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Infection and Immunity, May 2006, p. 2587-2595, Vol. 74, No. 5
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.5.2587-2595.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

OspE2 of Shigella sonnei Is Required for the Maintenance of Cell Architecture of Bacterium-Infected Cells

Masashi Miura,1,2 Jun Terajima,1 Hidemasa Izumiya,1 Jiro Mitobe,1 Teruya Komano,2 and Haruo Watanabe1*

Department of Bacteriology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan,1 Department of Biology, Tokyo Metropolitan University, Hachioji, Tokyo, Japan2

Received 31 August 2005/ Returned for modification 24 October 2005/ Accepted 7 February 2006


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ABSTRACT
 
The OspE2 product of Shigella spp., the expression of which is regulated by the mxiE gene, is secreted through a type III secretion system into host cells. We investigated the function of OspE2 of Shigella sonnei by using cultured epithelial cells. Cells invaded by an ospE2 deletion mutant altered their morphology into the rounding shape, which was not due to cell death, whereas cells invaded by the wild-type strain kept their cell shape intact. The ospE2 mutation did not affect initial cell entry and multiplication in cells, but the mutant formed smaller-than-normal plaques on cell monolayers, indicating a deficiency in cell-to-cell spread by the bacteria. An mxiE deletion mutant also showed changes in cell morphology and deficiency in bacterial spread to adjacent cells. In cells invaded by the ospE2 mutant, disturbance of actin stress fibers was prominent at 3 h after invasion. Analysis of OspE2 localization indicated that the OspE2 protein accumulated on focal contact-like structures in the infected host cells. These results suggest that colocalization of the OspE2 protein in the focal contacts of infected cells may function to maintain an intact cell morphology. The morphological change induced by invasion of the ospE2 mutant may affect secondary bacterial transmission.


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INTRODUCTION
 
Shigella spp., which are able to invade epithelial cells, are the causative agents of bacillary dysentery in humans (21). A large, 230-kb virulence plasmid in virulent Shigella is involved in invasion (43, 56). After internalization, Shigella strains lyse the phagocytic vacuole and enter the cell cytosol, where they multiply and move to invade adjacent cells (10, 43, 44). An outer membrane protein, VirG (IcsA), encoded on the virulence plasmid is involved in intra- and intercellular movements, which it accomplishes by polymerizing an actin tail that propels the bacterium through the cytoplasm and into adjacent cells (3, 24, 25). In this process, the moving bacterium forms protrusions from the cytoplasmic membrane as it enters adjacent cells (3, 17, 36, 40). During reinfection, bacteria reside within two membranes that belong to the protrusion and to the newly infected cell and subsequently lyse these two membranes by using IcsB (1), which is also involved in the escape of intracellular Shigella cells from autophagy (33).

The large virulence plasmid of Shigella contains the mxi-spa and ipa gene cluster, termed the "31-kb entry region" (for a review, see reference 39). mxi-spa comprises about 20 genes mostly coding for a type III secretion (TTS) apparatus, which is a bacterial multiprotein complex that forms a needle-like structure (45, 48). The ipa operon encodes effector proteins such as IpaB, IpaC, and IpaD. These proteins are translocated into the intracellular milieu of the host cells through the apparatus and act on various components of cytoskeletal architecture or eukaryotic signal transduction pathways to modulate host cell function (7, 51, 59).

Secretion of effector proteins from Shigella cells is induced by their contact with host cells (29) or by addition of the dye Congo red to the growth medium (2, 38). Inactivation of ipaB or ipaD gives rise to constitutive secretion of effector proteins (38). Under these conditions, 14 other TTS apparatus-secreted proteins were identified in Shigella flexneri, including the Osp proteins (for "outer Shigella") OspB, OspC1 to OspC4, OspD1 to OspD3, OspE1 and OspE2, OspF, and OspG (6). Genes for these proteins are mainly located outside the 31-kb entry region in the virulence plasmid (6, 9) and are regulated by MxiE, an activator protein of the AraC family, which is encoded within the mxi-spa operon and requires IpgC as a coactivator (18, 26). Most of the promoters for osp genes contain a 17-bp motif, the MxiE box, which is essential for activation of these promoters by MxiE (27). The mxiE mutant is invasive to cultured cells but completely avirulent in in vivo animal models (18). Hence, the functions of genes regulated by MxiE are thought to be essential for later steps in infection. The virA, ipaH1.4, ipaH2.5, ipaH4.5, ipaH7.8, and ipaH9.8 genes have also been found to be regulated by MxiE (23). The functions of some of the MxiE-regulated genes have been revealed. For example, OspG negatively controls innate immune responses by targeting ubiquitin-conjugating enzymes (20). IpaH9.8 is transported to the nucleus of mammalian cells and binds to a splicing factor, U2AF (35), to modulate immune responses of the host cells (34, 50). IpaH7.8 promotes the escape of Shigella cells from vacuoles of macrophages (10). VirA is capable of interacting with tubulin to promote microtubule destabilization and bacterial internalization (53, 58). The secreted antiactivator OspD1 and coantiactivator Spa15 are involved in the control of transcription by MxiE-IpgC (37). However, the functions of the other MxiE-regulated genes remain to be elucidated.

In this study, we focused on the function of the ospE2 gene, which is one of the MxiE-regulated genes. We have demonstrated that both an ospE2 deletion mutant and an mxiE deletion mutant caused morphological change in bacterially infected cells. OspE2 protein was secreted in a TTS system (TTSS)-dependent manner and was located in the focal contact-like structures of infected host cells. We discuss a role of OspE2 in maintenance of host cell morphology.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, cell culture, and media. The bacterial strains and plasmids used in this study are listed in Table 1. HeLa cells and HEp2 cells were grown in minimal essential medium (MEM) with 10% fetal calf serum (FCS) in a 5% CO2 incubator. Bacterial cells were grown in LB broth or on LB plates containing 1.5% Bacto agar. Antibiotic concentrations were as follows: chloramphenicol (Cm), 25 µg/ml; ampicillin (Amp), 100 µg/ml; kanamycin (Km), 50 µg/ml.


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  		TABLE 1. Bacterial strains and plasmids used in this study

Cloning of the ospE2 gene. For cloning of the Shigella sonnei ospE1 and ospE2 genes, shotgun cloning of pHW1273 DNA was performed. DNA resulting from a partial digestion of pHW1273 with Sau3AI was fractionated by agarose gel electrophoresis, and fragments (5 to 10 kb) were ligated into the BamHI site of pHW745. The ligation mixture was used for electroporation. The transformants were colony hybridized with an ospE2 gene probe of S. flexneri under conditions of low stringency, and positive colonies were sequenced. The clone pF3, carrying the ospE2 gene, was selected. The clone of ospE1 was isolated in a similar way.

Construction of deletion mutants. The ospE2 deletion mutant in S. sonnei was constructed by the PCR-based gene disruption method (8, 30). MS390 harboring the {lambda} red helper plasmid pKD46 was used for construction of the mutant. The plasmid pKD13 was used as a template for PCR using primers appropriate to amplify a Kmr marker flanked by ospE2-specific sequences. After replacement of the target gene by the Kmr marker, flipase (FLP)-mediated recombination was performed to remove the resistance marker. The deletion mutation was confirmed by Southern analysis and DNA sequencing. The construction of S. sonnei mxiE and spa40 mutant strains was performed in a similar way.

Construction of plasmids encoding OspE2. For subcloning of the ospE2 gene, a 744-bp DNA fragment resulting from partial digestion of pF3 with Sau3AI was ligated with BamHI-digested pUC19 in the orientation opposite the lac promoter, yielding pMM102. Plasmids pMM500, pMM700, and pMM800 were constructed as follows: the ospE2 gene was amplified by PCR with TaKaRa Ex Taq DNA polymerase (Takara Bio Inc.) and pMM102 DNA as a template, using a forward primer containing an EcoRI site and a reverse primer containing a PstI site for construction of pMM500, a forward primer containing an NdeI site and a reverse primer containing an EcoRI site for construction of pMM700, and a forward primer containing an EcoRI site and a reverse primer containing a SalI site for construction of pMM800. The PCR products were digested with the appropriate restriction enzymes and cloned into pTH18cr, pET-28a, or pIRES-hrGFP-2a, resulting in pMM500, pMM700, or pMM800, respectively. The plasmids pMM800 and pIRES-hrGFP-2a were inserted by Cmr gene exchange (Exchanger System; Stratagene) using the loxP site on the vector and Cre recombinase in vitro, resulting in pMM800Cm and pIRES-Cm, respectively. For construction of pMM819K, the ospE2 gene with a hemagglutinin (HA) tag (ospE2-HA) was amplified by PCR with pMM800 DNA as a template, using a forward primer containing a PstI site and a reverse primer containing an EcoRI site, and was cloned into pK19. The structure of each construct was confirmed by DNA sequencing and restriction enzyme analysis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Electrophoresis of proteins on polyacrylamide gels in the presence of SDS was performed as described by Laemmli (22) with slight modifications. Immunoblotting analyses were carried out with convalescent-phase serum of an S. flexneri-infected monkey (54) and rabbit anti-VirG antiserum, which was obtained by immunization of rabbits with a synthesized peptide (unpublished data). For the preparation of rabbit anti-OspE2 antiserum, rabbits were immunized with His-tagged OspE2, which was purified from Escherichia coli BL21 harboring pMM700. The monospecific rabbit polyclonal anti-IpaC antibody used for immunoblotting was described previously (46). Anti-rabbit immunoglobulin G conjugated to horseradish peroxidase was obtained from Jackson Laboratories. Immunoblot detection was carried out with the ECL kit (Amersham).

Plaque assay. The intra- and intercellular dissemination of bacteria was evaluated with the plaque formation assay (32). HEp2 cells were seeded onto six-well plates and grown for 2 days to confluent monolayers. Bacteria were inoculated into the cells at a multiplicity of infection of 30, and the cells were incubated for 2 h at 37°C. After three washes with phosphate-buffered saline (PBS), MEM containing 5% FCS and 100 µg/ml of gentamicin was added, and the cells were grown for 2 or 4 days at 37°C in the presence of CO2. After Giemsa staining, plaque diameters were measured.

Invasion assay. Bacterial invasion of HeLa or HEp2 cells was tested with the gentamicin protection assay (49). HeLa or HEp2 cells were grown on six-well tissue culture plates to semiconfluent monolayers in MEM with 10% FCS in a 5% CO2 incubator. Cells were infected with bacteria grown to log phase in LB at 37°C at a multiplicity of infection of 100, centrifuged at 900 x g for 5 min at 37°C, and further incubated at 37°C for 20 min. Cells were washed three times with PBS, and gentamicin was added to the medium at a final concentration of 200 µg/ml, followed by further incubation at 37°C for 20 min (19). For the invasion assay, the cells were lysed in 1 ml of PBS containing 0.1% Triton X-100 per well, and the number of bacteria invaded was counted after plating onto LB agar. For intracellular growth, cells were further incubated for 20 or 290 min and then lysed in 1 ml of PBS containing 0.1% Triton X-100 per well. After incubation at room temperature for 10 min, the lysates containing bacterial cells were diluted and plated onto LB agar plates in triplicate. Colonies grown on LB plates were counted. Assays were repeated three times. The results of the assays were expressed as the percentage of the number of surviving bacteria relative to that of bacteria initially added to the cell culture.

For estimating the frequency of host cell rounding, cells having more than five bacteria after Giemsa staining were grouped by the following criteria: nonspreading host cells were defined as rounded cells, and spreading cells with normal morphology were defined as nonrounded cells. The percentage of bacterium-invaded cells adopting rounded morphology was determined by counting at least 300 infected cells from three randomly selected light microscopy fields in each of three independent wells (>80 cells/field).

Analysis of secreted proteins. For assays of Ipa and OspE2 production and secretion, bacterial cells were grown at 37°C in LB to log phase. After centrifugation at 14,000 x g for 2 min, the bacterial pellet was resuspended in 3 ml of PBS. Congo red was added to a bacterial suspension at a final concentration of 0.002%, and the suspension was further incubated at 37°C for 10 min. The sample was centrifuged at 14,000 x g for 2 min to give the bacterial pellet and supernatant fraction. The bacterial pellet was solubilized in SDS-PAGE sample buffer, boiled for 5 min, and subjected to SDS-PAGE. The supernatant was passed through a 0.45-µm-pore-size membrane filter, and proteins were precipitated with 6% trichloroacetic acid. The precipitates were washed with acetone and solubilized in SDS sample buffer, boiled for 5 min, and subjected to SDS-PAGE.

Immunofluorescence microscopy. HeLa or HEp2 cells grown on coverslips were infected with the indicated bacterial strains for 3 or 3.5 h on a six-well plate. The cells were washed, fixed with 3.7% paraformaldehyde in PBS for 20 min, and treated for 5 min with 50 mM NH4Cl in PBS. Cells were rendered permeable to the antibody reagent with 0.1% Triton X-100 in PBS overnight at 4°C and treated with 1% bovine serum albumin in PBS for 1 h. To visualize F-actin, focal adhesion kinase (FAK), talin, or HA-tagged OspE2, permeabilized cells were labeled with 1/100-diluted Alexa 488-labeled phalloidin (Molecular Probes Inc.), 1/400-diluted rabbit anti-FAK antibody (Santa Cruz Biotechnology), 1/400-diluted rabbit anti-talin antibody (Santa Cruz Biotechnology), or 1/400-diluted mouse monoclonal antibody to HA (Sigma), respectively. For staining of bacterial cells, 1/100-diluted monospecific rabbit polyclonal anti-IpaC antibody (45) or 1/100-diluted rabbit anti-form I serum (Denka Seiken) was used. Cells were washed three times in PBS containing 0.1% Triton X-100 and treated with a 1/400 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody (Molecular Probes Inc.), Texas Red-conjugated sheep anti-rabbit immunoglobulin antibody (Molecular Probes Inc.), or Texas Red-conjugated sheep anti-mouse immunoglobulin antibody (Molecular Probes Inc.). Cells were washed three times in PBS containing 0.1% Triton X-100, washed twice in PBS, and then mounted. Samples were analyzed by fluorescence microscopy using an Olympus BX51 microscope equipped with a 40x lens objective. Images were captured with an Olympus E-20 digital camera and processed with Adobe Photoshop 5.5.

Assays for cell death. For extraction of cellular DNA and gel electrophoresis, HeLa cells (3.6 x 106) infected with the indicated strains or treated with 30 ng/ml tumor necrosis factor alpha (TNF-{alpha}) (Wako) plus 60 µg/ml cycloheximide (Sigma-Aldrich) for 3 h or 5 h were washed with PBS and lysed for 20 h at 37°C in lysis buffer (1% SDS, 100 mM NaCl, 10 mM Tris, 1 mM EDTA [pH 8.0]) supplemented with proteinase K (1 mg/ml). After three phenol and chloroform/isoamylalcohol (24:1) extractions, the DNA was precipitated with ethanol, dried, resolved in 10 mM Tris-1 mM EDTA (pH 8.0) supplemented with 1 µg/ml RNase A, separated on a 1.5% agarose gel, and visualized by ethidium bromide staining. Pictures were taken under UV light.

For terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), HeLa cells infected with the indicated strains or treated with 30 ng/ml TNF-{alpha} plus 60 µg/ml cycloheximide for 3 h or 5 h were washed with PBS, fixed, and permeabilized, and biotinylated nucleotide was incorporated enzymatically (TACS 2 TdT-Fluor In Situ Apoptosis Detection kit; Trevigen) into fragmented chromosomal DNAs, which were detected with a fluorescein conjugate of streptavidin. Nuclear fluorescence in HeLa cells was evaluated by fluorescence microscopy with the use of an Olympus BX51 microscope as described above.

Cytotoxicity was analyzed with the CytoTox 96 kit (Promega). The degree of cytotoxicity was evaluated by quantifying the amount of cytoplasmic lactate dehydrogenase (LDH) was released into supernatants from HeLa cells infected with the indicated strains or treated with 30 ng/ml TNF-{alpha} plus 60 µg/ml cycloheximide for 1, 3, or 5 h in 96-well plates. The percentage of cytotoxicity was determined by the following formula: [(released LDH activity – basal LDH activity in the supernatant)/(total LDH activity in the cells and supernatant – basal LDH activity in the supernatant)] x 100.

Nucleotide sequence accession numbers. The nucleotide sequences of ospE1 and ospE2 of S. sonnei were deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB185406 and AB185407, respectively.


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RESULTS
 
Construction and characterization of an ospE2 deletion mutant of S. sonnei. While the ospE gene family, designated ospE1 and ospE2, is known to be found in the S. flexneri large virulence plasmid (6), it is not known how many copies of ospE genes there are in the S. sonnei large virulence plasmid pHW1273. We first cloned ospE2 of S. sonnei as described in Materials and Methods. Sequence analysis demonstrated that the ospE2 gene of S. sonnei was 267 bp in length (88 amino acids), which was the same length as that of S. flexneri (6) but with 6 nucleotide substitutions, resulting in 5 amino acid substitutions in the C-terminal region of the product (A75V, D78V, Y85H, D87N, and F88I [S. flexneri versus S. sonnei]). The promoter region of the S. sonnei ospE2 gene contains the MxiE box, which is essential for the activation by MxiE, suggesting that ospE2 of S. sonnei is regulated by MxiE, as is the case in S. flexneri (27). ospE2 of S. sonnei encodes a small protein of 10 kDa, which exhibits considerable similarities in amino acid sequence to ECs1567 and ECs1821 of enterohemorrhagic E. coli O157 (14), although their functions have not been revealed so far. Southern blotting experiments using an S. sonnei ospE2 gene probe indicated that only one band was seen in pHW1273 under high stringency conditions, while two bands (ospE1 and ospE2) were detected under low stringency conditions (data not shown). However, sequencing of the ospE1 gene of pHW1273 indicated that ospE1 is inactive in S. sonnei, since deduced translation of the ospE1 gene stopped at the 14th codon, yielding a putative protein of 13 amino acids. Thus, only the ospE2 gene was expected to be active in S. sonnei.

To characterize the role of ospE2 in infection, we constructed a deletion mutant of the ospE2 gene on the large virulence plasmid of S. sonnei MS390 by using the PCR-based gene disruption method (30). The ospE2 mutant, designated MS1303, was used for cell assays. HEp2 cells of semiconfluent monolayers were infected with S. sonnei MS390 (wild type), MS1303 ({Delta}ospE2), and MS1303/pMM500 ({Delta}ospE2/ospE2+) for 3 h. HEp2 cells infected with MS1303 ({Delta}ospE2) displayed a prominent morphological change, a rounded shape with a retracted plasma membrane in which no clear protrusions were observed (Fig. 1A). In contrast, host cells infected with MS390 (wild type) exhibited a spreading cell shape, which was similar to that of mock infection.


Figure 1
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  		FIG. 1. Morphological changes in HEp2 cells after infection with S. sonnei {Delta}ospE2 and {Delta}mxiE mutants. (A) HEp2 cells were infected with S. sonnei HW506 (mock), MS390 (wild type), MS1303 ({Delta}ospE2), MS1303/pMM500 ({Delta}ospE2/ospE2+), MS1307 ({Delta}mxiE), or MS1307/pMM500 ({Delta}mxiE/ospE2+). After 3 h, cells were fixed with methanol and stained with Giemsa reagent. Scale bar, 10 µm. (B) Frequency of host cell rounding (panel A) quantified. The frequency of rounding of mock-infected cells was less than 3% at both 1 and 3 h after infection. Error bars indicate standard deviations.

Since the rounded shape was also observed less frequently in host cells infected with wild-type bacteria, the frequency of host cell rounding was estimated (Fig. 1B). The number of rounded host cells infected with MS1303 ({Delta}ospE2) was greater than that with MS390 (wild type) at both 1 and 3 h after infection. The frequency of host cell rounding of MS1303 ({Delta}ospE2) increased to nearly 80% by 3 h, whereas that of MS390 (wild type) remained almost half that of MS1303 ({Delta}ospE2) throughout the infection. Complementation of MS1303 ({Delta}ospE2) by pMM500 (ospE2+) restored the frequency of morphological changes to the wild-type level. These results indicate that the ospE2 gene is involved in the maintenance of host cell morphology.

We next examined the relationship between the morphological change caused by the ospE2 mutation and virulence properties in the host cells (Table 2). The invasion efficiency of MS1303 ({Delta}ospE2) into HEp2 cells in semiconfluent monolayers was almost the same as that of MS390 (wild type) or MS1303/pMM500 ({Delta}ospE2/ospE2+). MS1303 ({Delta}ospE2) produced and secreted Ipa proteins at levels similar to that of MS390 (wild type), suggesting that MS1303 ({Delta}ospE2) retains the same ability to invade host cells as the wild type. Intracellular multiplication of MS1303 ({Delta}ospE2) within 5.5 h after infection was also similar to that of MS390 (wild type). No distinct changes were noted in the VirG (IcsA) levels of S. sonnei MS390 (wild type) and MS1303 ({Delta}ospE2) (Table 2). These results indicate that the host cell rounding caused by the ospE2 mutant and the ospE2 mutation itself do not affect cell invasiveness and multiplication.


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  		TABLE 2. Characterization of ospE2 mutants related to virulence in S. sonnei

In gentamicin protection assays, the increase in the frequency of host cell rounding caused by the ospE2 mutation during the course of infection suggests that the morphological changes are caused by intracellular, not extracellular, bacteria. However, a factor(s) in culture supernatants may be involved in the cell rounding. Thus, we performed experiments using bacterial culture supernatants of both the wild type and the ospE2 mutant. However, no morphological changes in HEp2 cells were observed, even after 5.5 h of incubation with bacterial culture supernatants (data not shown). Therefore, we conclude that bacterial invasion is required for the changes into the rounded cell morphology.

Effects of the S. sonnei mxiE mutation on cell morphology similar to those of the ospE2 mutation. Recently, Kane et al. (18) reported that transcription of ospE2 in S. flexneri was activated by MxiE after infection and that the mxiE mutant showed a reduced plaque size phenotype. This led us to speculate that an mxiE mutant might have effects on infected cells similar to those of the ospE2 mutant. In fact, HEp2 cells infected with MS1307 ({Delta}mxiE) displayed a rounded phenotype similar to that caused by the ospE2 mutant (Fig. 1A and B). Furthermore, introduction of the ospE2+ gene with the lac promoter (pMM500) in trans into MS1307 ({Delta}mxiE) completely rescued the morphology (Fig. 1A and B). The host cell rounding phenotype of the ospE2 and mxiE mutants was also observed after infection of HeLa cells. These results suggest that ospE2 and mxiE mutations affect host cell morphology similarly after infection and that the host rounding phenotype is dependent on OspE2 but independent of other effector proteins that are regulated by MxiE.

Host cell rounding without cell death. We next investigated the involvement of cell death in the morphological changes after infection with the ospE2 mutant for 3 or 5 h. Fragmentation of cellular DNA into oligonucleosomes can occur in both types of cell death, apoptosis and necrosis (28, 35). Therefore, we tried to detect the chromosomal laddering in extracted DNA of MS1303 ({Delta}ospE2)-infected cells by gel electrophoresis and ethidium bromide staining (Fig. 2A). An apoptotic effect on HeLa cells is known to be achieved by treatment with TNF-{alpha} in combination with cycloheximide (31). The chromosomal DNA of HeLa cells treated with TNF-{alpha} plus cycloheximide was fragmented extensively into oligonucleosomes, clearly visible as a characteristic DNA ladder at 3 h after treatment (Fig. 2A, lane 2), whereas neither HeLa cells infected with MS1303 ({Delta}ospE2) and MS390 (wild type) nor HeLa cells untreated with TNF-{alpha} plus cycloheximide displayed fragmented DNAs (Fig. 2A, lanes 1, 3, and 4). Similar DNA patterns in each sample were also observed at 5 h after treatment or infection (data not shown).


Figure 2
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  		FIG. 2. Host cell rounding caused by the S. sonnei {Delta}ospE2 mutant without cell death. (A) Absence of fragmentation of cellular DNA into oligonucleosomes after infection with the S. sonnei {Delta}ospE2 mutant. DNA was extracted from 3.6 x 106 HeLa cells, separated on a 1.5% agarose gel, and visualized by ethidium bromide staining. Lane 1, no treatment (negative control); lane 2, treatment with 30 ng/ml TNF-{alpha} and 60 µg/ml cycloheximide (CHX) for 3 h to induce apoptosis (positive control); lane 3, infection with MS390 (wild type) for 3 h; lane 4, infection with MS1303 ({Delta}ospE2) for 3 h. (B) Host cell rounding caused by the S. sonnei {Delta}ospE2 mutant without DNA fragmentation. HeLa cells were infected with S. sonnei MS1303 ({Delta}ospE2) for 3 h or treated with 30 ng/ml TNF-{alpha} plus 60 µg/ml CHX to induce apoptosis for 3 h (positive control). Untreated HeLa cells were used as a negative control. Cells were labeled by the TUNEL method, and reacted cells were detected by fluorescence microscopy. Phase-contrast and fluorescence pictures were taken of the same section. (C) Infection with the S. sonnei {Delta}ospE2 mutant did not kill host cells. HeLa cells were infected with S. sonnei MS390 (wild type) or MS1303 ({Delta}ospE2) or treated with 30 ng/ml TNF-{alpha} plus 60 µg/ml CHX to induce apoptosis. Supernatants were taken at the indicated time points, and LDH released into the supernatants from host cells was assayed to measure cytotoxicity. The percentage of cytotoxicity was calculated as described in Materials and Methods. Each result represents the mean ± standard deviation from triplicate measurements.

Next, a TUNEL method was employed to check whether individual rounded cells infected with the ospE2 mutant were free from fragmentation of host DNA (Fig. 2B). HeLa cells exhibiting morphological changes were stained by the TUNEL method at 3 h after treatment with TNF-{alpha} plus cycloheximide, whereas neither rounded HeLa cells infected with MS1303 ({Delta}ospE2) nor untreated cells showed any response (Fig. 2B). Similar staining patterns in each sample were also observed at 5 h after treatment or infection (data not shown).

To quantify the viability of rounded host cells infected with the ospE2 mutant, we measured the activity of LDH released from the cells due to loss of host cell membrane integrity (Fig. 2C). HeLa cells treated with the drugs released about 40% and 70% LDH after 3 h and 5 h of incubation, respectively, whereas HeLa cells infected with MS390 (wild type) or MS1303 ({Delta}ospE2) for 5 h did not significantly release LDH. All of the above results suggest that rounded host cells infected with the ospE2 mutant are viable at least within 5 h of infection and show no signs of apoptosis or necrosis.

Accumulation of OspE2 protein at focal contact-like structures in infected cells. To investigate how OspE2 affects host cell shape, we examined the localization of the OspE2-HA protein within infected host cells. S. sonnei OspE2 and OspE2-HA protein secretion via TTSS in vitro was confirmed by immunoblotting analysis; OspE2-HA was secreted in MS390 but not in the spa40 mutant MS1330 (data not shown). HEp2 cells infected with S. sonnei strain MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) or MS1303/pK19 ({Delta}ospE2/– [i.e., the plasmid does not carry the ospE2 gene]) for 3 h were processed for immunofluorescence microscopy using an anti-HA monoclonal antibody to reveal OspE2-HA (green signals) and anti-S. sonnei form I antibody to reveal bacterial cells (red signals), respectively (Fig. 3A). While HEp2 cells infected with MS1303/pK19 ({Delta}ospE2/–) changed to a rounded morphology, host cells infected with MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) restored the spreading morphology seen in uninfected HEp2 cells (the infected cells are indicated by red arrows in panels c and f in Fig. 3A), indicating that HA labeling of OspE2 did not affect its activity. HEp2 cells infected with MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) showed numerous elongated green dots throughout the infected cell, while those without infection did not (white arrows in panel a in Fig. 3A). Some elongated dots were located at the cell periphery, where the bacteria were not colocalized (panel b). The elongated dot structures seemed to bear some resemblance to focal contacts (also known as focal adhesions or adhesion plaques) in their distribution and morphology. Such structures were not observed in mock-infected cells (data not shown). These results suggest that the OspE2 protein is secreted into the host cell cytosol after infection and accumulates at the focal contact-like structures.


Figure 3
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  		FIG. 3. OspE2-HA accumulates at focal contact-like structures in infected cells. (A) HEp2 cells were infected with S. sonnei MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) or MS1303/pK19 ({Delta}ospE2/–). After 3 h, the cells were fixed and immunostained. OspE2-HA was stained with anti-HA monoclonal antibody and secondary fluorescein isothiocyanate-tagged antibody. Bacterial cells were stained with anti-S. sonnei form I antibody and secondary Texas Red-tagged antibody. Single-color and phase-contrast images are shown for each strain. White arrows indicate the sites of accumulation of OspE2-HA signals. Red arrows in panels c and f indicate host cells with invaded bacteria. Scale bar, 10 µm. (B) HEp2 cells were infected with S. sonnei MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) or MS1303/pK19 ({Delta}ospE2/–) for 3 h and then subjected to immunofluorescence staining. F-actin was stained with Alexa 488-labeled phalloidin; OspE2-HA was stained with anti-HA monoclonal antibody and secondary Texas Red-tagged antibody. Single-color, merged, and phase-contrast images are shown. Actin stress fibers terminate at the site of OspE2-HA signals (white arrows in the inset of panel c). Red arrows in panels d and h indicate the infected cells. Scale bars represent 10 µm for panels d and h and 5 µm for the inset in panel c. (C) HEp2 cells infected with S. sonnei MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) for 3 h were stained with antibody against FAK (red) or talin (red) and OspE2-HA (green). Single-color, merged, and phase-contrast images are shown. White arrows indicate focal contact-like structures. Red arrows in panels d and h indicate infected cells. Scale bar, 10 µm.

To confirm that OspE2-HA accumulates at the focal contact-like structures of the host cells, we examined the spatial relationship between elongated dots of OspE2-HA and actin stress fibers, since actin stress fibers are known to terminate at the focal contacts (15). HEp2 cells infected with S. sonnei MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) or MS1303/pK19 ({Delta}ospE2/–) for 3 h were examined by immunofluorescence analysis using Alexa 488-conjugated phalloidin to visualize F-actin (green) and anti-HA antibody to visualize OspE2-HA (red) (Fig. 3B). The infected HEp2 cells are indicated by red arrows (panels d and h in Fig. 3B). Actin stress fibers were retained intact in the HEp2 cells infected with MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) but not in the cells infected with MS1303/pK19 ({Delta}ospE2/–) (panels a and e in Fig. 3B). HEp2 cells infected with MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) contained bacterial cells bearing actin comet tails (white arrowheads in panel a in Fig. 3B), which were not merged on localization of elongated dots of OspE2-HA (panel b in Fig. 3B). On the other hand, most of the visible actin stress fibers terminated at the sites where OspE2-HA accumulated (white arrows in the inset of panel c in Fig. 3B). These results suggest that the localization of elongated dots of OspE2-HA corresponds to that of focal contacts.

To ensure that the sites of OspE2-HA accumulation corresponded to the sites of protein complexes of focal contacts in the host cells, colocalization experiments by immunofluorescence microscopy using antibodies against FAK or talin, as indicators of focal contact proteins, were performed. HEp2 cells infected with MS1303/pMM819K ({Delta}ospE2/ospE2-HA+) for 3 h exhibited signals for FAK or talin (both red) and OspE2-HA (green) (Fig. 3C). The infected HEp2 cells are indicated by red arrows in panels d and h (Fig. 3C). The focal contacts were observed in the infected host cells (white arrows in panels b and f in Fig. 3C), while in uninfected cells, small punctuated dots which were known as focal complexes and thought to be precursors of mature focal contacts were faintly observed at the cell periphery. The signals of OspE2-HA (green signals indicated by white arrows in panels a and e in Fig. 3C) were colocalized with the sites of these focal contacts in the infected cells (yellow signals in panels c and g in Fig. 3C). These results strongly suggest that OspE2 proteins accumulate at focal contact structures in the host cells.

Small plaque formation of the ospE2 mutant. To test whether the ospE2 mutant retains the ability of intra- or intercellular spread, plaque assays using confluent monolayers of HEp2 cells were performed. After HEp2 cells were infected with MS390 (wild type), MS1303 ({Delta}ospE2), and MS1303/pMM500 ({Delta}ospE2/ospE2+), the sizes of plaques were measured (Table 2 and Fig. 4). MS1303 ({Delta}ospE2) produced smaller plaques than did MS390 (wild type). The diameter of the plaques developed by infection with MS1303 ({Delta}ospE2) was approximately 0.3 mm at 4 days after infection, while that of the plaques developed by MS390 (wild type) or MS1303/pMM500 ({Delta}ospE2/ospE2+) was over 0.8 mm. These results suggest that the ospE2 mutation affects the processes of secondary transmission of Shigella cells into neighboring host cells.


Figure 4
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  		FIG. 4. Formation of small plaques in confluent monolayers of HEp2 cells after infection with the S. sonnei {Delta}ospE2 mutant. Confluent monolayers of HEp2 cells were infected with S. sonnei MS390 (wild type) (A), MS1303 ({Delta}ospE2) (B), or MM1303/pMM500 ({Delta}ospE2/ospE2+) (C) and incubated for 2 h. After a wash, medium containing gentamicin was added and culturing was continued at 37°C. At 4 days after infection, monolayers were stained with Giemsa to observe plaque formation by bacterial spreading. (D) Values in the bar graph represent mean ± standard deviation diameter for a total of 150 plaques from three independent wells.


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DISCUSSION
 
The ospE2 gene has been identified as one of the MxiE-regulated genes in S. flexneri (18, 23). Southern blotting analysis of the virulence plasmid indicated that S. sonnei also has two ospE genes, as is the case for S. flexneri. Sequence analysis of the ospE1 gene of S. sonnei demonstrated that ospE1 is a pseudogene. Hence, use of S. sonnei seemed to be more advantageous for functional analysis of ospE2 gene. In this study, the role of the S. sonnei ospE2 gene in infection was investigated. We found that the ospE2 mutant causes morphological changes in infected cells and smaller plaque formation in cultured epithelial cells, without effects on initial cell entry and multiplication in cells. Cultured supernatant of the ospE2 mutant did not affect cell morphology. Only bacterium-invaded cells had the rounded phenotype, which was not due to the cell death. We also demonstrated that host cells infected with the mxiE mutant changed their morphology into the rounded shape, as host cells infected with the ospE2 mutant did. This is another phenotypic similarity between ospE2 and mxiE mutants, because the mxiE mutant is known to be deficient in plaque formation (18). For this reason, the morphological change of the host cells into rounding may correlate with the deficiency in plaque forming, namely, in the deficiency of secondary transmission of bacteria to adjacent cells. One explanation may be that the reduction of tight cell-to-cell contact caused by cell rounding affects the efficient spread of bacteria to adjacent cells.

Wild-type S. sonnei and the ospE2+ complemented mutant did not cause the rounding phenotype of invaded cells; only the ospE2 mutant did. This indicated that wild-type Shigella does not usually induce morphological changes even after invasion of the cells but that in the ospE2 mutant, another factor(s) besides OspE2 seems to be involved in the induction of cellular morphological change. Since infection of HeLa cells with an E. coli K-12 strain carrying the S. sonnei 230-kb large virulence plasmid without the ospE2 gene was sufficient to cause the cell rounding phenotype (data not shown), the large plasmid should contain the factors necessary for cell rounding. In the cells invaded by the S. sonnei ospE2 mutant, the disturbance of protein complexes with FAK, talin, and actin stress fibers in the focal contact sites was prominent at 3 h after invasion (Fig. 3B and data not shown). This morphological change seems to be different from the membrane ruffling induced by the Ipa proteins of Shigella (51) or SopE of Salmonella (12) at the early stage of bacterial cell entry. Infection of Yersinia pseudotuberculosis is known to cause rounding of HeLa cells. YopH (4, 16), one of the effector proteins of Yersinia, which targets focal contacts of host cells and interacts with p130Cas, is involved in cell rounding. Yersinia YopE, a GTPase-activating protein, induces disruption of the host cell-actin microfilament structure, allowing the bacterium to resist phagocytosis (5, 42). S. sonnei may have such factors for causing cell rounding, which should be elucidated in the near future.

A TTSS effector protein is mainly defined as the protein which is delivered into the cytoplasm of the host cell through the TTS apparatus. Accumulation of OspE2 to focal contact-like structures in infected host cells suggests that OspE2 acts as an effector protein. Because of the relatively low molecular size (approximately 10 kDa) of OspE2, it may be possible for OspE2 to diffuse throughout the cytoplasm of the cell immediately after bacterial invasion and to accumulate at the site of a putative target(s) near or at the focal contacts in order to fulfill its function. Therefore, the phenotype of the ospE2 mutant to cause morphological change in host cells is more than coincidence, since some host cell proteins at the focal contacts are important determinants for the maintenance of cell morphology (52). This leads us to speculate that OspE2 protein colocalized in the focal contacts of infected cells may stimulate the function of focal contact proteins in order to keep the morphology of cells intact or to inhibit the above-described factor(s) which disturbs the focal contact-like structure. It is known that Salmonella SptP, which functions as a GTPase-activating protein of Rho-family GTPases, restores the change in cellular architecture (11). We did not find any similarity in nucleotide sequences between ospE2 and other TTS effector protein-associated genes involved in the maintenance of cellular architecture.

We have described here the finding that OspE2 prevents the alteration of host cell shape at the postinvasion stage of infection. This remarkable adaptation to the modulation of host cell response may be advantageous for intracellular Shigella cells to disseminate into adjacent cells.


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ACKNOWLEDGMENTS
 
We thank E. Arakawa, K. Hirose, and S. Iyoda for helpful advice and discussions. The cloning vectors pTH18cr and pK19 were gifts from the cloning vector collection of the National Institute of Genetics (Japan).

This work was supported by a grant from the Ministry of Education, Science and Technology.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1337. Fax: 81-3-5285-1193. E-mail: haruwata{at}nih.go.jp. Back

Editor: J. B. Bliska


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Infection and Immunity, May 2006, p. 2587-2595, Vol. 74, No. 5
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