Department of Molecular Biosciences, University of Adelaide, Adelaide, South Australia, Australia, 5005
Correspondence
Renato Morona
renato.morona{at}adelaide.edu.au
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ABSTRACT |
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Present address: Program in Cell and Lung Biology, Hospital for Sick Children Research Institute, University of Toronto, McMaster Building Room 3026C, 555 University Ave, Toronto, Ontario M5G 1X8, Canada.
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INTRODUCTION |
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In this study, we investigated the role of LPS Oag modal chain length distribution in S. flexneri virulence by assessing its impact on a number of in vivo and in vitro correlates of virulence. In addition, we investigated IcsA localization on the cell surface of intracellular S. flexneri bacteria. Our data suggest that Oag modal chain length distribution has been optimized for S. flexneri virulence, and we hypothesize that Oag chains can mask IcsA and affect its function in ABM.
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METHODS |
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Bacterial strains and plasmids.
The bacterial strains and plasmids used in this study are described in Table 1.
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DNA methods and sequencing.
General DNA manipulation, plasmid DNA isolation, dye terminator DNA sequencing, PCR, DNA transformation and electroporation were performed as described previously (Baker et al., 1999; Daniels & Morona, 1999
). The location of mini-Tn5-CmR in pRMCD72 was determined by digestion with SmaI, and cloning three SmaI fragments (
2·8,
1·95 and
0·4 kb) into the SmaI site of pBluescript-SK (Stratagene). DNA sequencing using dye-labelled M13 forward and reverse primers was then undertaken.
Protein analysis.
Analysis of IcsA proteins by SDS-PAGE and Western immunoblotting was performed as described previously (Morona et. al., 1995; Daniels & Morona, 1999
). After transfer to a nitrocellulose membrane and incubation with a rabbit anti-IcsA antibody, bound antibody was detected using horseradish peroxidase-conjugated goat antibodies specific for rabbit immunoglobulins and enhanced chemiluminescence (Roche). Polyclonal rabbit anti-IcsA serum was used at 1 : 1000. The molecular mass markers used in this study were pre-stained (New England Biolabs; catalogue no. P7708S).
LPS analysis.
LPS was prepared by proteinase K treatment of whole-cell lysates, electrophoresed on SDS 15 or 20 % polyacrylamide gels and detected by silver-staining as described previously (Morona et al., 1991; Daniels & Morona, 1999
).
Construction of pRMCD76.
Plasmid pRMCD77, a derivative of pET17b (Novagen) but with its BglII site removed by end-filling with dNTPs and Klenow, was the base vector used for cloning and expression of wzz genes (Daniels & Morona, 1999). pRMCD76, harbouring the wzzO139 gene (also called otnB) from Vibrio cholerae O139, was constructed as follows. A 1519 bp EcoRIBglII fragment (corresponding to nucleotides 1065412173 of the otn gene region, GenBank accession no. X90547) was isolated from pBSotn and ligated to EcoRI- and BamHI-digested pRMCD77. After transformation into Escherichia coli DH5
, plasmids from ampicillin-resistant colonies were screened by restriction enzyme digestion, resulting in the isolation of pRMCD76. DNA sequencing using T7 promoter and T7 terminator primers confirmed that the clone had the wzzO139 ORF.
Cloning, overexpression and purification of IcsA.
Plasmid pRMA920 has the icsA coding region under IPTG-inducible control and was used to overexpress IcsA. Oligonucleotides #2156 (5'-GCCG AAT TCA ACG GAA TCT TTT CAG GGG-3', EcoRI site) and #2170 (5'-GCG GAT CCC CAT GTG ATT TGC CTC G-3', BamHI site) were used in a PCR with pD10; the resulting product was purified (Qiagen), digested with EcoRI and BamHI, and ligated to likewise digested pTTQ181. After transformation into JM105, screening of ampicillin-resistant colonies resulted in pRMA920. Plasmid pRMA920 was electroporated into RMA763, resulting in RMA943. IcsA was overexpressed and purified as follows. A 1 l culture of RMA943 in LB with ampicillin was incubated with aeration at 37 °C; at an OD600 value of 1·0, IPTG was added to a final concentration of 0·4 mM, and the culture was incubated for a further 3 h. The cells were collected by centrifugation at 7000 r.p.m. (10 min, 4 °C, Beckman JA-10 rotor), resuspended in 40 ml of 20 % (w/v) sucrose/30 mM Tris/HCl (pH 8·1) and incubated on ice for 30 min. The cells were centrifuged as above, and the cell pellet was frozen in an ethanol/dry-ice bath for 30 min, thawed and dispersed vigorously in 3 ml of 3 mM EDTA (pH 7·3). Cells were lysed with a Branson ultra-sonifier (50 % cycle, intermittent), and by several cycles of freezethawing. Unlysed cells and inclusion bodies were removed by slow-speed centrifugation (7000 r.p.m., 10 min, 4 °C) and the supernatant containing whole-cell membranes and cytoplasm was centrifuged at 35 000 r.p.m. using a 80 Ti rotor for 90 min at 20 °C in a Beckman L8-80 ultracentrifuge. The pellet was resuspended in 1 ml of water, an equal volume of Triton solution [4 % (v/v) Triton X-100, 2 mM MgCl2, 50 mM Tris, pH 7·5] was added and the sample was vortex-mixed intermittently for 30 min at room temperature. The sample was then centrifuged at 35 000 r.p.m. for 90 min at 20 °C. The resulting pellet was mixed with 2 % (v/v) Triton X-100 in 10 mM Tris (pH 8·0)/8 M urea, and centrifuged at 18 000 r.p.m. for 20 min (Beckman centrifuge). A Rosenbusch-type extraction was then performed (Rosenbusch, 1974). The pellet was resuspended in SDS-PAGE sample buffer (Lugtenberg et al., 1975
) without bromophenol blue and incubated at 37 °C for 40 min. The sample was centrifuged in a Beckman centrifuge at 15 000 r.p.m. for 60 min, at room temperature. The resulting supernatant was then applied to an S-200 Sephacryl column equilibrated with 10 mM Tris (pH 7·4)/2 % (w/v) SDS/5 mM EDTA/0·02 % (w/v) sodium azide, and the column was eluted with the same buffer. Fractions were collected and analysed on an SDS 12 % polyacrylamide gel. Under these conditions, IcsA was eluted close to the void volume. Fractions containing only the
120 kDa IcsA protein were pooled; the final preparation was free of any other protein, as determined by Coomassie blue staining.
Preparation of rabbit anti-IcsA serum.
IcsA, isolated by column chromatography, was further purified by two rounds of SDS-PAGE. The gel slice containing IcsA was homogenized in Freund's complete adjuvant and used to immunize a rabbit by injection at multiple sites. The resulting antiserum was adsorbed four times with RMA763 bacterial cells. After the addition of an equal volume of sterile glycerol (100 %), antibodies were stored at -20 °C. Western blotting against S. flexneri 2457T whole cells, trichloracetic acid (8 %, w/v, final) precipitated culture supernatant and whole-cell membranes showed that the antiserum only detected the amino terminal -domain of IcsA (data not shown).
Indirect immunofluorescence detection of IcsA on LB-grown bacteria.
The technique is a minor modification of a method by Klauser et al. (1990). Round glass coverslips were boiled for 1 min in 0·1 M HCl before storage in 95 % ethanol prior to air-drying for use. Poly-L-lysine (Sigma, catalogue no. P8920) [100 µl, 0·01 % (w/v) in H2O] was pipetted onto coverslips placed in a 24-well, flat-bottomed tissue culture tray (Costar) and incubated at room temperature for 5 min before being washed with PBS. Cells were formalin-killed by treating 10 ml of pelleted culture (5000 r.p.m., 10 min, IEC Centra 4X centrifuge) with 1 ml of 2 % (w/v) paraformaldehyde (Sigma) for 20 min. The cells were washed twice in PBS and resuspended in a final volume of 1 ml PBS. One-hundred microlitres of cells were added to each well, then centrifuged at 800 r.p.m. for 10 min (Hereaus Labofuge 400R). The wells were aspirated dry and washed three times with PBS. The primary antibody (1 : 100) in PBS with 10 % (w/v) fetal calf serum (FCS; IMVS, Adelaide) was added and incubated at 37 °C for 60 min. After three washes with PBS, the secondary antibody (1 : 80) (FITC-conjugated goat anti-rabbit antibody; Silenus, catalogue no. RDAF) in PBS with 10 % (w/v) FCS was added and left at 37 °C for 30 min, followed by a further two PBS washes. The coverslips were gently aspirated dry and mounted upside down on a clean glass microscope slide with 3 µl of Mowiol 4-88 (Calbiochem, catalogue no. 475904) containing 20 µg p-phenylenediamine ml-1 (Sigma, catalogue no. P6001) and sealed with acrylic nail polish. Bacteria were photographed with Kodak TMAX400 film using an Olympus B2 microscope equipped with phase-contrast and epifluorescence illumination, standard FITC filters and a 100xachromatic oil immersion lens.
Virulence assays.
The ability of S. flexneri strains to form plaques on HeLa cell (laboratory stock) and BHK-26 cell (IMVS) monolayers was performed exactly as described by Oaks et al. (1985). The Sereny keratoconjunctivitis test was performed using guinea pigs, as directed by the Animal Ethics Committee of the University of Adelaide, and the severity of the reaction was scored according to Sereny reaction ratings (Sereny, 1957
; Formal et al., 1958
).
Invasion of HeLa cells and immunofluorescence (IF) staining.
HeLa cells were infected as follows. An 18 h-old culture of an S. flexneri strain was diluted 1 in 50 in LB (10 ml), grown for 1·5 h, centrifuged at 5000 r.p.m. for 10 min (IEC Centra 4X centrifuge), washed and resuspended at approximately 109 bacteria ml-1 in D-PBS [PBS with 0·1 % (w/v) CaCl2/0·1 % (w/v) MgCl2]. HeLa cells, seeded on glass coverslips in a 24-well tray and grown to give semi-confluence, were washed twice with D-PBS, overlaid with the 100 µl bacterial suspension and centrifuged at 2000 r.p.m. for 10 min (Hereaus Labofuge 400R, 8177 rotor). Bacterial entry was allowed for 60 min incubation at 37 °C in a humidified CO2 incubator (5 % CO2). Infected cells were washed with D-PBS three times and incubated with 0·5 ml MEM (Gibco) containing 50 µg gentamicin ml-1 (Gibco) for a further 2 h at 37 °C in a CO2 incubator, to allow intracellular spread. For IF staining, two different fixation methods were used. To detect F-actin comet tails, infected cells were fixed for 10 min in 3·7 % (w/v) paraformaldehyde in PBS and then permeabilized 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 room temperature with 80 % acetone, incubated with PBS for 1 min, then permeabilized with 0·1 % (v/v) Triton X-100 in PBS for 1 min. After blocking in 1 % (w/v) FCS in PBS for 10 min, the infected cells were incubated at 37 °C for 30 min with either polyclonal anti-Shigella LPS (Denka Seiken, Japan, catalogue no. 310 111) (1 in 100) or rabbit anti-IcsA antiserum (1 in 100) as described above. After washing three times in PBS, coverslips were incubated for 15 min at 37 °C with either FITC-conjugated goat anti-rabbit or Texas-red-conjugated goat anti-rabbit (Amersham, catalogue no. N2034) secondary antibodies (1 in 100) as required. F-actin was visualized by staining with FITCphalloidin (0·1 µg ml-1; Sigma, catalogue no. P5282), and propidium iodide (10 µg ml-1; Sigma, catalogue no. P4170) was used to counter-stain bacteria and cellular nuclei; FITCphalloidin and propidium iodide were included with the secondary antibody incubation as required. The coverslips were mounted as described above. Preparations were examined with a Bio-Rad MRC-600 confocal laser-scanning microscope, using an Olympus IMT-2 inverted microscope and a 100xoil immersion, apochromatic, 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.
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RESULTS |
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Plasmids harbouring wzzO139 (pRMCD76), wzzSF (pRMCD78), wzzST (pRMCD80) and the control vector (pRMCD77) were electroporated into RMA696 (2457T wzzSF : : KmR) and RMA982 (2457T wzzSF : : KmR wzzpHS2 : : mini-Tn5-CmR). The LPS molecules produced by the resulting strains were analysed by SDS-PAGE and silver-staining (Fig. 3), and the Oag modal chain lengths observed are summarized in Table 2
. As expected, pRMCD78 (wzzSF) complemented the wzzSF : : KmR mutation in RMA982 and RMA696, resulting in LPS with either one (S-type) or two (S- and VL-type) Oag modal chain lengths, respectively, with the latter profile being almost identical to that of the parental strain 2457T (Fig. 3a
, lanes 1 and 5). Strains RMA982 and RMA696 harbouring pRMCD80 (wzzST) produced LPS with either one (L-type) or two (L- and VL-type) Oag modal lengths, respectively, as expected (Daniels & Morona, 1999
). RMA982 and RMA696 harbouring pRMCD76 (wzzO139) produced LPS with either one [very short (VS) type, 27 RUs] or two (VS- and VL-type) Oag modal lengths, respectively (Fig. 3
, Table 2
). One notable difference between the strains was that RMA3108 [RMA696(pRMCD76), wzzO139] (Fig. 3a
, lane 3) and RMA3231 [RMA982(pRMCD76), wzzO139] (Fig. 3b
, lane 3) had reduced levels of LPS with a single Oag RU (semi-rough LPS), and of LPS with VL-type Oag chains (RMA3108 only) compared with wzzSF- and wzzST-complemented strains.
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Effect of LPS Oag modal chain length variation on Sereny reaction
To confirm the impact of LPS Oag modal length on S. flexneri virulence, we compared the abilities of strains 2457T, RMA696 (2457T wzzSF : : KmR), RMA3107 [RMA696(pRMCD77), control], RMA3108 [RMA696(pRMCD76), wzzO139], RMA3109 [RMA696(pRMCD78), wzzSF], RMA3110 [RMA696(pRMCD80), wzzST] and RMA982 (2457T wzzSF : : KmR wzzpHS2 : : mini-Tn5-CmR) to provoke keratoconjunctivitis in the guinea pig eye (Sereny assay) (Table 2). Strains RMA696, RMA3107 and RMA3110 gave a reduced Sereny positive reaction, correlating with their inability to from plaques on HeLa cell monolayers, while strains RMA3108 and RMA3109 were fully virulent. RMA982 was also highly virulent in this assay and was identical to 2457T. Overall, the severity of the Sereny reaction correlated with the strains' plaque-forming ability. These results highlight the importance of LPS Oag modal length distribution for S. flexneri virulence.
Infection of HeLa cells and F-actin comet tail formation
We investigated if the LPS Oag modal length distribution had any affect on S. flexneri intracellular spread within HeLa cells, by comparing the abilities of RMA696 (2457T wzzSF : : KmR), RMA982 (2457T wzzSF : : KmR wzzpHS2 : : mini-Tn5-CmR) and derivatives carrying either the control vector or plasmids encoding different wzz genes to form F-actin comet tails. The latter is indicative of the ability of IcsA to function in ABM. In particular, we wanted to determine if the reduced virulence and ability to plaque on HeLa cell monolayers shown by RMA3110 [RMA696(pRMCD80), wzzST] correlated with a defect in IcsA function.
HeLa cells were infected with 2457T, RMA696 (2457T wzzSF : : KmR), RMA3107 [RMA696(pRMCD77), control], RMA3108 [RMA696(pRMCD76), wzzO139], RMA3109 [RMA696(pRMCD78), wzzSF], RMA3110 [RMA696(pRMCD80), wzzST], RMA982 (2457T wzzSF : : KmR wzzpHS2 : : mini-Tn5-CmR), RMA3232 [RMA982(pRMCD77), control], RMA3231 [RMA982(pRMCD76), wzzO139], RMA3233 [RMA982(pRMCD78), wzzSF], RMA3234 [RMA982(pRMCD80), wzzST] and RMA2041 (2457T icsA : : TcR), which was included as a negative control. All strains tested were able to invade HeLa cells with similar efficiency (data not shown). 2457T formed F-actin comet tails (approx. 20 % of bacteria) (Fig. 4a
) and as expected, RMA2041 (Fig. 4b
) did not form F-actin comet tails nor did it show any intracellular spread. RMA696, RMA3107 and RMA3110 (Fig. 4c, e, h
) rarely formed F-actin comet tails (<1 % of bacteria) but did exhibit some intracellular spread when compared to the IcsA-null mutant RMA2041, which formed a micro-colony clustered near the nucleus (Fig. 4b
). RMA3108, RMA3109, RMA982 and all RMA982 derivatives were able to form F-actin comet tails (approx. 20 % of bacteria) (Fig. 4f, g, i, j, k, l
). These data suggest that the inability of RMA3110 (like RMA696) to form plaques was a consequence of the IcsA in this strain being unable to efficiently nucleate actin polymerization and thus initiate ABM.
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DISCUSSION |
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We used plasmids carrying wzzO139, wzzSF and wzzST to complement the wzz mutations in RMA696 and RMA982. The ability of the V. cholerae wzzO139 gene (carried on pRMCD76) to fully complement the S. flexneri wzzSF : : KmR mutation, resulting in a novel LPS with a VS-type Oag modal chain length, was surprising since the WzzO139 protein has a low level of sequence identity with WzzSF and functions in V. cholerae O139 capsule polysaccharide (CPS) biosynthesis (Bik et. al., 1995, 1996
; Morona et al., 2000
; Attridge et al., 2001
). Confirming this function, pRMCD76 was recently shown to complement a V. cholerae O139 otnB (wzzO139) mutant and restored production of O139 CPS (Attridge et al., 2001
). While the ability of wzzO139 to complement the S. flexneri wzzSF : : KmR defect and regulate LPS Oag chain length clearly shows that WzzO139 can act as a Wzz protein, its role in V. cholerae O139 LPS/capsule biosynthesis may be altered such that it is involved in O139 CPS biosynthesis rather than LPS Oag synthesis.
Like RMA982, the strains that were unable to produce the VL-type Oag chains formed plaques and F-actin comet tails regardless of their LPS Oag modal length status (Table 2). Different results were obtained when the complemented strains were able to produce the VL-type Oag chains. In this situation, VS- and S-type Oag chains but not L-type Oag chains were permissive for IcsA function in ABM. Our data, combined with those of Sandlin et al. (1996)
, who found that semi-rough LPS with a single Oag RU does not support plaque formation, show that IcsA function in ABM is compatible with and requires LPS with at least two Oag RUs but less than 90 RUs, a relatively broad range. It should be noted that we did not test Oag modal lengths between 35 and 90 Oag RUs. In the presence of LPS with VL-type Oag chains this requirement becomes more stringent, and the LPS must have Oag chains with two to less than 18 RUs.
While unknown, different types of LPS molecules may be randomly distributed on the S. flexneri cell surface. Alternatively, rough LPS molecules (LPS lacking Oag chains) and LPS molecules with particular Oag chain lengths may be preferentially localized to certain regions of the cell (e.g. polar versus lateral regions), and there might be a preferred association between LPS molecules of different size and particular outer-membrane proteins. Based on our observations, LPS with VL-type Oag chains in the absence of LPS with S-type Oag chains (as in RMA696 and RMA3107) place IcsA molecules in an environment where they cannot function, perhaps due to the VL-type Oag chains masking and/or interfering with the interaction of IcsA with host-cell proteins (see below). While the WzzST-determined L-type Oag chains on their own are permissive for IcsA function, unlike the VS- and S-type Oag chains, they cannot counteract the inhibitory effect of VL-type Oag chains. Hence, RMA3110 LPS, which has L-type and VL-type Oag chains, behaves like the RMA696 LPS, which has VL-type Oag chains, and affects IcsA function in ABM.
Overall, we found that detection of IcsA produced by various S. flexneri strains when examined by indirect IF microscopy did not correlate with the observed impact of LPS Oag chain length on the ability of the strains to form plaque and F-actin comet tails (Figs 6 and 7). Presumably, the epitopes recognized by IcsA antibodies are more accessible than the sequences that interact with host-cell proteins involved in initiating ABM. With the exceptions of RMA696 and RMA3107 (which have only VL-type Oag chains), detection of IcsA on LB-grown S. flexneri strains was unaffected by Oag modal chain length; this was also the case for S. flexneri strains producing LPS with VL-type Oag chains resident within HeLa cells. However, when S. flexneri strains unable to produce VL-type Oag chains were growing inside HeLa cells, we found that detection of IcsA was influenced by LPS Oag chain length. In this situation, a reduction in IcsA detection was observed which correlated with an increase in LPS Oag chain length. These results suggest that VL-type Oag chains can mask and interfere with the binding of IcsA antibodies. One possible explanation for the differences observed between LB-grown and intracellular bacteria is that different fixation conditions were used to prepare the samples for IF staining, and these may have affected the detection of IcsA epitopes which can be masked by the VL-type Oag chains.
Interestingly, strains RMA982, RMA3232 [RMA982(pRMCD77), control] and RMA3231 [RMA982(pRMCD76), wzzO139], when resident within HeLa cells, had IcsA at their cell poles and on lateral regions of their cell surfaces (Fig. 7), and these strains were also able to form plaques and F-actin comet tails. The cell surface localization of IcsA in these strains and their phenotype is inconsistent with the hypothesis that strict polar IcsA localization is needed for ABM within cells (Goldberg, 2001
; Suzuki & Sasakawa, 2001
).
Our data show a correlation between LPS Oag modal length distribution and the ability of S. flexneri strains to form plaques and F-actin comet tails, and the results are consistent with strains RMA696 (2457T wzzSF : : KmR), RMA3107 [RMA696(pRMCD77), control] and RMA3110 [RMA696(pRMCD80), wzzST] having an ABM defect. However, indirect IF detection of IcsA revealed no major difference between these three strains and the related ABM-competent strains RMA3108 [RMA696(pRMCD76), wzzO139] and RMA3109 [RMA696(pRMCD78), wzzSF]. To explain these observations, we hypothesize that plaque formation only occurs when a minimum number of IcsA proteins are simultaneously active to initiate F-actin comet tail formation. Below this threshold level, sustained ABM sufficient to form plaques is not achieved. This is consistent with our observations that RMA696, RMA3107 and RMA3110 can form F-actin comet tails, albeit very infrequently. Additionally, Magdalena & Goldberg (2002) have recently estimated that a threshold level of 4000 molecules of IcsA is needed to achieve efficient ABM. In the model to be described below, we suggest that Oag chains influence the availability of these IcsA molecules to act in ABM.
A model to explain our observations is shown in Fig. 8. In the wild-type strain 2457T, IcsA epitopes and ABM initiation domains are accessible to antibodies and host proteins due to the presence of LPS with S- and VL-type Oag chains. The presence of LPS with shorter Oag chains (e.g. VS- to S-type) in the outer membrane counteracts the effect of the VL-type Oag chains, thereby allowing IcsA to function in ABM, while LPS with VL-type Oag chains can still confer serum resistance (Hong & Payne, 1997
). In strain RMA696 (2457T wzzSF mutant), the LPS only has VL-type Oag chains. These mask and interfere with the IcsA domain(s) that interact(s) with host-cell protein(s) (e.g. N-WASP) (Goldberg, 2001
; Suzuki & Sasakawa, 2001
), and also slightly reduce antibody binding, presumably due to a decrease in epitope accessibility. In this situation, initiation of ABM is inefficient. In strain RMA982 (2457T wzzSF wzzpHS2 double mutant), relatively short Oag chains are present on the cell surface. In this situation, IcsA can interact with host proteins and hence, ABM can be efficiently initiated. In strain RMA3110 [RMA696(pRMCD80), wzzST], the VL-type Oag chains still interfere with the ability of IcsA to initiate ABM as the WzzST-determined L-type Oag chains are too long to compete with and counteract the effect of the VL-type Oag chains. The L-type Oag chains by themselves do not interfere with the ability of IcsA to initiate ABM (strain RMA3234). Indirect support for this model comes from our observation that detection of IcsA on intracellular S. flexneri bacteria unable to co-express VL-type Oag chains is affected by Oag modal chain length. We have also directly demonstrated, using in situ enzymic hydrolysis, that Oag chains can mask and sterically hinder detection of IcsA by indirect IF (R. Morona & L. Van Den Bosch, unpublished data). This model does not take into account any conformational differences between Oag chains with different modal length distributions or any interactions that may occur between the Oag chains.
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ACKNOWLEDGEMENTS |
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Received 21 November 2002;
revised 14 January 2003;
accepted 22 January 2003.
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