BACKGROUND
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  <cit>  and growth temperature  <cit> .
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   <cit> .
virf, an arac-type transcriptional regulator, activates the transcription of inve   <cit> .
inve is a homologue of a plasmid-partitioning factor, parb  <cit> , and possesses dna binding activity  <cit> .
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  <cit> .
recently, we reported that the temperature-dependent expression of ttss is controlled at the post-transcriptional level, through the regulation of inve synthesis  <cit> .
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.
RESULTS
osmolarity and ttss expression
the expression of ttss in shigella is markedly reduced in low-salt lb medium  <cit> .
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  and presence  of  <dig> mm nacl.
to control for differences in growth rate in lb medium, we used yeast extract and nutrient broth  medium  <cit> , since growth rate in yenb in the absence  and presence  of  <dig> mm nacl was similar at 37°c.
the osmotic pressure of yenb medium without and with  <dig> mm nacl was  <dig> ±  <dig> and  <dig> ±  <dig> mosm/kg• h2o, respectively.
when  <dig> mm nacl was replaced with  <dig> mm kcl, the osmotic pressure was  <dig> ±  <dig> mosm/kg• h2o, whereas when nacl was replaced with  <dig> mm sorbitol, osmotic pressure was  <dig> ±  <dig> mosm/kg• h2o.
to monitor the expression of ttss, we measured the expression of the effector protein ipab and the regulatory molecule inve.
the expression of ipab and inve was tightly repressed in low osmotic conditions, whereas in the presence of either  <dig> mm nacl or  <dig> mm kcl, the level of both proteins increased to a similar extent .
a linear relationship was observed between salt concentration and the levels of inve and ipab , which indicated that there is no threshold for the effective induction of ttss synthesis.
in the presence of  <dig> mm sorbitol, the levels of both inve and ipab were approximately 50% lower than in the presence of nacl and kcl .
when the concentration of sorbitol was increased to  <dig> mm, inve and ipab levels increased to the level of the nacl and kcl growth conditions.
these results indicated that in addition to salt concentration, osmolarity regulates the expression of ttss, although the optimum concentration for maximum induction differed among osmolytes .
transcription of virf and inve under low osmotic conditions
both ipab and inve are under the control of the upstream transcriptional regulator virf  <cit> .
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 , although the level of mrna expression was approximately  <dig>  ±  <dig> % of the maximum level observed in the presence of  <dig> 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  <cit> .
in wild-type s. sonnei carrying the virf-lacz reporter gene, the level of β-galactosidase activity in the absence of salt was  <dig> % of that in the presence of  <dig> mm nacl , 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 , whereas under the same conditions, there was a significant level of inve mrna detectable by rt-pcr .
real-time rt-pcr analysis indicated that the amount of inve mrna in the absence of nacl was  <dig>  ±  <dig> % of the level in the presence of  <dig> mm nacl.
we carried out a reporter gene assay to examine the expression of inve at both the transcriptional and translational levels  <cit> .
in low osmolarity, β-galactosidase activity in wild-type s. sonnei that expressed the transcriptional fusion gene invetx-lacz was moderately decreased, to  <dig> % of that seen in the presence of  <dig> mm nacl .
in contrast, β-galactosidase activity in cells that expressed the translational fusion gene invetl-lacz was  <dig> % of the level in the presence of  <dig> mm nacl .
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.
post-transcriptional regulation of inve
to examine the mechanism of post-transcriptional regulation of inve expression more directly, we replaced the native inve promoter with a promoter cassette containing the e. coli arac repressor and the arabad promoter region  <cit> .
in this system, we were able to examine virf-independent expression of inve under the control of the arac-dependent arabad promoter.
strain ms <dig> carrying Δpinve::parabad  <cit>  was cultured in the presence or absence of  <dig> mm nacl, and the synthesis of inve protein was induced by increasing the concentration of arabinose.
similar levels of inve mrna were detected in the presence of  <dig>  and  <dig>  mm arabinose, independently of the presence or absence of nacl .
however, the synthesis of inve protein was significantly decreased in the absence of nacl , as was inve-dependent synthesis of the ttss effector protein ipab .
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  carrying an expression plasmid for inve   <cit>  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 .
by comparison, inve mrna decayed within  <dig> minutes  after rifampicin treatment, and the rate of decay was much faster in low nacl than in  <dig> mm nacl .
these results indicated that inve protein is metabolically stable once it is synthesized.
involvement of hfq in the post-transcriptional regulation of inve synthesis
previously, we showed that the rna-binding protein hfq  <cit>  is involved in the temperature-dependent regulation of inve expression, and that this regulation occurs at the post-transcriptional level  <cit> .
we next examined the expression of inve in an hfq deletion mutant strain of s. sonnei  under low osmotic conditions.
as in the case of temperature-dependent regulation, the level of expression of inve and ipab in an hfq mutant strain in the absence of nacl was approximately 33% of that seen in the presence of  <dig> mm nacl , which suggested that hfq is involved in the osmolarity-dependent post-transcriptional regulation of inve and ipab synthesis.
real-time analysis of virf mrna in the hfq mutant in the absence of nacl indicated that the level of expression of virf was  <dig>  ±  <dig> % of that seen in the wild-type strain in the presence of  <dig> mm nacl, which suggested that the level of virf transcription in the hfq mutant parallels the level of inve protein synthesis.
thus, in the absence of hfq, the level of inve protein in low osmotic conditions correlated with the level of virf and inve transcription .
to confirm these results, we introduced an hfq expression plasmid, ptrc-hfq, into the hfq deletion mutant.
ectopic expression of hfq in the mutant strain resulted in the repression of inve expression in low osmotic conditions , and abolished the expression of inve and ipab even in physiological osmotic conditions .
stability of inve mrna
we examined the stability of inve mrna in the hfq mutant by rt-pcr and real-time pcr analysis.
under physiological osmotic conditions, inve mrna levels in the wild-type strain were high, and remained stable for at least  <dig> min after rifampicin treatment .
under low osmotic conditions, inve mrna levels were low , and inve mrna was rapidly degraded within the first  <dig> min after rifampicin treatment .
by comparison, the stability of inve mrna was markedly increased in the hfq deletion mutant even under low osmotic conditions  .
this increase in inve mrna stability correlated with increased inve protein levels in cells.
these results further support the prediction that the stability of inve mrna is intimately coupled with the expression of inve protein.
hfq-inve mrna interaction in vitro under low-salt conditions
in low osmotic conditions, bacteria maintain intracellular osmotic homeostasis through the rapid release of small intracellular molecules, such as ions and amino acids  <cit> .
since potassium ion is a major cation in bacteria  <cit> , we measured intracellular k+ concentrations in s. sonnei under low osmotic conditions.
in s. sonnei strain ms <dig> grown in the absence and presence of  <dig> mm nacl, the intracellular k+ concentration was  <dig> ±  <dig> mmoles/mg cell and  <dig> ±  <dig> 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.
since interactions between proteins and nucleic acids are influenced by salt concentration, we examined the effect of salt concentration on the interaction of hfq and inve rna in vitro, using an rna gel-shift assay and surface plasmon resonance .
hfq-inve rna complex formation was examined by gel-shift assay using a binding buffer that contained  <dig> mm nh4cl  <cit> .
to control for the decrease in intracellular k+ concentration in the absence of physiological concentrations of nacl, we also performed the gel-shift assay in buffer that contained  <dig> mm nh4cl.
the rna probe  was mixed with increasing concentrations of purified hfq hexamer complex  at 37°c for  <dig> min.
in the presence of  <dig> mm nh4cl, we observed an initial shift of the rna probe upon the addition of  <dig> nm hfq hexamer , whereas the corresponding shift in the presence of  <dig> mm nh4cl required  <dig> nm hexamer .
the apparent binding constant, as determined by the disappearance of half of the free rna probe, was  <dig>  nm hfq in the presence of  <dig> mm nh4cl and  <dig>  nm in the presence of  <dig> mm nh4cl.
we also examined the interaction between hfq and inve rna by surface plasmon resonance .
similar to the gel-shift assay, we examined the interaction in the presence of either  <dig> mm or  <dig> mm nh4cl at 37°c.
the  <dig> 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  <dig> nm hfq under both buffer conditions  when the hfq protein was used up to  <dig> nm .
thus, the apparent binding affinity based on surface plasmon resonance was higher than that  determined by gel-shift analysis.
distinct differences in the rna binding properties of hfq were observed in the presence of  <dig> mm and  <dig> mm nh4cl.
the minimum concentration of hfq required for initial binding was  <dig> nm in the presence of  <dig> mm nh4cl and  <dig> nm in the presence of  <dig> mm nh4cl.
in the presence of  <dig> mm nh4cl, sequential binding of hfq complexes was observed in an hfq concentration-dependent manner, whereas in the presence of  <dig> mm nh4cl, there was a sudden increase in hfq binding at a concentration of  <dig> 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.
effect of hfq mutation on invasion and virulence in vivo
to determine whether the repression of ttss expression in low osmotic conditions influenced invasion by s. sonnei, we performed an invasion assay using s. sonnei strains that were grown in the absence of nacl.
when grown in low-salt conditions, the ability of the wild-type strain to invade hela cells was tightly repressed.
the hfq mutant strain ms <dig> was highly invasive, and invasion was markedly repressed by the addition of iptg, which induced the expression of hfq .
these results indicated that hfq is intimately involved in synthesis of ttss-associated genes in s. sonnei.
in the case of shigella, hfq mutation has been shown to increase invasion efficiency in cultured cell lines  <cit> .
however, hfq mutations have also been shown to reduce the virulence of other gram-negative bacteria in a variety of animal models  <cit>  through the regulation of expression of stress response genes  <cit> .
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.
guinea pigs infected with either the wild-type or hfq mutant strain developed keratoconjunctivitis within three days of infection.
the symptoms, including swelling of the cornea, development of conjunctivitis and excretion of pus, appeared to be more severe in animals infected with the wild-type strain .
the recovery period for animals infected with the wild-type strain was significantly longer on average than for animals infected with the hfq mutant strain .
the production of serum antibodies against ttss-associated secretary effector molecules was significantly higher in animals that were infected with the wild-type strain .
similar results were also observed when using an hfq mutant of s. flexneri mf <dig> .
thus, hfq mutation appeared to diminish the virulence of s. sonnei in vivo, independently of ttss-associated gene expression.
effect of h-ns on virf expression in low osmotic conditions
the nucleoid protein h-ns is involved in the expression of ttss through its ability to regulate virf expression  <cit> .
the effect of h-ns on virf expression in low osmotic conditions was examined using the β-galactosidase reporter gene assay.
although the hns mutation of shigella has been reported as transposon insertion, deletion of the full-length hns gene resulted in the loss of the virulence plasmid in our experiment using s. sonnei.
since the transcription of virf is regulated by chromosomal factors, the effect of h-ns on virf transcription was examined in the hns deletion mutant strain ms <dig>  in hns mutants carrying the virf-lacz reporter gene  <cit> , the β-galactosidase activity under low osmotic conditions was  <dig> % of that under physiological osmotic conditions .
in the s. sonnei wild-type strain, it was  <dig> % .
these results indicated that the nucleoid protein h-ns is involved, at least in part, in the osmolarity-dependent regulation of virf expression.
the level of h-ns protein and that of the two-component regulator cpxr, which is a critical activator of virf transcription  <cit> , were similar under both low and physiological osmotic conditions at 30°c and 37°c .
discussion
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  <cit> .
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 ; 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 ; 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 ; 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 ; 5) inve production was reduced upon over-expression of hfq protein, even in physiological osmotic conditions ; and 6) the stability of inve mrna decreased under low osmotic conditions in the wild-type strain, but was increased in the hfq mutant .
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  and invasion  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  <cit> , plays a regulatory role in inve expression  <cit> .
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  <cit> .
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  <cit> .
in case of inve mrna, a change of the signal that represents thermodynamic alteration of the structure was actually detected in circular dichroism spectroscopy  <cit>  for the  <dig> nucleotides inve rna  <cit> .
furthermore, the characteristics of the binding of inve mrna to hfq in low-salt  and low-temperature  <cit>  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  <cit> .
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  <cit> .
in fact, one of the mechanisms of post-transcriptional regulation is the regulation of mrna stability  <cit> .
the degradosome is a well-characterized mrna degradation system that consists of rnasee, as well as hfq .
we examined the role of rnasee in ttss synthesis using a deletion mutant  of the c-terminal region of rnasee and e. coli rne-3071ts strain n <dig>  <cit>  carrying expression plasmids for virf, inve and ttss genes   <cit> .
ttss synthesis was unaffected in either of the two strains , 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  <cit> .
in fact, a shigella hfq mutant is currently under evaluation for use as a vaccine in the guinea pig model  <cit> .
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.
CONCLUSIONS
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.

 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. 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. virf, an arac-type transcriptional regulator, activates the transcription of inve. 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.  osmolarity and ttss expression 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 and presence of mm nacl. to control for differences in growth rate in lb medium, we used yeast extract and nutrient broth medium, since growth rate in yenb in the absence and presence of mm nacl was similar at 37°c. the osmotic pressure of yenb medium without and with mm nacl was ± and ± mosm/kg• h2o, respectively. when mm nacl was replaced with mm kcl, the osmotic pressure was ± mosm/kg• h2o, whereas when nacl was replaced with mm sorbitol, osmotic pressure was ± mosm/kg• h2o. to monitor the expression of ttss, we measured the expression of the effector protein ipab and the regulatory molecule inve. the expression of ipab and inve was tightly repressed in low osmotic conditions, whereas in the presence of either mm nacl or mm kcl, the level of both proteins increased to a similar extent. a linear relationship was observed between salt concentration and the levels of inve and ipab, which indicated that there is no threshold for the effective induction of ttss synthesis. in the presence of mm sorbitol, the levels of both inve and ipab were approximately 50% lower than in the presence of nacl and kcl. when the concentration of sorbitol was increased to mm, inve and ipab levels increased to the level of the nacl and kcl growth conditions. these results indicated that in addition to salt concentration, osmolarity regulates the expression of ttss, although the optimum concentration for maximum induction differed among osmolytes. transcription of virf and inve under low osmotic conditions both ipab and inve are under the control of the upstream transcriptional regulator virf. 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, although the level of mrna expression was approximately ± % of the maximum level observed in the presence of 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 % of that in the presence of mm nacl, 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, whereas under the same conditions, there was a significant level of inve mrna detectable by rt-pcr. real-time rt-pcr analysis indicated that the amount of inve mrna in the absence of nacl was ± % of the level in the presence of 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 % of that seen in the presence of mm nacl. in contrast, β-galactosidase activity in cells that expressed the translational fusion gene invetl-lacz was % of the level in the presence of mm nacl. 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. post-transcriptional regulation of inve to examine the mechanism of post-transcriptional regulation of inve expression more directly, we replaced the native inve promoter with a promoter cassette containing the e. coli arac repressor and the arabad promoter region. in this system, we were able to examine virf-independent expression of inve under the control of the arac-dependent arabad promoter. strain ms carrying Δpinve::parabad was cultured in the presence or absence of mm nacl, and the synthesis of inve protein was induced by increasing the concentration of arabinose. similar levels of inve mrna were detected in the presence of and mm arabinose, independently of the presence or absence of nacl. however, the synthesis of inve protein was significantly decreased in the absence of nacl, as was inve-dependent synthesis of the ttss effector protein ipab. 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 carrying an expression plasmid for 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. by comparison, inve mrna decayed within minutes after rifampicin treatment, and the rate of decay was much faster in low nacl than in mm nacl. these results indicated that inve protein is metabolically stable once it is synthesized. involvement of hfq in the post-transcriptional regulation of inve synthesis previously, we showed that the rna-binding protein hfq is involved in the temperature-dependent regulation of inve expression, and that this regulation occurs at the post-transcriptional level. we next examined the expression of inve in an hfq deletion mutant strain of s. sonnei under low osmotic conditions. as in the case of temperature-dependent regulation, the level of expression of inve and ipab in an hfq mutant strain in the absence of nacl was approximately 33% of that seen in the presence of mm nacl, which suggested that hfq is involved in the osmolarity-dependent post-transcriptional regulation of inve and ipab synthesis. real-time analysis of virf mrna in the hfq mutant in the absence of nacl indicated that the level of expression of virf was ± % of that seen in the wild-type strain in the presence of mm nacl, which suggested that the level of virf transcription in the hfq mutant parallels the level of inve protein synthesis. thus, in the absence of hfq, the level of inve protein in low osmotic conditions correlated with the level of virf and inve transcription. to confirm these results, we introduced an hfq expression plasmid, ptrc-hfq, into the hfq deletion mutant. ectopic expression of hfq in the mutant strain resulted in the repression of inve expression in low osmotic conditions, and abolished the expression of inve and ipab even in physiological osmotic conditions. stability of inve mrna we examined the stability of inve mrna in the hfq mutant by rt-pcr and real-time pcr analysis. under physiological osmotic conditions, inve mrna levels in the wild-type strain were high, and remained stable for at least min after rifampicin treatment. under low osmotic conditions, inve mrna levels were low, and inve mrna was rapidly degraded within the first min after rifampicin treatment. by comparison, the stability of inve mrna was markedly increased in the hfq deletion mutant even under low osmotic conditions. this increase in inve mrna stability correlated with increased inve protein levels in cells. these results further support the prediction that the stability of inve mrna is intimately coupled with the expression of inve protein. hfq-inve mrna interaction in vitro under low-salt conditions 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 ms grown in the absence and presence of mm nacl, the intracellular k+ concentration was ± mmoles/mg cell and ± 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. since interactions between proteins and nucleic acids are influenced by salt concentration, we examined the effect of salt concentration on the interaction of hfq and inve rna in vitro, using an rna gel-shift assay and surface plasmon resonance. hfq-inve rna complex formation was examined by gel-shift assay using a binding buffer that contained mm nh4cl. to control for the decrease in intracellular k+ concentration in the absence of physiological concentrations of nacl, we also performed the gel-shift assay in buffer that contained mm nh4cl. the rna probe was mixed with increasing concentrations of purified hfq hexamer complex at 37°c for min. in the presence of mm nh4cl, we observed an initial shift of the rna probe upon the addition of nm hfq hexamer, whereas the corresponding shift in the presence of mm nh4cl required nm hexamer. the apparent binding constant, as determined by the disappearance of half of the free rna probe, was nm hfq in the presence of mm nh4cl and nm in the presence of mm nh4cl. we also examined the interaction between hfq and inve rna by surface plasmon resonance. similar to the gel-shift assay, we examined the interaction in the presence of either mm or mm nh4cl at 37°c. the 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 nm hfq under both buffer conditions when the hfq protein was used up to nm. thus, the apparent binding affinity based on surface plasmon resonance was higher than that determined by gel-shift analysis. distinct differences in the rna binding properties of hfq were observed in the presence of mm and mm nh4cl. the minimum concentration of hfq required for initial binding was nm in the presence of mm nh4cl and nm in the presence of mm nh4cl. in the presence of mm nh4cl, sequential binding of hfq complexes was observed in an hfq concentration-dependent manner, whereas in the presence of mm nh4cl, there was a sudden increase in hfq binding at a concentration of 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. effect of hfq mutation on invasion and virulence in vivo to determine whether the repression of ttss expression in low osmotic conditions influenced invasion by s. sonnei, we performed an invasion assay using s. sonnei strains that were grown in the absence of nacl. when grown in low-salt conditions, the ability of the wild-type strain to invade hela cells was tightly repressed. the hfq mutant strain ms was highly invasive, and invasion was markedly repressed by the addition of iptg, which induced the expression of hfq. these results indicated that hfq is intimately involved in synthesis of ttss-associated genes in s. sonnei. 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 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. guinea pigs infected with either the wild-type or hfq mutant strain developed keratoconjunctivitis within three days of infection. the symptoms, including swelling of the cornea, development of conjunctivitis and excretion of pus, appeared to be more severe in animals infected with the wild-type strain. the recovery period for animals infected with the wild-type strain was significantly longer on average than for animals infected with the hfq mutant strain. the production of serum antibodies against ttss-associated secretary effector molecules was significantly higher in animals that were infected with the wild-type strain. similar results were also observed when using an hfq mutant of s. flexneri mf. thus, hfq mutation appeared to diminish the virulence of s. sonnei in vivo, independently of ttss-associated gene expression. effect of h-ns on virf expression in low osmotic conditions the nucleoid protein h-ns is involved in the expression of ttss through its ability to regulate virf expression. the effect of h-ns on virf expression in low osmotic conditions was examined using the β-galactosidase reporter gene assay. although the hns mutation of shigella has been reported as transposon insertion, deletion of the full-length hns gene resulted in the loss of the virulence plasmid in our experiment using s. sonnei. since the transcription of virf is regulated by chromosomal factors, the effect of h-ns on virf transcription was examined in the hns deletion mutant strain ms in hns mutants carrying the virf-lacz reporter gene, the β-galactosidase activity under low osmotic conditions was % of that under physiological osmotic conditions. in the s. sonnei wild-type strain, it was %. these results indicated that the nucleoid protein h-ns is involved, at least in part, in the osmolarity-dependent regulation of virf expression. the level of h-ns protein and that of the two-component regulator cpxr, which is a critical activator of virf transcription, were similar under both low and physiological osmotic conditions at 30°c and 37°c. discussion 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; 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; 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; 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; 5) inve production was reduced upon over-expression of hfq protein, even in physiological osmotic conditions; and 6) the stability of inve mrna decreased under low osmotic conditions in the wild-type strain, but was increased in the hfq mutant. 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 and invasion 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. 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 nucleotides inve rna. furthermore, the characteristics of the binding of inve mrna to hfq in low-salt 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. we examined the role of rnasee in ttss synthesis using a deletion mutant of the c-terminal region of rnasee and e. coli rne-3071ts strain n carrying expression plasmids for virf, inve and ttss genes. ttss synthesis was unaffected in either of the two strains, 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. 
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. osmolarity-dependent changes in ttss synthesis in shigella involve the post-transcriptional regulation of inve expression, in addition to partial transcriptional activation by virf. 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. 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 are stable at 37°c, but mrna stability markedly decreases at low temperatures where the ttss synthesis is tightly repressed. 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. deletion of an rna chaperone gene 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. 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. the stability of inve mrna is reduced under low osmotic conditions, similar to the effect of temperature. to date, the molecular details of the regulation of ttss expression in response to osmotic pressure are not known. transcription of virf, which encodes the master regulator of ttss expression, was partially repressed under low osmotic conditions. in the current study, we investigated the mechanism of regulation of ttss by osmotic pressure.
 the expression of type iii secretion system 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 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.  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 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. 