regions of the cell surface. Since there was little differences between the distribution of IcsAp on smooth LPS and IcsA on R-LPS bacteria, we further compared their cell surface localisation on intracellular bacteria. HeLa cells infected with 2457T‘smooth þ IcsAWT’, 2457T‘smooth þ IcsAp’, 2457T‘rough’, 2457T ‘rough þ IcsAWT’, 2457T‘rough þ IcsAp’ were immunostained to detect IcsA/IcsAp. Consistent with the result obtained using LB grown 2457T‘smooth þ IcsAp’ bacteria (Fig. 2, panel b), intracellular 2457T‘smooth þ IcsAp’ bacteria also had IcsAp localised on lateral and polar regions of the cell (Fig. 3, panel c). Similar to LB grown bacteria, intracellular 2457T‘smooth þ IcsAWT’ bacteria had polarly localised IcsA (Fig. 3, panel b). 2457T‘rough’, 2457T‘rough þ IcsAWT’, 2457T‘rough þ IcsAp’ had IcsA on lateral and polar regions of their cell surfaces (Fig. 3, panels d, e, f). This was similar to the localisation of IcsAp on intracellular 2457T‘smooth þ IcsAp’, as described above (Fig. 3, panel c). Labelling of IcsA p on the cell surface of 2457T‘smooth þ IcsAp’ was not as intense as that observed for the isogenic 2457T‘rough þ IcsAp’ strain (Fig. 3,

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panel f). This difference may be due to the absence of Oag chains in the R-LPS mutant that can mask IcsA on the lateral regions of the cells surface of S-LPS strains [28].

3. Conclusion In this study, we found little or no difference between the localisation of IcsA on the cell surface of a R-LPS S. flexneri strain and that of IcsAp on the cell surface of a S-LPS strain; in either strain it was located on lateral and polar regions of the cell surface. Like IcsA, IcsAp was fully functional in a smooth LPS background but was non-functional in a R-LPS background. We conclude that strict polar localisation is not needed for efficient IcsA function in ABM. This is consistent with observation that E. coli K-12 ompT producing IcsA has it on lateral and polar regions of the cell surface and exhibits ABM in assays using cell extracts and purified proteins [29,30]. The inability of the R-LPS strain to plaque on HeLa cell monolayer and form F-actin comet tails with wild type morphology was unlikely to be due to loss of IcsA polar localisation. We conclude that the LPS Oag chains are directly required in some way for IcsA to function in ABM. We speculate that the absence of Oag chains affects the conformation and/or orientation of the alpha domain of IcsA, which results in inefficient nucleation of ABM and altered F-actin comet tail formation. We do not exclude the possibility that production and/or activity of other factors are also altered in the absence of Oag chains [31].

4. Materials and methods 4.1. Growth media and growth conditions

Fig. 2. Detection of IcsA on the cell surface of LB grown S. flexneri bacteria. S. flexneri strains were grown to early logarithmic phase in LB, fixed, and immuno-stained to detect IcsA on the cell surface with rabbit anti-IcsA antibody and an FITC-conjugated secondary antibody. The strains in each panel are as follows: a, 2457T DicsA < TcR (pIcsA) [RMA2090]; b, 2457T DicsA < TcR (pIcsAp) [RMA2092]; c, 2457T rmlD < KmR [RMA723], d, 2457T rmlD < KmR DicsA < TcR (pIcsA) [RMA2107]; e, 2457T rmlD < KmR DicsA < TcR (pIcsAp) [RMA2108]. For each panel, the phase contrast image is on the left hand side and corresponding immunofluorescent image is on the right hand side of the S. flexneri strains indicated in the figure. An enlargement of a typical bacterium is shown in each immunofluorescent image (Insert). Strains 2457T DicsA < TcR [RMA2041] and 2457T rmlD < KmR RMA723 DicsA < TcR [RMA2043] showed no detectable staining and are not shown. The experiment was repeated on three occasions and typical results are shown.

Strains were routinely grown in Luria Bertani (LB) broth and agar as previously described [25]. Tryptic Soya Broth (TSB) (Difco) was used to grow strains used to infect tissue culture cells. S. flexneri strains were re-isolated on Congo Red agar [25] every 2 months to verify virulence status. Unless otherwise stated, S. flexneri bacteria were grown from a Congo Red positive colony in LB for 16 h, then diluted 1 in 50 into fresh LB and grown for 2 h. Under these growth conditions, IcsA protein was almost entirely in the intact 116 kDa form, as determined by Western immunoblotting. Antibiotics were used at the following concentrations: ampicillin (Ap), 50 mg/ml; chloramphenicol (Cm), 25 mg/ml, kanamycin (Km) (50 mg/ml); and tetracycline (Tc), 10 mg/ml. Unless stated otherwise, strains were grown at 37 8C. 4.2. Bacterial strains and plasmids The bacterial strains used are described in Table 1.

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Fig. 3. Detection of IcsA on the cell surface of intracellular S. flexneri bacteria. HeLa cell monolayers were infected with S. flexneri strains and stained to detect IcsA. Production of IcsA by S. flexneri bacteria was detected by indirect immunofluorescence staining with a rabbit anti-IcsA antibody and a FITC-conjugated secondary antibody (seen here as Green), and S. flexneri bacteria were detected by counter-staining with propidium iodide (seen here as Red). The strains in each panel are: a, 2457T DicsA < TcR [RMA2041]; b, 2457T DicsA < TcR (pIcsA) [RMA2090]; c, 2457T DicsA < TcR (pIcsAp) [RMA2092]; d, 2457T rmlD < KmR [RMA723]; e, 2457T rmlD < KmR DicsA < TcR (pIcsA) [RMA2107]; f, 2457T rmlD < KmR DicsA < TcR (pIcsAp) [RMA2108]. The large, oval-shaped, red-staining bodies are the HeLa cell nuclei. Within each panel, an enlargement of a typical bacterium is shown. The experiment was repeated on three occasions and typical results are shown.

4.3. DNA methods DNA manipulations, PCR, transformation, and electroporation into S. flexneri was performed as recently described [37,38]. DH5a was used for all cloning. 4.4. Construction of DicsA < TetR mutant strains The icsA gene was inactivated in various S. flexneri strains by allelic exchange mutagenesis using plasmid pRMA2039 which is based on the suicide vector pCACTUS [35]. Plasmid pRMA2039 was constructed as follows. Plasmid pRMA915, which contains the icsA gene on a 3.5 kb Eco RI –Sal I fragment from pD10 cloned into pBCKS (Stratagene) [33], was digested with Eco RI and Xho I and cloned into similarly digested pLitmus29, resulting in plasmid pRMA2020. The entire icsA coding region within pRMA2020 was deleted by digesting it with Xmn I and religating the plasmid. After transforming into DH5a, screening of plasmids from Ap resistant colonies by restriction enzyme digestion resulted in pRMA2021 which had the expected deletion (DicsA). Plasmid pRMA2021 was digested with Bam HI and Bgl II, and the mutated icsA gene was cloned into Bam HI digested pCACTUS. Cm resistant

colonies were selected at 30 8C and plasmid pRMA2022 having the expected insert was recovered. A Tc resistance cassette (TcR) was inserted into the unique Xmn I site within the deleted icsA region by digesting pRMA2022 with Xmn I and blunt-end ligating it with Sma I digested pSBA383 which was the source of the 2.5 kb tetAR(B) TcR cassette. Transformation into DH5a at 30 8C, and screening of Cm and Tc resistant colonies by restriction digestion resulted in pRMA2039. Allelic exchange mutagenesis was performed as previously described (35). In brief, pRMA2039 was electroporated into various S. flexneri strains, and Tc and Cm resistant colonies were selected at 30 8C. The pRMA2039 containing strains were grown at 42 8C with Tc selection, then plated on LB agar lacking NaCl but containing 6% (w/v) sucrose and Tc to select colonies which had undergone allelic exchange. Colonies were confirmed to be Cm sensitive, and PCR was used to confirm the presence of the mutation. 4.5. Construction of IcsA plasmids Plasmid pIcsA containing the 3.5 kb Eco RI –Sal I icsA fragment cloned into Eco RI– Sal I digested pBR322 is

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4.8. Immunofluorescence detection of IcsA on LB grown bacteria

Table 1 Bacterial strains and plasmids used Relevant characteristicsa

Reference /Source

Cloning host

Gibco BRL

Shigella flexneri 2a wild type 2457T DicsA < TcR RMA2041 [pIcsA] RMA2041 [pIcsAp] 2457T rmlD < KmR RMA723 DicsA < TcR RMA2043 [pIcsA] RMA2043 [pIcsAp]

[25] This study This study This study [25] This study This study This study Ref. [4] Ref. [14]

pBR322 pRMA915 pIcsA pIcsAp pCACTUS pSBA383 pLitmus29

Source of virG/icsA gene pD10 with virG203 (called icsA p in this paper) ApR, TcR icsA gene cloned in pBC-KS icsA gene cloned in pBR322 icsA p gene cloned in pBR322 Suicide vector, CmR Source of tetAR(B) cassette (TcR) Cloning vector; ApR

pRMA2020 pRMA2021 pRMA2022 pRMA2039

icsA gene cloned in pLitmus29 pRMA2020 with DicsA pCACTUS with DicsA; CmR pCACTUS with DicsA < TcR, CmR

Strain/Plasmid

Escherichia coli K-12 DH5a Shigella flexneri 2457T RMA2041 RMA2090 RMA2092 RMA723 RMA2043 RMA2107 RMA2108 Plasmids pD10 pTSG203

a

Ref. [32] Ref. [33] Ref. [34] This study Ref. [35] Ref. [36] New England Biolabs This study This study This study This study

R, Resistance.

described elsewhere [34]. Plasmid pIcsAp, which encodes a mutant IcsA protein (VirG203) having two amino acids changes (R758D, R759D) inactivating the IcsP/SopA protease cleavage site, was constructed by sub-cloning the 3.5 kb Eco RI– Sal I fragment from plasmid pTSG203 into pBR322.

4.6. IcsA antibody A rabbit anti-IcsA antibody was prepared as described elsewhere [25,33]. The antibody recognises epitopes within the alpha domain but not the beta domain of IcsA.

4.7. Formalin-fixation of bacteria Bacteria (10 ml culture) were centrifuged (IEC Centra 4X, 5,000 rpm, 10 min), washed twice in saline by centrifugation, resuspended in 5 ml 2% (w/v) paraformaldehyde (Sigma) in saline (formalin-saline) and incubated at 37 8C for 1 h with agitation every 15 min. The bacteria were then washed three times in saline, and resuspended in 1 ml saline.

The method used was recently described [25,33], and is a modification of the method of Klauser et al. [39]. In brief, formalin-fixed bacterial cells were centrifuged onto poly-Llysine treated, acid-washed, round glass cover-slips at 2000 rpm (Hereaus Labofuge 400R) for 10 min. After incubation with primary antibody (anti-IcsA, 1:100) in PBS with 10% (w/v) foetal calf serum (FCS) at 37 8C for 60 min, the cover-slips were washed with PBS, and incubated at 37 8C for 30 min with the secondary antibody (1:80) (fluorescein-isothiocyanate (FITC) conjugated goat antirabbit, Silenus (cat. No. RDAF)) in PBS with 10% (w/v) FCS. After washing in PBS, the cover-slips were air dried, mounted on glass microscope slides with Mowiol 4– 88 (Calbiochem) containing 20 mg/ml p-phenylenediamine (Sigma), and sealed with acrylic nail polish. Bacteria were photographed with Kodak TMAX400 film using an Olympus B2 microscope equipped with phase contrast and epi-fluorescence illumination, standard FITC filters, and a 100X achromatic oil immersion lens. 4.9. Plaque assay Plaque assays were performed using HeLa cells (laboratory stock, IMVS) as described by Oaks et al. [40]. 4.10. Invasion of HeLa cells and immunofluorescence staining Infection of HeLa cells and immunofluorescence staining were performed as recently described [25,33]. In brief, 18 h cultures of a S. flexneri strains were diluted 1:50, grown for 1.5 h, centrifuged (IEC Centra 4X, 5,000 rpm, 10 min), washed, and resuspended at approximately 109 bacteria/ml in D-PBS (PBS with 0.1% (w/v) CaCl2, 0.1% (w/v) MgCl2). HeLa cells, seeded on glass cover-slips in a 24 well tray and grown for 16 h to give semi-confluence, were washed with D-PBS, overlaid with 100 ml of bacterial suspension, and centrifuged for 10 min at 2000 rpm (Hereaus Labofuge 400R). After 60 min incubation at 37 8C in humidified CO2 incubator (5% CO2), the infected cells were washed five times with D-PBS and incubated with 0.5 ml MEM (Gibco) containing 50 mg/ml of gentamicin (Gibco) for a further 1.5 h at 37 8C in a CO2 incubator. To detect F-actin comet tails, infected cells were fixed for 10 min in 3.7% (w/v) paraformaldehyde in PBS, and then permeabilised with 0.2% (v/v) Triton X-100 in PBS for 1 min. To detect IcsA on the cell surface of intracellular bacteria, infected cells were fixed for 5 min at RT with 80% (v/v) acetone, incubated with PBS for 1 min, then permeabilised with 0.1% (v/v) Triton X-100 in PBS for 1 min. After blocking in 1% (w/v) FBS in PBS for 10 min, the infected cells were incubated at 37 8C for 30 min with either polyclonal antiShigella LPS (Denka Seiken Co, Japan; Polyvalent B, Cat.

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No. 310 111) (1 in 100), or rabbit anti-IcsA antiserum (1 in 100). After washing in PBS, cover-slips were incubated for 15 min at 37 8C with either FITC-conjugated goat antirabbit or Texas Red-conjugated goat anti-rabbit (Amersham, UK) secondary antibodies (1 in 100), as required. F-actin was visualised by staining with FITC-phalloidin (0.1 mg/ml, Sigma), and propidium iodide (10 mg/ml, Sigma) was used to counter-stain bacteria and cellular nuclei, and were included with the secondary antibody incubation, as required. The cover-slips were then mounted as described above, and examined with a Bio-Rad MRC-600 confocal laser scanning microscope, using a 100 £ oil immersion, objective lens. FITC and Texas Red images were collected simultaneously, and false colour merged using Confocal Assistant 4.02. Each image shown is from a single plane. Statistical analysis was performed using Student’s unpaired, two tailed t-test (PRISM 3.03, GraphPad Software).

Acknowledgements This work was supported by a Project Grant from the National Health and Medical Research Council of Australia to RM. Elizabeth Anderson is thanked for technical support. Chihiro Sasakawa is thanked for providing pTSG203. Judy Morona is thanked for help her comments on the manuscript.

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