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Multicopy icsA is able to suppress the virulence defect caused by the wzzSF mutation in Shigella flexneri
FEMS Microbiology Letters 221 (2003) 213^219
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Multicopy icsA is able to suppress the virulence defect caused by the wzzSF mutation in Shigella £exneri Renato Morona , Luisa Van Den Bosch School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia Received 9 January 2003; received in revised form 13 February 2003; accepted 26 February 2003 First published online 1 April 2003
Abstract The lipopolysaccharides (LPS) of Shigella flexneri are important for virulence and their O antigen (Oag) polysaccharide chains affect IcsA (VirG)-mediated actin-based motility (ABM) within mammalian cells. S. flexneri 2a 2457T has smooth LPS whose Oag chains have two modal lengths (short (S)-type and very long (VL)-type), and has IcsA predominantly located at one pole on its cell surface. A S. flexneri 2457T wzzSF mutant (RMA696) has VL-type Oag but not S-type Oag chains, less IcsA detectable by immunofluorescence on its cell surface, reduced virulence and defective ABM. Introduction of a plasmid encoding IcsA into S. flexneri wzzSF showed that multicopy icsA could suppress the virulence defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation) caused by the wzzSF mutation suggesting that the VL-type Oag chains were masking IcsA and limiting the amount available to initiate ABM. 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lipopolysaccharide ; O antigen; wzz; Virulence; IcsA; Shigella £exneri
1. Introduction Shigella £exneri bacteria cause bacillary dysentery in humans. A key aspect of pathogenesis is the ability of S. £exneri bacteria to use actin-based motility (ABM) to move inside cells, and to spread to adjacent uninfected intestinal cells [1]. ABM is initiated by the outer membrane protein IcsA (also called VirG) [2,3] and the cell lipopolysaccharide (LPS) molecules also impact on this process. The 116 kDa IcsA protein is predominantly located at one of the cell poles [4^6]. Sequences located within the amino-terminal K-domain (herein termed IcsAP, 85^95 kDa) have been shown to target synthesis and export of IcsA to the old cell pole [7^9]. Intact LPS molecules, also referred to as smooth LPS, have three regions: lipid A, core sugars, and O antigen (Oag) polysaccharide chains. Mutations in the large number of genes involved in LPS core sugars region and Oag biosynthesis can a¡ect virulence, and the cell surface expression and localisation of IcsA [10^18]. We are partic-
ularly interested in the mutations that a¡ect the LPS Oag chain length distribution. The LPS of many S. £exneri 2a strains such as 2457T have LPS Oag chains whose length are non-randomly distributed such that they have two modal lengths. The modal length of 11^17 repeat units (RU) (termed S-type) is determined by the chromosomally located wzzSF gene [19], and that of s 90 RU (termed VLtype) is determined by the pHS-2 plasmid located wzzpHS2 gene [10,20]. Mutation in either gene results in loss of LPS with the respective Oag modal chain length. A S. £exneri 2457T wzzSF mutant (RMA696) was unable to form plaques on HeLa cells, had reduced virulence in a Sereny assay, reduced levels of IcsA on its cell surface, and markedly reduced ability to form F-actin comet tails [18]. In this paper we investigate the relationship between Oag chain length regulation and IcsA function, and provide evidence that the wzzSF virulence defect is caused by a limiting availability of IcsA.
2. Materials and methods * Corresponding author : Tel. : +61 (8) 8303 4151; Fax : +61 (8) 8303 7532. E-mail address : [email protected] (R. Morona).
2.1. Growth media and growth conditions Strains were grown in Luria Bertani (LB) broth and
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00217-9
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agar as previously described [18]. Unless otherwise stated, S. £exneri bacteria were grown from a Congo red agar [18] positive colony in LB for 16 h, then diluted 1 in 50 into fresh LB and grown for 2 h. Under these growth conditions, the IcsA protein was almost entirely in the intact 116 kDa form. Antibiotics used were: ampicillin (Ap), 50 Wg ml31 ; chloramphenicol (Cm), 25 Wg ml31 ; kanamycin (Km), 50 Wg ml31 ; and tetracycline (Tc), 10 Wg ml31 . Unless stated otherwise, strains were grown at 37‡C.
2.5. IcsA antibody
2.2. Bacterial strains and plasmids
Whole cell lysates were prepared by resuspending bacteria (approximately 2U108 cells) in 0.05 ml sample bu¡er [25] and heating for 5 min at 100‡C. Analysis of proteins by SDS^PAGE and Western immunoblotting was performed as recently described [24]. After transfer to nitrocellulose membrane, and incubation with rabbit anti-IcsA, bound antibody was detected using a goat anti-rabbit, horseradish peroxidase conjugate, and enhanced chemiluminescence (Roche). Polyclonal rabbit anti-IcsA serum was used at 1:250. Molecular mass markers (prestained, Bio-Rad cat. no. 161-0305) were: phosphorylase B (116 kDa), bovine serum albumin (80 kDa), ovalbumin (51.8 kDa), carbonic anhydrase (34.7 kDa), soybean trypsin inhibitor (30 kDa), lysozyme (22 kDa).
The bacterial strains and plasmids used are described in Table 1. The icsA gene was inactivated in 2457T wzzSF : :KmR by allelic exchange mutagenesis using plasmid pRMA2039 (Van Den Bosch, L. and Morona, R., submitted for publication) which is based on the suicide vector pCACTUS [19]. This resulted in deletion of the entire icsA gene (vicsA) and its replacement with a tetracycline resistance cassette (TcR ) [22]. 2.3. DNA methods DNA manipulations and electroporation into S. £exneri were performed as recently described [23,24]. DH5K was used for all cloning. 2.4. Construction of pIcsA Plasmid pIcsA was constructed as follows. A 3.5 kb EcoRI^SalI fragment containing icsA was subcloned from pD10 into similarly digested pBC-KS(+), resulting in plasmid pRMA915. The 3.5 kb EcoRI^SalI icsA fragment was then subcloned from pRMA915 into EcoRI^ SalI-digested pBR322. Transformation into DH5K and screening of Ap-resistant, Tc-sensitive colonies resulted in the isolation of pIcsA.
A rabbit anti-IcsA antibody was prepared as described elsewhere ([18]; Morona, R., Daniels, C. and Van Den Bosch, L., submitted for publication). 2.6. Preparation of IcsA samples, and sodium dodecyl sulphate^polyacrylamide gel electrophoresis (SDS^PAGE) and Western immunoblotting
2.7. Sereny reaction and plaque assay The Sereny assay to assess ability to cause keratoconjunctivitis was performed using guinea pigs as directed by the Animal Ethics Committee of the University of Adelaide. The severity of the reaction was scored according to the Sereny reaction ratings [26,27]. Plaque assays were performed using HeLa cells (laboratory stock, Institute of Medical and Veterinary Science, IMVS) as described by Oaks et al. [28].
Table 1 Bacterial strains and plasmids Strain/plasmid Escherichia coli K12 DH5K S. £exneri 2457T RMA696 RMA2095 RMA2042 RMA723 RMA2103 Plasmids pD10 pBC-KS(+) pRMA915 pBR322 pIcsA pRMA2039 a
Relevant characteristicsa
Reference/source
cloning host
Gibco-BRL
S. £exneri 2a wild-type 2457T wzzSF : :KmR 2457T wzzSF : :KmR (pIcsA) 2457T wzzSF : :KmR vicsA: :TcR 2457T rmlD: :KmR 2457T rmlD: :KmR (pIcsA)
[18] [18] this study this study [18] this study
source of virG/icsA gene CmR icsA gene cloned in pBC-KS(+); CmR ApR , TcR icsA gene cloned in pBR322 pCACTUS with vicsA : :TcR ; CmR
[3] Stratagene this study [21] this study Van Den Bosch, L. and Morona, R., submitted for publication
R, resistance.
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2.8. Immuno£uorescence detection of IcsA on LB grown bacteria The method used was recently described ([18,29], Morona, R., Daniels, C. and Van Den Bosch, L., submitted for publication). In brief, formalin-¢xed bacterial cells (0.1 ml) were centrifuged onto round glass coverslips in a 24well £at-bottomed tissue culture tray. After washing in phosphate-bu¡ered saline (PBS), primary antibody (antiIcsA, 1:100) in PBS with 10% (w/v) foetal calf serum (FCS) was added, incubated at 37‡C for 60 min, washed with PBS, then incubated with £uorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (1:80) (Silenus) at 37‡C for 30 min. The coverslips were washed in PBS, air dried, mounted on glass microscope slides with Mowiol 4-88 (Calbiochem) containing 20 Wg ml31 p-phenylenediamine (Sigma), and sealed with acrylic nail polish. Bacteria were photographed with Kodak TMAX400 ¢lm using an Olympus B2 microscope equipped with phase contrast and epi£uorescence illumination, standard FITC ¢lters, and a 100U achromatic oil immersion lens. 2.9. Invasion of HeLa cells, detection of F-actin, and immuno£uorescence staining Infection of HeLa cells and immuno£uorescence staining were performed as recently described ([18] ; Morona, R., Daniels, C. and Van Den Bosch, L., submitted for publication). In brief, S. £exneri strains were grown in LB and resuspended at approximately 109 bacteria ml31 in D-PBS (PBS with 0.1% (w/v) CaCl2 , 0.1% (w/v) MgCl2 ). 100 Wl of bacterial suspension was then centrifuged onto HeLa cells grown to semicon£uence on glass coverslips. After 60 min incubation at 37‡C in humidi¢ed CO2 incubator (5% CO2 ), the infected cells were washed ¢ve times with D-PBS and incubated with 0.5 ml minimal Eagle’s medium (MEM) (Gibco) containing 50 Wg ml31 of gentamicin (Gibco) for a further 1.5 h at 37‡C in a CO2 incubator. To detect F-actin comet tails, infected cells were ¢xed for 10 min in 3.7% (w/v) paraformaldehyde in PBS, and then permeabilised with 0.2% Triton X-100 in PBS for 1 min. To detect IcsA on intracellular bacteria, infected cells were ¢xed for 5 min at room temperature with 80% (v/v) acetone, incubated with PBS for 1 min, then permeabilised with 0.1% Triton X-100. After blocking in 1% foetal bovine serum (FBS), the infected cells were incubated at 37‡C for 30 min with either polyclonal anti-Shigella LPS (Denka Seiken Co, Japan) or rabbit anti-IcsA antibody (1:100). After washing in PBS, coverslips were incubated with either FITC-conjugated goat anti-rabbit or Texas red-conjugated goat anti-rabbit (Amersham, UK) secondary antibodies (1:100), as required. F-actin was visualised by staining with FITC-phalloidin (0.1 Wg ml31 , Sigma), and propidium iodide (PI) (10 Wg ml31 , Sigma) was used to counterstain bacteria and cellular nuclei, and was included with the secondary anti-
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body incubation, as required. The coverslips were mounted as described above, and were examined with a Bio-Rad MRC-600 confocal laser scanning microscope using a 100U oil immersion, objective lens. FITC and Texas red/PI images were false colour merged using Confocal Assistant 4.02. Each image shown is from a single plane.
3. Results 3.1. E¡ect of pIcsA on IcsA production in S. £exneri Since S. £exneri wzzSF : :KmR [RMA696] had decreased levels of IcsA detectable on its cell surface [18], this suggested to us that the VL-type Oag chains produced by the wzzSF mutant may be masking IcsA and thereby decreasing its ability to act in ABM. We investigated if the virulence-related defects due to the wzzSF mutation could be suppressed by using multicopy icsA to increase the amount of IcsA present on the cell surface. In S. £exneri, icsA is located on the large virulence plasmid. To increase the IcsA dosage, plasmid pIcsA was electroporated into 2457T wzzSF : :KmR [RMA696] resulting in 2457T wzzSF : :KmR (pIcsA) [RMA2095]. Western immunoblotting with anti-IcsA antibody was
Fig. 1. IcsA production by S. £exneri RMA696 derived strains. The S. £exneri strains indicated were grown to early logarithmic phase in LB, whole cell lysates were prepared, electrophoresed on an SDS 12% polyacrylamide gel, and after transfer to nitrocellulose proteins were detected with a rabbit anti-IcsA antibody. Lane 1, 2457T ; lane 2, 2457T wzz : : KmR [RMA696]; lane 3, 2457T wzz: :KmR vicsA : :TcR [RMA2042] ; lane 4, 2457T wzz : :KmR (pIcsA) [RMA2095]. The positions of the fulllength 116 kDa IcsA protein, and the minor, contaminating 85 kDa IcsAP (secreted form of IcsA), are indicated on the left-hand side of the ¢gure. Lanes contain approximately 2U108 bacterial cells.
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then used to compare the level of IcsA produced by 2457T and 2457T wzzSF : :KmR with that produced by 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 1). The whole cell lysate of 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 1, lane 5) had a higher level of IcsA (1.5^2U) than that of 2457T and 2457T wzzSF : :KmR (Fig. 1, lanes 1 and 2), and as expected 2457T wzzSF : :KmR vicsA: :TcR [RMA2042] did not produce IcsA (Fig. 1, lane 3). 3.2. E¡ect of multicopy icsA on wzzSF virulence phenotypes The Sereny assay, plaque formation on HeLa cell monolayers, and F-actin comet tail formation were used to compare virulence, and intracellular and intercellular spreading ability of 2457T wzzSF : :KmR [RMA696] with its pIcsA harbouring derivative. 2457T wzzSF : :KmR carrying pIcsA [RMA2095] gave a strongly positive Sereny reaction, identical to that of 2457T (Table 2), unlike 2457T wzzSF : :KmR [RMA696] which gave a weak Sereny reaction (Table 2) as previously reported [18]. Furthermore, strain 2457T wzzSF : :KmR (pIcsA) [RMA2095] was able to form plaques on HeLa cell monolayers that were indistinguishable from those produced by 2457T (Fig. 2a,c) whereas 2457T wzzSF : : KmR [RMA696] (Fig. 2b) was unable to plaque on HeLa cell monolayers as previously reported [18]. The ability of pIcsA to restore the HeLa cell monolayer plaquing ability to 2457T wzzSF : :KmR [RMA696] was further investigated by staining with FITC-phalloidin to detect F-actin comet tail formation. 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 3d) formed F-actin comet tails similar to those produced by 2457T (Fig. 3a). While 2457T wzzSF : :KmR [RMA696] (Fig. 3c) only rarely formed F-actin comet tails, as previously reported [18], it did exhibit some ABM as 2457T wzzSF : :KmR [RMA696] bacteria were dispersed throughout the cytoplasm of infected cells in contrast to 2457T wzzSF : :KmR vicsA: :TcR [RMA2042] bacteria which clustered near the cell nucleus (Fig. 3b). These results show that multicopy icsA was able to suppress the virulence and ABM defect due to the
Fig. 2. Plaque formation by S. £exneri strains. This ¢gure shows plaques formed on HeLa cell monolayers by S. £exneri strains. Strains are: a: 2457T ; b: 2457T wzz: :KmR [RMA696]; c: 2457T wzz : :KmR (pIcsA) [RMA2095]. Arrowhead indicates a plaque. The experiment was performed on three occasions and representative results are shown.
wzzSF : :KmR mutation, suggesting that masking of IcsA by the VL-type Oag chains could be overcome by increased levels of cell surface IcsA. 3.3. Detection of IcsA on the cell surface of LB grown S. £exneri strains We investigated if the e¡ect of multicopy icsA on wzzSF : :KmR mutant phenotype correlated with increased detection of cell surface IcsA. Production of IcsA on the cell surface of 2457T, 2457T wzzSF : :KmR [RMA696], and 2457T wzzSF : :KmR vicsA: :Tc (pIcsA) [RMA2095] either
Table 2 Ability of S. £exneri strains to plaque on HeLa cells, and virulence Strain
Chromosome
Relevant plasmidsa
LPS typeb
Plaque assayc
Sereny reactiond
2457T RMA696 RMA2095 RMA2042 RMA723 RMA2103
wild-type wzzSF : :KmR wzzSF : :KmR wzzSF : :KmR vicsA: :TcR rmlD: :KmR rmlD: :KmR
VP VP VP+pIcsA VP VP VP+pIcsA
S+VL VL VL VL R R
+ 3 + 3 3 3
+++ + +++ nd 3 nd
a
VP, virulence plasmid. pIcsA, pBR322 encoded icsA. S, strain produces LPS with O antigen chains having a modal length of 11^17 RU ; VL, strain produces LPS with O antigen chains having a modal length of s 90 RU; R, strain produces LPS with lacking O antigen chains. c + means that the strain was able to form plaques on HeLa cell monolayers ; 3 means that the strain was unable to form plaques on HeLa cell monolayers. d 3, no Sereny reaction or mild irritation ; +, mild keratoconjunctivitis or late development and/or clearing; +++, fully developed keratoconjunctivitis ; nd, not done. b
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was not seen when intracellular 2457T wzzSF : :KmR [RMA696] and 2457T wzzSF : :KmR (pIcsA) [RMA2095] bacteria were compared (Fig. 5b,c), and did not correlate with the ability of these strains to form F-actin comet tails (Fig. 4). One possible explanation is that to detect IcsA on intracellular bacteria we used di¡erent ¢xation conditions to that used for LB grown bacteria (see Section 2). Under these conditions, it may be that VL-type Oag chains produced in the wzzSF mutant background had a greater e¡ect on detection of IcsA on the intracellular S. £exneri bacteria than on LB grown bacteria. 3.4. E¡ect of multicopy icsA on the rough LPS mutant phenotype While unlikely, it was possible that pIcsA was having some e¡ect on 2457T wzzSF : :KmR [RMA696] which was unrelated to Oag but which suppressed its virulence-re-
Fig. 3. Detection of F-actin comet tails produced by S. £exneri growing within HeLa cells. HeLa cell monolayers were infected with S. £exneri strains and stained to detect F-actin comet tails. S. £exneri bacteria were detected by indirect immuno£uorescence staining with rabbit antiOag antibody and a Texas red-conjugated 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 ; b: RMA2042 [2457T wzzSF : :KmR vicsA: :TcR ]; c: 2457T wzzSF : :KmR [RMA696]; d: 2457T wzzSF : :KmR (pIcsA) [RMA2095]. In each panel, the arrowheads indicate the location of an F-actin comet tail. The experiment was performed on three occasions and representative results are shown.
grown in LB or inside HeLa cells were compared by immuno£uorescence staining using an anti-IcsA antibody. IcsA was detected at the cell pole of LB grown 2457T, 2457T wzzSF : :KmR [RMA696], and 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 4a,b,c), however the intensity of IcsA labelling on 2457T and 2457T wzzSF : :KmR (pIcsA) [RMA2095] was greater than that seen for 2457T wzzSF : :KmR [RMA696]. These results indicate that pIcsA increased the amount of IcsA detected on the cell surface of 2457T wzzSF : :KmR [RMA696] which correlated with the e¡ect on F-actin comet tail and plaque formation, and hence ABM. When resident within HeLa cells, IcsA was detected at the cell poles of 2457T, 2457T wzzSF : :KmR [RMA696] and 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 5), however the intensity of IcsA staining was weaker for the latter two strains. The di¡erence in IcsA labelling observed between LB grown 2457T wzzSF : :KmR [RMA696] and 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 4b,c)
Fig. 4. Detection of IcsA on the cell surface of S. £exneri strains with LPS mutations. S. £exneri strains were grown to early logarithmic phase in LB, ¢xed, and immuno£uorescently 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 ; b: 2457T wzzSF : :KmR [RMA696] ; c: 2457T wzzSF : :KmR (pIcsA) [RMA2095]. For each panel, the phase contrast image is on the lefthand side and corresponding immuno£uorescent image is on the righthand side of the S. £exneri strains indicated in the ¢gure. Strain 2457T wzzSF : :KmR vicsA : :TcR [RMA2042] showed no detectable staining and is not shown. The experiment was performed on three occasions and representative results are shown.
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Fig. 5. Detection of IcsA on the cell surface of intracellular S. £exneri bacteria. HeLa cell monolayers were infected with S. £exneri strains and stained to detect IcsA. Production of IcsA by S. £exneri bacteria was detected by indirect immuno£uorescence staining with a rabbit anti-IcsA antibody and an FITC-conjugated secondary antibody (seen here as green), and S. £exneri bacteria were detected by counterstaining with propidium iodide (seen here as red). The strains in each panel are: a: 2457T; b: 2457T wzzSF : :KmR [RMA696] ; c: 2457T wzzSF : :KmR (pIcsA) [RMA2095]. The large, oval-shaped, red-staining bodies are the HeLa cell nuclei. Within each panel, an enlargement of typical bacteria is shown. Strain 2457T wzzSF : :KmR vicsA: :TcR [RMA2042] showed no detectable IcsA staining and is not shown. The experiment was performed on three occasions and representative results are shown.
lated defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation). To assess this possibility, we investigated if pIcsA had an e¡ect on the virulence-related defects associated with the rough LPS (Oagde¢cient) phenotype due to the rmlD mutation [18]. We transformed pIcsA into the rough LPS strain 2457T rmlD : :KmR [RMA723], and then investigated the ability of the resulting strain to plaque on HeLa cells and form F-actin comet tails. Both 2457T rmlD: :KmR [RMA723] and 2457T rmlD : :KmR (pIcsA) [RMA2103] were unable to plaque on HeLa cells (Table 1). HeLa cells infected with 2457T, 2457T rmlD: :KmR [RMA723], and 2457T rmlD: : KmR (pIcsA) [RMA2103] were stained with FITC-phalloidin. Compared to 2457T, both rmlD mutants exhibited a low frequency of F-actin comet tails that had an altered morphology (data not shown). Hence, unlike its e¡ect on the wzzSF phenotype, multicopy icsA was unable to suppress the ABM defects caused by the rmlD : :KmR mutation.
4. Discussion We have previously reported that 2457T wzzSF : :KmR [RMA696] had reduced cell surface expression of IcsA and decreased virulence [18]. The VL-type LPS Oag chains produced in the wzzSF mutant may mask IcsA thereby interfering with its function and/or limiting the amount of IcsA protein available to initiate ABM. This prompted us to investigate the impact of multicopy icsA on the wzzSF phenotype by introducing pIcsA into 2457T wzzSF : :KmR [RMA696]. We found that multicopy icsA was able to suppress the virulence defect due to the wzzSF mutation as determined
using the Sereny reaction, HeLa cell monolayer plaquing ability, and F-actin comet tail formation; all were restored to wild-type (Table 1, Figs. 2 and 3). When grown in LB, 2457T wzzSF : :KmR (pIcsA) had increased levels of cellular IcsA (Fig. 1) and higher levels of IcsA on its cell surface (Fig. 4) as compared to 2457T wzzSF : :KmR [RMA696]. However, this di¡erence could not be shown by immuno£uorescence detection of IcsA on intracellular bacteria (Fig. 5). The ability of multicopy icsA to suppress the wzz mutant phenotype suggests that the VL-type Oag chains exclusively present in 2457T wzzSF : :KmR [RMA696] are in some way limiting the amount of IcsA available to nucleate ABM. Increasing the amount of IcsA on the cell surface was able to compensate for the e¡ect of these VL-type Oag chains. This interpretation is consistent with the recent results of Magdalena and Goldberg [30] who determined that a threshold level of approximately 4000 IcsA molecules per bacterium was needed for e⁄cient ABM. In support of our results, inactivation of wzzpHS2 in 2457T wzzSF : :KmR [RMA696] and in SA100rol (a wzzSF mutant, [10]), resulted in LPS Oag chains that are of random length and restoration of virulence and plaque forming ability ([10] ; Morona, R., Daniels, C. and Van Den Bosch, L., submitted for publication). Hence, our data suggest that VL-type Oag chains when present as the sole LPS Oag modal chain length can prevent and/or sterically hinder IcsA function in ABM.
Acknowledgements This work was supported by a Project Grant from the National Health and Medical Research Council of Aus-
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tralia to R.M. Elizabeth Anderson is thanked for technical support. Judy Morona is thanked for reading the manuscript.
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[15] Rajakumar, K., Jost, B.H., Sasakawa, C., Okada, N., Yoshikawa, M. and Adler, B. (1994) Nucleotide sequence of the rhamnose biosynthetic operon of Shigella £exneri 2a and role of lipopolysaccharide in virulence. J. Bacteriol. 176, 2362^2373. [16] Sandlin, R.C., Lampel, K.A., Keasler, S.P., Goldberg, M.B., Stolzer, A.L. and Maurelli, A.T. (1995) Avirulence of rough mutants of Shigella £exneri: requirement of O Antigen for correct unipolar localisation of IcsA in the bacterial outer membrane. Infect. Immun. 63, 229^237. [17] Sandlin, R.C., Goldberg, M.B. and Maurelli, A.T. (1996) E¡ect of O side-chain length and composition on the virulence of Shigella £exneri 2a. Mol. Microbiol. 22, 63^73. [18] Van Den Bosch, L., Manning, P.A. and Morona, R. (1997) Regulation of O-antigen chain length is required for Shigella £exneri virulence. Mol. Microbiol. 23, 765^775. [19] Morona, R., Van Den Bosch, L. and Manning, P.A. (1995) Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella £exneri. J. Bacteriol. 177, 1059^1068. [20] Stevenson, G., Kessler, A. and Reeves, P.R. (1995) A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants. FEMS Microbiol. Lett. 125, 25^30. [21] Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heynecker, H.L. and Boyer, H.W. (1977) Construction and characterisation of new cloning vehicles. II. A multi-purpose cloning system. Gene 2, 95^ 113. [22] Rajakumar, K., Sasakawa, C. and Adler, B. (1997) Use of a novel approach, termed island probing, identi¢es the Shigella £exneri she pathogenicity island which encodes a homolog of the immunoglobulin A protease-like family of proteins. Infect. Immun. 65, 4606^ 4614. [23] Baker, S.J., Gunn, J.S. and Morona, R. (1999) The Salmonella typhi melittin resistance gene pqaB a¡ects intracellular growth in PMAdi¡erentiated U937 cells, polymyxin B resistance, and lipopolysaccharide. Microbiology 145, 367^378. [24] Daniels, C. and Morona, R. (1999) Analysis of Shigella £exneri Wzz (Rol) function by mutagenesis and cross-linking : Wzz is able to oligomerise. Mol. Microbiol. 34, 181^194. [25] Lugtenberg, B., Meijers, J., Peters, R., van der Hoek, P. and van Alphen, L. (1975) Electrophoretic resolution of the major outer membrane proteins of Escherichia coli K12 into four bands. FEBS Lett. 58, 254^258. [26] Formal, S.B., Dammin, G.J., LaBrec, E.H. and Schneider, H. (1958) Experimental Shigella infections : characteristics of a fatal infection produced in guinea pigs. J. Bacteriol. 75, 604^610. [27] Sereny, B. (1957) Experimental keratoconjunctivitis shigellosa. Acta Microbiol. Acad. Sci. Hung. 4, 367^376. [28] Oaks, E.V., Wing¢eld, M.E. and Formal, S.B. (1985) Plaque formation by virulent Shigella £exneri. Infect. Immun. 48, 124^129. [29] Klauser, T., Pohlner, J. and Meyer, T.F. (1990) Extracellular transport of cholera toxin B subunit using Neisseria IgA protease betadomain : conformation-dependent outer membrane translocation. EMBO J. 9, 1991^1999. [30] Magdalena, J. and Goldberg, M.B. (2002) Quanti¢cation of Shigella IcsA required for bacterial actin polymerization. Cell. Motil. Cytoskeleton 51, 187^196.
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Multicopy icsA is able to suppress the virulence defect caused by the wzzSF mutation in Shigella £exneri Renato Morona , Luisa Van Den Bosch School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia Received 9 January 2003; received in revised form 13 February 2003; accepted 26 February 2003 First published online 1 April 2003
Abstract The lipopolysaccharides (LPS) of Shigella flexneri are important for virulence and their O antigen (Oag) polysaccharide chains affect IcsA (VirG)-mediated actin-based motility (ABM) within mammalian cells. S. flexneri 2a 2457T has smooth LPS whose Oag chains have two modal lengths (short (S)-type and very long (VL)-type), and has IcsA predominantly located at one pole on its cell surface. A S. flexneri 2457T wzzSF mutant (RMA696) has VL-type Oag but not S-type Oag chains, less IcsA detectable by immunofluorescence on its cell surface, reduced virulence and defective ABM. Introduction of a plasmid encoding IcsA into S. flexneri wzzSF showed that multicopy icsA could suppress the virulence defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation) caused by the wzzSF mutation suggesting that the VL-type Oag chains were masking IcsA and limiting the amount available to initiate ABM. 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Lipopolysaccharide ; O antigen; wzz; Virulence; IcsA; Shigella £exneri
1. Introduction Shigella £exneri bacteria cause bacillary dysentery in humans. A key aspect of pathogenesis is the ability of S. £exneri bacteria to use actin-based motility (ABM) to move inside cells, and to spread to adjacent uninfected intestinal cells [1]. ABM is initiated by the outer membrane protein IcsA (also called VirG) [2,3] and the cell lipopolysaccharide (LPS) molecules also impact on this process. The 116 kDa IcsA protein is predominantly located at one of the cell poles [4^6]. Sequences located within the amino-terminal K-domain (herein termed IcsAP, 85^95 kDa) have been shown to target synthesis and export of IcsA to the old cell pole [7^9]. Intact LPS molecules, also referred to as smooth LPS, have three regions: lipid A, core sugars, and O antigen (Oag) polysaccharide chains. Mutations in the large number of genes involved in LPS core sugars region and Oag biosynthesis can a¡ect virulence, and the cell surface expression and localisation of IcsA [10^18]. We are partic-
ularly interested in the mutations that a¡ect the LPS Oag chain length distribution. The LPS of many S. £exneri 2a strains such as 2457T have LPS Oag chains whose length are non-randomly distributed such that they have two modal lengths. The modal length of 11^17 repeat units (RU) (termed S-type) is determined by the chromosomally located wzzSF gene [19], and that of s 90 RU (termed VLtype) is determined by the pHS-2 plasmid located wzzpHS2 gene [10,20]. Mutation in either gene results in loss of LPS with the respective Oag modal chain length. A S. £exneri 2457T wzzSF mutant (RMA696) was unable to form plaques on HeLa cells, had reduced virulence in a Sereny assay, reduced levels of IcsA on its cell surface, and markedly reduced ability to form F-actin comet tails [18]. In this paper we investigate the relationship between Oag chain length regulation and IcsA function, and provide evidence that the wzzSF virulence defect is caused by a limiting availability of IcsA.
2. Materials and methods * Corresponding author : Tel. : +61 (8) 8303 4151; Fax : +61 (8) 8303 7532. E-mail address : [email protected] (R. Morona).
2.1. Growth media and growth conditions Strains were grown in Luria Bertani (LB) broth and
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00217-9
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agar as previously described [18]. Unless otherwise stated, S. £exneri bacteria were grown from a Congo red agar [18] positive colony in LB for 16 h, then diluted 1 in 50 into fresh LB and grown for 2 h. Under these growth conditions, the IcsA protein was almost entirely in the intact 116 kDa form. Antibiotics used were: ampicillin (Ap), 50 Wg ml31 ; chloramphenicol (Cm), 25 Wg ml31 ; kanamycin (Km), 50 Wg ml31 ; and tetracycline (Tc), 10 Wg ml31 . Unless stated otherwise, strains were grown at 37‡C.
2.5. IcsA antibody
2.2. Bacterial strains and plasmids
Whole cell lysates were prepared by resuspending bacteria (approximately 2U108 cells) in 0.05 ml sample bu¡er [25] and heating for 5 min at 100‡C. Analysis of proteins by SDS^PAGE and Western immunoblotting was performed as recently described [24]. After transfer to nitrocellulose membrane, and incubation with rabbit anti-IcsA, bound antibody was detected using a goat anti-rabbit, horseradish peroxidase conjugate, and enhanced chemiluminescence (Roche). Polyclonal rabbit anti-IcsA serum was used at 1:250. Molecular mass markers (prestained, Bio-Rad cat. no. 161-0305) were: phosphorylase B (116 kDa), bovine serum albumin (80 kDa), ovalbumin (51.8 kDa), carbonic anhydrase (34.7 kDa), soybean trypsin inhibitor (30 kDa), lysozyme (22 kDa).
The bacterial strains and plasmids used are described in Table 1. The icsA gene was inactivated in 2457T wzzSF : :KmR by allelic exchange mutagenesis using plasmid pRMA2039 (Van Den Bosch, L. and Morona, R., submitted for publication) which is based on the suicide vector pCACTUS [19]. This resulted in deletion of the entire icsA gene (vicsA) and its replacement with a tetracycline resistance cassette (TcR ) [22]. 2.3. DNA methods DNA manipulations and electroporation into S. £exneri were performed as recently described [23,24]. DH5K was used for all cloning. 2.4. Construction of pIcsA Plasmid pIcsA was constructed as follows. A 3.5 kb EcoRI^SalI fragment containing icsA was subcloned from pD10 into similarly digested pBC-KS(+), resulting in plasmid pRMA915. The 3.5 kb EcoRI^SalI icsA fragment was then subcloned from pRMA915 into EcoRI^ SalI-digested pBR322. Transformation into DH5K and screening of Ap-resistant, Tc-sensitive colonies resulted in the isolation of pIcsA.
A rabbit anti-IcsA antibody was prepared as described elsewhere ([18]; Morona, R., Daniels, C. and Van Den Bosch, L., submitted for publication). 2.6. Preparation of IcsA samples, and sodium dodecyl sulphate^polyacrylamide gel electrophoresis (SDS^PAGE) and Western immunoblotting
2.7. Sereny reaction and plaque assay The Sereny assay to assess ability to cause keratoconjunctivitis was performed using guinea pigs as directed by the Animal Ethics Committee of the University of Adelaide. The severity of the reaction was scored according to the Sereny reaction ratings [26,27]. Plaque assays were performed using HeLa cells (laboratory stock, Institute of Medical and Veterinary Science, IMVS) as described by Oaks et al. [28].
Table 1 Bacterial strains and plasmids Strain/plasmid Escherichia coli K12 DH5K S. £exneri 2457T RMA696 RMA2095 RMA2042 RMA723 RMA2103 Plasmids pD10 pBC-KS(+) pRMA915 pBR322 pIcsA pRMA2039 a
Relevant characteristicsa
Reference/source
cloning host
Gibco-BRL
S. £exneri 2a wild-type 2457T wzzSF : :KmR 2457T wzzSF : :KmR (pIcsA) 2457T wzzSF : :KmR vicsA: :TcR 2457T rmlD: :KmR 2457T rmlD: :KmR (pIcsA)
[18] [18] this study this study [18] this study
source of virG/icsA gene CmR icsA gene cloned in pBC-KS(+); CmR ApR , TcR icsA gene cloned in pBR322 pCACTUS with vicsA : :TcR ; CmR
[3] Stratagene this study [21] this study Van Den Bosch, L. and Morona, R., submitted for publication
R, resistance.
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2.8. Immuno£uorescence detection of IcsA on LB grown bacteria The method used was recently described ([18,29], Morona, R., Daniels, C. and Van Den Bosch, L., submitted for publication). In brief, formalin-¢xed bacterial cells (0.1 ml) were centrifuged onto round glass coverslips in a 24well £at-bottomed tissue culture tray. After washing in phosphate-bu¡ered saline (PBS), primary antibody (antiIcsA, 1:100) in PBS with 10% (w/v) foetal calf serum (FCS) was added, incubated at 37‡C for 60 min, washed with PBS, then incubated with £uorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (1:80) (Silenus) at 37‡C for 30 min. The coverslips were washed in PBS, air dried, mounted on glass microscope slides with Mowiol 4-88 (Calbiochem) containing 20 Wg ml31 p-phenylenediamine (Sigma), and sealed with acrylic nail polish. Bacteria were photographed with Kodak TMAX400 ¢lm using an Olympus B2 microscope equipped with phase contrast and epi£uorescence illumination, standard FITC ¢lters, and a 100U achromatic oil immersion lens. 2.9. Invasion of HeLa cells, detection of F-actin, and immuno£uorescence staining Infection of HeLa cells and immuno£uorescence staining were performed as recently described ([18] ; Morona, R., Daniels, C. and Van Den Bosch, L., submitted for publication). In brief, S. £exneri strains were grown in LB and resuspended at approximately 109 bacteria ml31 in D-PBS (PBS with 0.1% (w/v) CaCl2 , 0.1% (w/v) MgCl2 ). 100 Wl of bacterial suspension was then centrifuged onto HeLa cells grown to semicon£uence on glass coverslips. After 60 min incubation at 37‡C in humidi¢ed CO2 incubator (5% CO2 ), the infected cells were washed ¢ve times with D-PBS and incubated with 0.5 ml minimal Eagle’s medium (MEM) (Gibco) containing 50 Wg ml31 of gentamicin (Gibco) for a further 1.5 h at 37‡C in a CO2 incubator. To detect F-actin comet tails, infected cells were ¢xed for 10 min in 3.7% (w/v) paraformaldehyde in PBS, and then permeabilised with 0.2% Triton X-100 in PBS for 1 min. To detect IcsA on intracellular bacteria, infected cells were ¢xed for 5 min at room temperature with 80% (v/v) acetone, incubated with PBS for 1 min, then permeabilised with 0.1% Triton X-100. After blocking in 1% foetal bovine serum (FBS), the infected cells were incubated at 37‡C for 30 min with either polyclonal anti-Shigella LPS (Denka Seiken Co, Japan) or rabbit anti-IcsA antibody (1:100). After washing in PBS, coverslips were incubated with either FITC-conjugated goat anti-rabbit or Texas red-conjugated goat anti-rabbit (Amersham, UK) secondary antibodies (1:100), as required. F-actin was visualised by staining with FITC-phalloidin (0.1 Wg ml31 , Sigma), and propidium iodide (PI) (10 Wg ml31 , Sigma) was used to counterstain bacteria and cellular nuclei, and was included with the secondary anti-
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body incubation, as required. The coverslips were mounted as described above, and were examined with a Bio-Rad MRC-600 confocal laser scanning microscope using a 100U oil immersion, objective lens. FITC and Texas red/PI images were false colour merged using Confocal Assistant 4.02. Each image shown is from a single plane.
3. Results 3.1. E¡ect of pIcsA on IcsA production in S. £exneri Since S. £exneri wzzSF : :KmR [RMA696] had decreased levels of IcsA detectable on its cell surface [18], this suggested to us that the VL-type Oag chains produced by the wzzSF mutant may be masking IcsA and thereby decreasing its ability to act in ABM. We investigated if the virulence-related defects due to the wzzSF mutation could be suppressed by using multicopy icsA to increase the amount of IcsA present on the cell surface. In S. £exneri, icsA is located on the large virulence plasmid. To increase the IcsA dosage, plasmid pIcsA was electroporated into 2457T wzzSF : :KmR [RMA696] resulting in 2457T wzzSF : :KmR (pIcsA) [RMA2095]. Western immunoblotting with anti-IcsA antibody was
Fig. 1. IcsA production by S. £exneri RMA696 derived strains. The S. £exneri strains indicated were grown to early logarithmic phase in LB, whole cell lysates were prepared, electrophoresed on an SDS 12% polyacrylamide gel, and after transfer to nitrocellulose proteins were detected with a rabbit anti-IcsA antibody. Lane 1, 2457T ; lane 2, 2457T wzz : : KmR [RMA696]; lane 3, 2457T wzz: :KmR vicsA : :TcR [RMA2042] ; lane 4, 2457T wzz : :KmR (pIcsA) [RMA2095]. The positions of the fulllength 116 kDa IcsA protein, and the minor, contaminating 85 kDa IcsAP (secreted form of IcsA), are indicated on the left-hand side of the ¢gure. Lanes contain approximately 2U108 bacterial cells.
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then used to compare the level of IcsA produced by 2457T and 2457T wzzSF : :KmR with that produced by 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 1). The whole cell lysate of 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 1, lane 5) had a higher level of IcsA (1.5^2U) than that of 2457T and 2457T wzzSF : :KmR (Fig. 1, lanes 1 and 2), and as expected 2457T wzzSF : :KmR vicsA: :TcR [RMA2042] did not produce IcsA (Fig. 1, lane 3). 3.2. E¡ect of multicopy icsA on wzzSF virulence phenotypes The Sereny assay, plaque formation on HeLa cell monolayers, and F-actin comet tail formation were used to compare virulence, and intracellular and intercellular spreading ability of 2457T wzzSF : :KmR [RMA696] with its pIcsA harbouring derivative. 2457T wzzSF : :KmR carrying pIcsA [RMA2095] gave a strongly positive Sereny reaction, identical to that of 2457T (Table 2), unlike 2457T wzzSF : :KmR [RMA696] which gave a weak Sereny reaction (Table 2) as previously reported [18]. Furthermore, strain 2457T wzzSF : :KmR (pIcsA) [RMA2095] was able to form plaques on HeLa cell monolayers that were indistinguishable from those produced by 2457T (Fig. 2a,c) whereas 2457T wzzSF : : KmR [RMA696] (Fig. 2b) was unable to plaque on HeLa cell monolayers as previously reported [18]. The ability of pIcsA to restore the HeLa cell monolayer plaquing ability to 2457T wzzSF : :KmR [RMA696] was further investigated by staining with FITC-phalloidin to detect F-actin comet tail formation. 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 3d) formed F-actin comet tails similar to those produced by 2457T (Fig. 3a). While 2457T wzzSF : :KmR [RMA696] (Fig. 3c) only rarely formed F-actin comet tails, as previously reported [18], it did exhibit some ABM as 2457T wzzSF : :KmR [RMA696] bacteria were dispersed throughout the cytoplasm of infected cells in contrast to 2457T wzzSF : :KmR vicsA: :TcR [RMA2042] bacteria which clustered near the cell nucleus (Fig. 3b). These results show that multicopy icsA was able to suppress the virulence and ABM defect due to the
Fig. 2. Plaque formation by S. £exneri strains. This ¢gure shows plaques formed on HeLa cell monolayers by S. £exneri strains. Strains are: a: 2457T ; b: 2457T wzz: :KmR [RMA696]; c: 2457T wzz : :KmR (pIcsA) [RMA2095]. Arrowhead indicates a plaque. The experiment was performed on three occasions and representative results are shown.
wzzSF : :KmR mutation, suggesting that masking of IcsA by the VL-type Oag chains could be overcome by increased levels of cell surface IcsA. 3.3. Detection of IcsA on the cell surface of LB grown S. £exneri strains We investigated if the e¡ect of multicopy icsA on wzzSF : :KmR mutant phenotype correlated with increased detection of cell surface IcsA. Production of IcsA on the cell surface of 2457T, 2457T wzzSF : :KmR [RMA696], and 2457T wzzSF : :KmR vicsA: :Tc (pIcsA) [RMA2095] either
Table 2 Ability of S. £exneri strains to plaque on HeLa cells, and virulence Strain
Chromosome
Relevant plasmidsa
LPS typeb
Plaque assayc
Sereny reactiond
2457T RMA696 RMA2095 RMA2042 RMA723 RMA2103
wild-type wzzSF : :KmR wzzSF : :KmR wzzSF : :KmR vicsA: :TcR rmlD: :KmR rmlD: :KmR
VP VP VP+pIcsA VP VP VP+pIcsA
S+VL VL VL VL R R
+ 3 + 3 3 3
+++ + +++ nd 3 nd
a
VP, virulence plasmid. pIcsA, pBR322 encoded icsA. S, strain produces LPS with O antigen chains having a modal length of 11^17 RU ; VL, strain produces LPS with O antigen chains having a modal length of s 90 RU; R, strain produces LPS with lacking O antigen chains. c + means that the strain was able to form plaques on HeLa cell monolayers ; 3 means that the strain was unable to form plaques on HeLa cell monolayers. d 3, no Sereny reaction or mild irritation ; +, mild keratoconjunctivitis or late development and/or clearing; +++, fully developed keratoconjunctivitis ; nd, not done. b
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was not seen when intracellular 2457T wzzSF : :KmR [RMA696] and 2457T wzzSF : :KmR (pIcsA) [RMA2095] bacteria were compared (Fig. 5b,c), and did not correlate with the ability of these strains to form F-actin comet tails (Fig. 4). One possible explanation is that to detect IcsA on intracellular bacteria we used di¡erent ¢xation conditions to that used for LB grown bacteria (see Section 2). Under these conditions, it may be that VL-type Oag chains produced in the wzzSF mutant background had a greater e¡ect on detection of IcsA on the intracellular S. £exneri bacteria than on LB grown bacteria. 3.4. E¡ect of multicopy icsA on the rough LPS mutant phenotype While unlikely, it was possible that pIcsA was having some e¡ect on 2457T wzzSF : :KmR [RMA696] which was unrelated to Oag but which suppressed its virulence-re-
Fig. 3. Detection of F-actin comet tails produced by S. £exneri growing within HeLa cells. HeLa cell monolayers were infected with S. £exneri strains and stained to detect F-actin comet tails. S. £exneri bacteria were detected by indirect immuno£uorescence staining with rabbit antiOag antibody and a Texas red-conjugated 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 ; b: RMA2042 [2457T wzzSF : :KmR vicsA: :TcR ]; c: 2457T wzzSF : :KmR [RMA696]; d: 2457T wzzSF : :KmR (pIcsA) [RMA2095]. In each panel, the arrowheads indicate the location of an F-actin comet tail. The experiment was performed on three occasions and representative results are shown.
grown in LB or inside HeLa cells were compared by immuno£uorescence staining using an anti-IcsA antibody. IcsA was detected at the cell pole of LB grown 2457T, 2457T wzzSF : :KmR [RMA696], and 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 4a,b,c), however the intensity of IcsA labelling on 2457T and 2457T wzzSF : :KmR (pIcsA) [RMA2095] was greater than that seen for 2457T wzzSF : :KmR [RMA696]. These results indicate that pIcsA increased the amount of IcsA detected on the cell surface of 2457T wzzSF : :KmR [RMA696] which correlated with the e¡ect on F-actin comet tail and plaque formation, and hence ABM. When resident within HeLa cells, IcsA was detected at the cell poles of 2457T, 2457T wzzSF : :KmR [RMA696] and 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 5), however the intensity of IcsA staining was weaker for the latter two strains. The di¡erence in IcsA labelling observed between LB grown 2457T wzzSF : :KmR [RMA696] and 2457T wzzSF : :KmR (pIcsA) [RMA2095] (Fig. 4b,c)
Fig. 4. Detection of IcsA on the cell surface of S. £exneri strains with LPS mutations. S. £exneri strains were grown to early logarithmic phase in LB, ¢xed, and immuno£uorescently 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 ; b: 2457T wzzSF : :KmR [RMA696] ; c: 2457T wzzSF : :KmR (pIcsA) [RMA2095]. For each panel, the phase contrast image is on the lefthand side and corresponding immuno£uorescent image is on the righthand side of the S. £exneri strains indicated in the ¢gure. Strain 2457T wzzSF : :KmR vicsA : :TcR [RMA2042] showed no detectable staining and is not shown. The experiment was performed on three occasions and representative results are shown.
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Fig. 5. Detection of IcsA on the cell surface of intracellular S. £exneri bacteria. HeLa cell monolayers were infected with S. £exneri strains and stained to detect IcsA. Production of IcsA by S. £exneri bacteria was detected by indirect immuno£uorescence staining with a rabbit anti-IcsA antibody and an FITC-conjugated secondary antibody (seen here as green), and S. £exneri bacteria were detected by counterstaining with propidium iodide (seen here as red). The strains in each panel are: a: 2457T; b: 2457T wzzSF : :KmR [RMA696] ; c: 2457T wzzSF : :KmR (pIcsA) [RMA2095]. The large, oval-shaped, red-staining bodies are the HeLa cell nuclei. Within each panel, an enlargement of typical bacteria is shown. Strain 2457T wzzSF : :KmR vicsA: :TcR [RMA2042] showed no detectable IcsA staining and is not shown. The experiment was performed on three occasions and representative results are shown.
lated defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation). To assess this possibility, we investigated if pIcsA had an e¡ect on the virulence-related defects associated with the rough LPS (Oagde¢cient) phenotype due to the rmlD mutation [18]. We transformed pIcsA into the rough LPS strain 2457T rmlD : :KmR [RMA723], and then investigated the ability of the resulting strain to plaque on HeLa cells and form F-actin comet tails. Both 2457T rmlD: :KmR [RMA723] and 2457T rmlD : :KmR (pIcsA) [RMA2103] were unable to plaque on HeLa cells (Table 1). HeLa cells infected with 2457T, 2457T rmlD: :KmR [RMA723], and 2457T rmlD: : KmR (pIcsA) [RMA2103] were stained with FITC-phalloidin. Compared to 2457T, both rmlD mutants exhibited a low frequency of F-actin comet tails that had an altered morphology (data not shown). Hence, unlike its e¡ect on the wzzSF phenotype, multicopy icsA was unable to suppress the ABM defects caused by the rmlD : :KmR mutation.
4. Discussion We have previously reported that 2457T wzzSF : :KmR [RMA696] had reduced cell surface expression of IcsA and decreased virulence [18]. The VL-type LPS Oag chains produced in the wzzSF mutant may mask IcsA thereby interfering with its function and/or limiting the amount of IcsA protein available to initiate ABM. This prompted us to investigate the impact of multicopy icsA on the wzzSF phenotype by introducing pIcsA into 2457T wzzSF : :KmR [RMA696]. We found that multicopy icsA was able to suppress the virulence defect due to the wzzSF mutation as determined
using the Sereny reaction, HeLa cell monolayer plaquing ability, and F-actin comet tail formation; all were restored to wild-type (Table 1, Figs. 2 and 3). When grown in LB, 2457T wzzSF : :KmR (pIcsA) had increased levels of cellular IcsA (Fig. 1) and higher levels of IcsA on its cell surface (Fig. 4) as compared to 2457T wzzSF : :KmR [RMA696]. However, this di¡erence could not be shown by immuno£uorescence detection of IcsA on intracellular bacteria (Fig. 5). The ability of multicopy icsA to suppress the wzz mutant phenotype suggests that the VL-type Oag chains exclusively present in 2457T wzzSF : :KmR [RMA696] are in some way limiting the amount of IcsA available to nucleate ABM. Increasing the amount of IcsA on the cell surface was able to compensate for the e¡ect of these VL-type Oag chains. This interpretation is consistent with the recent results of Magdalena and Goldberg [30] who determined that a threshold level of approximately 4000 IcsA molecules per bacterium was needed for e⁄cient ABM. In support of our results, inactivation of wzzpHS2 in 2457T wzzSF : :KmR [RMA696] and in SA100rol (a wzzSF mutant, [10]), resulted in LPS Oag chains that are of random length and restoration of virulence and plaque forming ability ([10] ; Morona, R., Daniels, C. and Van Den Bosch, L., submitted for publication). Hence, our data suggest that VL-type Oag chains when present as the sole LPS Oag modal chain length can prevent and/or sterically hinder IcsA function in ABM.
Acknowledgements This work was supported by a Project Grant from the National Health and Medical Research Council of Aus-
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tralia to R.M. Elizabeth Anderson is thanked for technical support. Judy Morona is thanked for reading the manuscript.
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