Involvement of RNA-binding protein Hfq in the osmotic-response regulation of invE gene expression in Shigella sonnei
© Mitobe et al. 2009
Received: 05 December 2008
Accepted: 28 May 2009
Published: 28 May 2009
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© Mitobe et al. 2009
Received: 05 December 2008
Accepted: 28 May 2009
Published: 28 May 2009
The expression of Type III secretion system (TTSS) in Shigella is regulated in response to changes in environmental osmolarity and temperature. Temperature-dependent regulation of virF, the master regulator of TTSS synthesis, is believed to occur at the transcriptional level. We recently demonstrated, however, that TTSS synthesis also involves post-transcriptional regulation of the synthesis of InvE, a target of virF and key regulator of TTSS synthesis. The mRNA levels of invE (virB) are stable at 37°C, but mRNA stability markedly decreases at low temperatures where the TTSS synthesis is tightly repressed. Deletion of hfq, which encodes an RNA chaperone in Gram-negative bacteria, results in the restoration of expression of invE and other TTSS genes at low temperature due to an increase in the stability of invE mRNA. To date, the molecular details of the regulation of TTSS expression in response to osmotic pressure are not known. In the current study, we investigated the mechanism of regulation of TTSS by osmotic pressure.
Transcription of virF, which encodes the master regulator of TTSS expression, was partially repressed under low osmotic conditions. Several lines of evidence indicated that osmolarity-dependent changes in TTSS synthesis are controlled at the post-transcriptional level, through the regulation of InvE synthesis. First, the expression InvE protein was tightly repressed under low osmotic growth conditions, even though invE mRNA transcripts were readily detectable. Second, under low osmotic conditions, invE mRNA was rapidly degraded, whereas deletion of hfq, which encodes an RNA chaperone, resulted in increased invE mRNA stability and the production of InvE protein. Third, the binding of purified Hfq in vitro to invE RNA was stronger in low-salt buffer, as assessed by gel-shift analysis and surface plasmon resonance (Biacore analysis).
Osmolarity-dependent changes in TTSS synthesis in Shigella involve the post-transcriptional regulation of InvE expression, in addition to partial transcriptional activation by virF. The stability of invE mRNA is reduced under low osmotic conditions, similar to the effect of temperature. Deletion of an RNA chaperone gene (hfq) abolished the repression of TTSS synthesis at low osmolarity through a mechanism that involved increased stability of invE mRNA. We propose that the expression of Shigella virulence genes in response to both osmolarity and temperature involves the post-transcriptional regulation of expression of InvE, a critical regulator of TTSS synthesis.
TTSS plays a major role in virulence determination in pathogenic Shigella. The expression of TTSS is regulated in response to environmental stimuli, such as changes in salt concentration  and growth temperature [2, 3]. This response to environmental factors is appropriate for the life cycle of Shigella, in which the expression of virulence genes is required for invasion and propagation in the host intestinal tract, but might be a potential burden for survival in the natural environment.
The genes that encode the components of TTSS in Shigella are located on the virulence plasmid, and are controlled by two regulator proteins, VirF and InvE (VirB) [4, 5]. VirF, an AraC-type transcriptional regulator, activates the transcription of invE (virB) [4, 6–8]. InvE is a homologue of a plasmid-partitioning factor, ParB , and possesses DNA binding activity . InvE activates the transcription of the mxi-spa and ipa genes, which encode the components of TTSS, through competition with the global repressor H-NS, a histone-like DNA binding protein .
Recently, we reported that the temperature-dependent expression of TTSS is controlled at the post-transcriptional level, through the regulation of InvE synthesis . The mRNA of invE is highly stable at 37°C, but stability decreases significantly at 30°C where the TTSS synthesis is tightly repressed. Deletion mutants of hfq, which encodes an RNA-binding protein in Gram-negative bacteria, restores the expression of invE and other TTSS genes at low temperature due to the increased stability of the invE mRNA.
To date, a detailed mechanism of osmolarity-dependent regulation of TTSS expression has yet to be elucidated. In the current study, we examined whether osmotic-dependent changes in TTSS expression involved post-transcriptional regulation. We present several lines of evidence that invE expression is regulated at the post-transcriptional level during TTSS synthesis in Shigella, and that the RNA chaperone Hfq plays a key role in regulating invE mRNA stability.
The expression of TTSS in Shigella is markedly reduced in low-salt LB medium . However, it is not clear whether the critical factor for the decreased expression of TTSS in LB medium is low osmolarity or low-salt concentration. We analysed the expression of TTSS in the presence of several different osmolytes, but similar osmotic pressures. There was a difference in the growth rate of S. sonnei in LB medium in the absence (doubling time, 42.1 minutes) and presence (doubling time, 30.6 minutes) of 150 mM NaCl. To control for differences in growth rate in LB medium, we used yeast extract and nutrient broth (YENB) medium , since growth rate in YENB in the absence (doubling time, 32.2 minutes) and presence (doubling time, 31.4 minutes) of 150 mM NaCl was similar at 37°C. The osmotic pressure of YENB medium without and with 150 mM NaCl was 96 ± 3 and 397 ± 3 mOsm/kg• H2O, respectively. When 150 mM NaCl was replaced with 155 mM KCl, the osmotic pressure was 391 ± 2 mOsm/kg• H2O, whereas when NaCl was replaced with 260 mM sorbitol, osmotic pressure was 384 ± 1 mOsm/kg• H2O.
Both ipaB and invE are under the control of the upstream transcriptional regulator VirF [4, 6–8]. To identify the level at which IpaB and InvE expression was regulated in response to changes in osmolarity, we analyzed the expression of virF. In the absence of salt, virF mRNA was detectable by RT-PCR (Fig. 1B, virF mRNA), although the level of mRNA expression was approximately 29.0 ± 4.6% of the maximum level observed in the presence of 150 mM NaCl. In an attempt to determine the mechanism of regulation of virF transcription, we performed a reporter gene assay in which the expression of lacZ was driven by the virF promoter . In wild-type S. sonnei carrying the virF-lacZ reporter gene, the level of β-galactosidase activity in the absence of salt was 20.6% of that in the presence of 150 mM NaCl (Fig. 1C, Graph 1), which indicated that the virF promoter is partially active even in the absence of NaCl.
We examined VirF-dependent expression of invE by Western blot and RT-PCR. The production of InvE protein was almost completely repressed under conditions of low osmolarity (Fig. 1B, α-InvE), whereas under the same conditions, there was a significant level of invE mRNA detectable by RT-PCR (Fig. 1B, invE mRNA). Real-time RT-PCR analysis indicated that the amount of invE mRNA in the absence of NaCl was 9.5 ± 1.6% of the level in the presence of 150 mM NaCl. We carried out a reporter gene assay to examine the expression of invE at both the transcriptional and translational levels . In low osmolarity, β-galactosidase activity in wild-type S. sonnei that expressed the transcriptional fusion gene invETx-lacZ was moderately decreased, to 28.9% of that seen in the presence of 150 mM NaCl (Fig. 1C, Graph 2). In contrast, β-galactosidase activity in cells that expressed the translational fusion gene invETL-lacZ was 7.3% of the level in the presence of 150 mM NaCl (Fig. 1C, Graph 3). These results indicated that the expression of InvE protein is repressed in the absence of salt, a condition under which genes for at least two regulatory proteins are still transcribed, albeit at reduced levels. Thus, the repression of InvE synthesis occurs primarily at the post-transcriptional level.
To determine whether the low level of InvE protein synthesis under conditions of low NaCl was due to decreased protein stability, we examined the metabolic stability of InvE in an invE deletion mutant strain (strain MS1632) carrying an expression plasmid for InvE (pBAD-invE)  at various times after treatment with rifampicin. The levels of InvE and IpaB were slightly lower in the absence of NaCl than in the presence of NaCl. Both proteins gradually degraded over time after rifampicin treatment, but the rate of degradation was essentially the same in the presence or absence of NaCl (Fig. 2B). By comparison, invE mRNA decayed within 10 minutes (min) after rifampicin treatment, and the rate of decay was much faster in low NaCl than in 150 mM NaCl (see below). These results indicated that InvE protein is metabolically stable once it is synthesized.
In low osmotic conditions, bacteria maintain intracellular osmotic homeostasis through the rapid release of small intracellular molecules, such as ions and amino acids . Since potassium ion is a major cation in bacteria , we measured intracellular K+ concentrations in S. sonnei under low osmotic conditions. In S. sonnei strain MS506 grown in the absence and presence of 150 mM NaCl, the intracellular K+ concentration was 131 ± 4 mmoles/mg cell and 316 ± 0 mmoles/mg cell, respectively. These results indicated that K+ concentration under low osmotic conditions decreases to nearly 40% of that seen under physiological osmotic conditions.
We also examined the interaction between Hfq and invE RNA by surface plasmon resonance (Biacore analysis). Similar to the gel-shift assay, we examined the interaction in the presence of either 40 mM or 100 mM NH4Cl at 37°C. The 140 nucleotide invE RNA probe that was used for the gel-shift assay was immobilized onto a sensor chip, and then increasing amounts of Hfq protein were added. The binding of Hfq hexamer to invE RNA reached a plateau at a concentration of nearly 8 nM Hfq under both buffer conditions (Fig. 5B) when the Hfq protein was used up to 32 nM (data not shown). Thus, the apparent binding affinity based on surface plasmon resonance was higher than that (16 nM) determined by gel-shift analysis. Distinct differences in the RNA binding properties of Hfq were observed in the presence of 40 mM and 100 mM NH4Cl. The minimum concentration of Hfq required for initial binding was 1 nM in the presence of 40 mM NH4Cl and 4 nM in the presence of 100 mM NH4Cl. In the presence of 40 mM NH4Cl, sequential binding of Hfq complexes was observed in an Hfq concentration-dependent manner, whereas in the presence of 100 mM NH4Cl, there was a sudden increase in Hfq binding at a concentration of 4 nM Hfq. These results confirmed the results of the gel-shift assay, and indicated that the binding of Hfq to invE RNA is influenced by salt concentration.
Invasion efficiency of bacteria grown in low-salt conditions
Rate of invasion
1 ± 1
2 ± 1
100 ± 29
MS390 (YENB+150 mM NaCl)
11 ± 3
In the case of Shigella, hfq mutation has been shown to increase invasion efficiency in cultured cell lines . However, hfq mutations have also been shown to reduce the virulence of other Gram-negative bacteria in a variety of animal models [20–25] through the regulation of expression of stress response genes . To investigate the role of Hfq in Shigella virulence in vivo, we performed a Sereny test, in which we monitored the development of keratoconjunctivitis in guinea pigs following inoculation with wild-type and hfq mutant strains of Shigella.
Virulence genes in Shigella are expressed in response to increases in temperature and/or osmolarity. Previously, we demonstrated that the temperature-dependent expression of virulence-related genes is regulated mainly at the post-transcriptional level, and that the RNA chaperone Hfq is involved in the translational control of virulence gene mRNA expression . At that time, however, precise details on the mechanism of osmolarity-dependent regulation of virulence gene expression in Shigella were unavailable.
The expression and synthesis of TTSS is controlled by the VirF-InvE regulator cascade. The expression of TTSS is markedly reduced by low osmolarity due to the repression of InvE synthesis. In the current study, several lines of evidence indicated that the repression of InvE occurs mainly at the post-transcriptional level: 1) there were significant, albeit low levels of invE mRNA in cells under low osmotic conditions, whereas InvE protein was barely detectable (Fig. 1B invE mRNA); 2) expression of the translational fusion gene invE-lacZ was fully repressed under low osmotic conditions, whereas expression of the corresponding transcriptional fusion gene was only partially repressed (Fig. 1C, Graphs 2 and 3); 3) in an arabinose-inducible promoter system, production of InvE protein decreased under low osmotic conditions even in the presence of sufficient amounts of invE mRNA (Fig. 2A); 4) in the absence of the RNA chaperone Hfq, the amount of InvE protein correlated with the level of virF transcription, even in low osmotic conditions (Fig. 3A); 5) InvE production was reduced upon over-expression of Hfq protein, even in physiological osmotic conditions (Fig. 3B); and 6) the stability of invE mRNA decreased under low osmotic conditions in the wild-type strain, but was increased in the hfq mutant (Fig. 4).
The synthesis of TTSS is induced in response to changes in osmolarity. While several osmolytes were able to induce TTSS synthesis, the response was weaker with the non-salt osmolyte sorbitol. Differences in TTSS synthesis in response to different osmolytes might be due to differences in permeability or influx through the bacterial membrane. Under physiological conditions, the contribution of non-salt osmolytes is likely to less relevant, because carbohydrates are almost completely absorbed in the ileum before reaching the colon, where infection and propagation of Shigella takes place. In the colon, Na+ ions and water are actively absorbed, and K+ ions are passively secreted, leading to an induction of TTSS synthesis. However, we did not observe significant differences in the expression of TTSS (Fig. 1A) and invasion (data not shown) in the presence of the two ions, which indicates that the trigger for TTSS induction is ionic strength, and not the nature of the ionic species.
In prokaryotes, the regulation of gene expression takes place mainly at the level of transcription. In the expression of a set of genes, however, regulation takes place at any one of several post-transcriptional stages, including the regulation of mRNA stability and translation, through a variety of mechanisms. We propose a model for the post-transcriptional repression of InvE expression in which the association of invE mRNA with the RNA chaperone Hfq controls mRNA stability. Recently, it was suggested that an iron-regulated small RNA, RyhB , plays a regulatory role in invE expression . At present, we cannot rule out the possibility that an interaction between invE mRNA and an as-yet unidentified RNA is involved in the temperature- and osmotic pressure-dependent activation of InvE synthesis. To date, various mechanisms have been proposed for the regulation of translation initiation through the modulation of RNA structure, including the structure of the initiation codon . For example, the temperature-dependent formation of a secondary structure within the 5'-untranslated element of the heat-shock operon mRNA of the plant bacterium Bradyrhizobium japonicum has been shown to regulate the level of translation of that mRNA [32–34]. In case of invE mRNA, a change of the signal that represents thermodynamic alteration of the structure was actually detected in circular dichroism spectroscopy  for the 140 nucleotides invE RNA . Furthermore, the characteristics of the binding of invE mRNA to Hfq in low-salt (Fig. 5) and low-temperature  conditions are consistent with an opening of the secondary structure of the RNA through the binding of multiple Hfq molecules. Of note, the pattern of binding of invE RNA to Hfq in low-salt buffer was remarkably similar to that seen in low temperature conditions . That indicates that the distribution of RNA-Hfq interaction strength upon the ionic circumstance exists in a similar range, which is defined by the thermodynamic distribution of Hfq binding between 30°C and 37°C. To date, specific molecular sensors of low osmotic conditions or mild temperature change have not been identified. Our results suggest that low osmotic conditions evoke a decrease in intracellular ionic strength, resulting in a similar effect on the strength of the RNA-Hfq interaction as that of decreased temperature. This raises the interesting possibility that post-transcriptional regulation itself represents a sensing system for changes in temperature and osmotic pressure.
The lack of active translation of invE mRNA could result in its destabilization . In fact, one of the mechanisms of post-transcriptional regulation is the regulation of mRNA stability . The degradosome is a well-characterized mRNA degradation system that consists of RNaseE, as well as Hfq (46). We examined the role of RNaseE in TTSS synthesis using a deletion mutant (Δrne 701–892) of the C-terminal region of RnaseE and E. coli rne-3071 ts strain N3431  carrying expression plasmids for virF, invE and TTSS genes (pJK1143 and pJK1142, respectively) . TTSS synthesis was unaffected in either of the two strains (data not shown), which indicates that an as-yet unidentified degradation pathway involving Hfq likely plays a role in the degradation of invE mRNA.
Similar to other bacterial species, hfq mutants of S. sonnei and S. flexneri exhibited decreased virulence in vivo. If the up-regulation of virulence gene expression due to hfq deletion leads to efficient antigen presentation for the host immune-system, then the hfq deletion is a potentially viable candidate for the development of a more effective Shigella vaccine, one that goes beyond the serotype-specific effects seen in current vaccine development . In fact, a Shigella hfq mutant is currently under evaluation for use as a vaccine in the guinea pig model . Shigella can survive in a range of environmental conditions, such as low osmotic pressure and low temperature, where strict repression of virulence gene expression is required. The development of a bi-functional sensing system for osmolarity and temperature represents an important adaptation for survival by this organism.
Changes in TTSS synthesis in response to osmotic pressure in Shigella involve in part the transcriptional regulation of the master regulator virF. In the current study, we demonstrated that post-transcriptional regulation of InvE expression is also involved in TTSS synthesis. This mechanism of post-transcriptional regulation of InvE synthesis was abolished in mutants that lacked hfq. The stability of invE mRNA was increased in the absence of Hfq, a major RNA chaperone in gram-negative bacteria. We propose that the synthesis of TTSS and pathogenesis of Shigella in varying temperature and osmolarity environments is dependent on the post-transcriptional regulation of InvE.
Luria-Bertani (LB) medium (LB Lenox, Difco Laboratory, Detroit MI) and YENB medium (0.75% Difco Yeast extract, 0.8% Difco Nutrient broth)  were used for the low osmotic media. YENB medium containing 150 mM NaCl (Wako Chemical, Tokyo Japan) was used as the physiological osmotic medium. YENB medium containing 155 mM KCl (Wako) or 260 mM sorbitol (Sigma Co., St. Louis MO) was used as a control for osmotic pressure. The osmotic pressure of each type of medium was measured by the decreasing freezing point method  in a clinical inspection facility (SRL Co., Tokyo Japan). The concentrations of antibiotics were as follows: ampicillin (Wako), 50 μg/ml; chloramphenicol (Wako), 12.5 μg/ml; rifampicin (R3501 Sigma), 200 μg/ml. Concentrations are also specified in the Figure legends for each experiment. For all experiments, the indicated strains were inoculated into 2 ml of LB medium and grown overnight at 30°C with shaking (150 rpm) in a water-bath. The cultures were diluted 100-fold in 5 ml of fresh YENB medium with or without salt. The samples were incubated at 37°C with shaking at 150 rpm, and monitored for turbidity at 600 nm (A 600) by spectroscopy (Spectronic™ 20+, Shimadzu Co., Kyoto Japan). Cells were harvested when they reached an A 600 of 0.8. Aliquots of the culture were used for measuring β-galactosidase activity (50 μl), as previously described , or subjected to 10% SDS-PAGE and Western blot analysis (10 μl) . The control experiments, indicated by black bars in Figure 1C (NC, negative control), were conducted by ΔcpxR strain MS2830 (Graph 1), or strain MS506 cured of virulence plasmid (Graphs 2 and 3) carrying the indicated reporter plasmid. All controls were grown in YENB plus 150 mM NaCl. The percentages indicated in the text were calculated after data was normalized to the negative control. Data represents the means and standard deviation of at least two independent experiments. IpaB and InvE proteins were detected using an anti-IpaB monoclonal antibody and an anti-InvE polyclonal antibody , respectively. For the detection of CpxR and H-NS, 5 μl of whole cell culture were separated by 15–20% tricine gradient gel electrophoresis (Wako), and then analysed by Western blot using an anti-CpxR  and anti-H-NS antibody, respectively, as previously described [42, 43].
Bacterial strains and plasmids used in this study
Bacterial strains and plasmids
rne-3071 ts , lacZ43, LAM-, relA1, spoT1 (CGSC#6975)
S. sonnei wild-type strain, (Tcr)
S. sonnei HW383 without pSS120 plasmid (Tcr, non invasive)
MS390ΔcpxR (cpxR: chromosomal activator of virF gene)
MS390Δhns (non invasive)
MS390Δrne 701–892 ::aphA
S. flexneri 2a wild-type strain,
2457T carrying mutation in virF gene (non-invasive)
PCR-amplified invE gene was cloned into pBAD24 (Apr)
virF-lacZ translational fusion plasmid (Cmr)
invE and ipa-mxi-spa (TTSS) genes encoding plasmid (Kmr)
virF-encoding plasmid (Cmr)
invE-lacZYA transcriptional fusion in pTH18cs5(Cmr)
invE-lacZYA translational fusion in pTH18cs5(Cmr)
IPTG inducible expression plasmid(Apr)
PCR-amplified hfq gene was cloned into pTrc99A(Apr)
Intracellular K+ ion concentration was measured by potassium-electrode, as described previously . An avirulent S. sonnei strain, MS506, was grown to an A 600 of 0.8 in 45 ml of YENB medium or YENB medium plus 150 mM NaCl at 37°C, and then the culture was chilled on ice for 15 min. The culture was divided into triplicate tubes (15 ml Falcon tubes, #430766, Corning Inc., Corning NY), and then bacterial cells were collected by centrifugation at 5000 × g for 15 min at 4°C. An aliquot of each culture was diluted and plated on LB agar for measuring colony counts. The bacterial cells were washed twice at 4°C with 5 ml of hypotonic buffer (20 mM Na-Phosphate pH7.0 for the YENB cultures) or isotonic buffer (20 mM Na-Phosphate pH7.0, 150 mM NaCl for the YENB plus 150 mM NaCl cultures). Cells were suspended in 2 ml of hypotonic buffer and then sonicated using a SONIFIER-250D (Branson Ultrasonic Co., Danbury CT) until microscopic examination confirmed that all the cells were completely disrupted. The samples were cleared by centrifugation at 12000 × g for 30 min at 4°C, and the K+ ion concentration of the supernatants was measured by potassium electrode  at SRL Co. (Tokyo Japan).
Two ml of whole cell culture were quickly mixed with 150 μl of 5% (v/v) water-saturated phenol in ethanol to prevent RNA degradation . virF and invE mRNAs were purified and analysed using a Titan™ one tube RT-PCR kit (Roche, Indianapolis IN) and Perfect Real-time™ (Takara Bio Co., Shiga Japan), as described previously . For the detection of virF mRNA by real-time PCR, virFc-314F (5'-GGAGACGTTTATTTGTATATTTCGCTCTA-3', 120 nM) and virFc-398R (5'-GACGGTTAGCTCAGGCAATGAT-3', 120 nM) primers and the fluorescent probe virFc-345T (5'-FAM-AAAGCAATTTGCCCTTCATCGAT-TAMRA-3', 32 nM) were designed by ABI primer design software (Applied Biosystems Inc., Foster CA) and synthesized by ABI Japan (Tokyo). Real-time PCR analysis was performed using an ABI PRISM 2000 Thermal Cycler, as described previously . RNA preparation and real-time PCR analysis were repeated at least 3 times with similar results.
The labelled RNA probe (20 fmoles), corresponding to 140 nucleotides of the invE gene (starting from the transcription start site at +1) , and purified Hfq protein (0, 1, 2, 4, 8, or 16 nM Hfq hexamer) were mixed in a volume of 10 μl in one of two RNA binding buffers (40 mM NH4Cl, 10 mM Tris-HCl pH7.5, 5 mM magnesium acetate, 0.1 mM dithiothreitol; or 100 mM NH4Cl, 10 mM Tris-HCl pH7.5, 5 mM magnesium acetate, 0.1 mM dithiothreitol) at 37°C for 10 min. Gel-shift analysis was performed at 37°C as described previously .
Surface plasmon resonance was performed with Biacore 2000 optical sensor device using the same 140 nucleotide invE RNA probe for the gel-shift assay as described previously . The probe was immobilized onto a sensor tip SA (GE Healthcare Co., Piscataway NJ), causing a change of nearly 150 resonance units. Purified Hfq protein was diluted to a final concentration of 0, 1, 2, 4 or 8 nM (Hfq hexamer) in one of two RNA binding buffers, as described for gel-shift assays, and then injected for 180 seconds through two flow cells (flow cell 1, blank; flow cell 2, invE RNA) at a flow rate of 20 ml/min at 37°C. Non-specific proteins were dissociated from the chip by washing (for 700 seconds). Bound Hfq protein was subsequently removed with 2 M NaCl. The response value of the reference cell (flow cell 1, blank) was subtracted from the response value of flow cell 2 (invE RNA) to correct for nonspecific binding, and the results are expressed as difference units (D.U.). The right panels of Figure 5A and 5B are reprinted from our previous issue  with the permission of the American Society for Biochemistry and Molecular Biology (Copyright © 2008), which were performed with the identical materials to the left panels in the same experimental period.
Pre-cultures in LB media were inoculated into 5 ml of YENB medium and then incubated for 2 hrs at 37°C with shaking. For strains carrying expression plasmids, IPTG was added to a final concentration of 0.1 mM 40 min after inoculation, and then the cultures were allowed to incubate for an additional 80 min at 37°C. Bacterial invasion into HeLa cells using the gentamicin protection assay was performed as previously described .
Three groups (6 total) of male Hartley guinea pigs (2 weeks old, SLC Co., Hamamatu Japan) were infected with S. sonnei and S. flexneri strains for the Sereny test, an experimental animal model of conjunctivitis . Fresh LB cultures of the indicated strains were harvested at an A 600 of 0.8 and then collected by centrifugation. Bacterial cells (5 × 108) in 10 μl of LB medium were deposited into the conjunctival sac of each eye of 2 animals for two consecutive days. Four day later, the symptoms of each animal were recorded by digital photography. Sera were obtained two weeks after infection, and the levels of antibodies against soluble effector molecules of TTSS were measured by ELISA using peroxidase-conjugated anti-guinea pig IgG as the secondary antibody (A5545 Sigma). The source of effector molecules was a culture supernatant of strain MS390 grown at 37°C in LB medium containing 10 μg/ml Congo Red (C6767 Sigma), with which the effector molecules of TTSS are known to be secreted . The culture supernatant (200 μl) was plated onto polystyrene microtiter plates (Costar #3369, Corning) and the plates were incubated at 4°C for 18 hours (hrs). Serial dilutions (25, 100, 400, 1600-fold in phosphate-buffered saline) of guinea pig sera were added to the plate and allowed to react for 2 hrs at 37°C, after which the secondary antibody (5000-fold dilution) was added for 1 hr at room temperature. Absorbance at 620 nm was measured using a Multiskan Ascent microplate reader (Thermo Labsystem, Helsinki Finland) after the addition of 1-Step™ ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) (#37615 Pierce, Rockford IL), as described by the manufacture. All animal experiments were conducted in compliance with the Animal Welfare Act, and adhered to the principles stated in the Guide for Care and Use of Laboratory Animals  after approval as #209002-2 by a board of experimental animals at the National Institute of Infectious Diseases (NIDD), Japan.
TTSS: Type three secretion system
This research was supported by a grant-in-aid for Exploratory Research 19657043 from the Ministry of Education, Science and Technology (KAKENHI), Ministry of Health, Labor and Welfare (H19·kokusai-igaku) of the Japanese Government. An E. coli strain N3431 was kindly provided from the Coli Genetic Stock Center (Yale University, CT). We thank Shu-ichi Nakayama for providing anti-CpxR antibody, Nobuo Koizumi and Ken Shimuta for assistance of the animal experiments.
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