article: Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.
  Salmonella enterica serovar Typhimurium is a prevalent gastrointestinal pathogen. Upon arrival in the intestinal lumen, Salmonella is able to both invade epithelial cells and survive within phagocytic cells. Genomic studies revealed the presence of several pathogenicity islands in the Salmonella chromosome (SPIs). Among them, SPI-1 and SPI-2 are the best studied and known to encode factors required for invasion of non-phagocytic cells and survival within macrophages, respectively [1,2]. Induction of virulence programs is generally associated with significant energetic costs for the bacterial cell. For example, induction of SPI-1 under non-infectious conditions in vitro has a negative impact on cell physiology, resulting in a deleterious effect on Salmonella’s growth [3]. Consequently, the expression of virulence programs is generally tightly regulated and induction occurs only upon sensing of a variety of defined environmental and physiological signals. The complex regulatory circuit that controls SPI-1 expression has attracted much attention [2,4] and become a model to understand how the activities of multiple molecular factors converge to achieve a precise timing of virulence gene activation. The majority of the multiple signal transduction systems that modulate SPI-1 regulation converge at the level of HilA expression, a SPI-1 encoded transcriptional regulator required for the expression of most SPI-1 genes [5]. Salmonella does not express HilA when it is growing exponentially in LB cultures, a condition stated as non-permissive in the present study. However, HilA expression is induced at early stationary phase, when growth conditions become nutrient-limiting [6], a condition here referred to as SPI-1-permissive. Transcription of hilA is controlled by three AraC-like transcriptional activators: HilD, HilC and RtsA. The first two are encoded within SPI-1 itself, while RtsA is encoded outside this locus [7]. HilD, HilC and RtsA form a feed-forward regulatory loop, whereby each activator induces the two other genes, but also auto-regulates its own expression [8]. This regulatory triad responds to a wide range of physiological and environmental stimuli that are sensed by a variety of cellular factors, including both global and specific regulators (as reviewed by Fabrega and Vila [2]). Within this triad, a prominent role has been attributed to HilD, the main target for signaling pathways controlling SPI-1 expression [8,9]. Regulatory mechanisms have been described, acting at all levels of hilD gene expression—transcriptional, post-transcriptional, translational and post-translational [10–13]. Most studies focused on the mechanisms required for full induction of SPI-1 genes, whereas very little is known on the regulatory pathways involved in the shutdown of the SPI-1 expression under non-permissive conditions. Here, we report that general transcription factor CRP is required to silence SPI-1 genes in exponential growing cells and propose a new regulatory axis formed by CRP and the broadly conserved small RNA (sRNA) Spot 42 that contributes to growth phase-specific activation of SPI-1 genes. CRP is a global transcriptional regulator that acts as a metabolic sensor upon binding of intracellular cAMP (cyclic adenosine monophosphate), which is synthesized by the adenylate cyclase CyaA [14]. CRP-cAMP-deficient Salmonella strains are unable to secrete SPI-1 T3SS effector proteins and are avirulent in a mouse model, suggesting a role for CRP-cAMP in the regulation of Salmonella virulence [15,16]. Indeed, CRP-cAMP indirectly regulates virulence by affecting the post-transcriptional regulation of hilD. The sRNAs CsrB and CsrC are under the transcriptional control of Bar/SirA and are upregulated in a crp knockout mutant. CsrB and CsrC are antagonists of CsrA, a post-transcriptional repressor of hilD mRNA [10,17,18]. Therefore, in early stationary phase (permissive conditions for SPI-1 expression), CRP-cAMP generally promotes SPI-1 expression by indirectly repressing the activity of CsrA [17–19]. Here, we report that in mid-logarithmic growth phase (non-permissive conditions), CRP-cAMP represses hilD expression by a mechanism requiring Hfq and the 3’UTR of hilD mRNA. Given the established primary role of Hfq in mediating the base pairing interactions of sRNAs [20,21], it is tempting to speculate that hilD may be post-transcriptionally regulated by a CRP-cAMP controlled sRNA; this control, however, would be unusual in light of the fact that almost all Hfq-associated sRNAs characterized to date recognize mRNAs in the 5’ region. Of several candidates for CRP-cAMP-dependent sRNAs known in enteric bacteria [22], Spot 42 has been best characterized in Escherichia coli, where together with CRP-cAMP, it forms a multi-output feedforward loop to enact catabolite repression [23–27]. In Salmonella, Spot 42 has been known as one of the most abundant Hfq-associated sRNAs in fast-growing cells [28], but except for a repression of the sugar-related mglB mRNA [29] its activity has not been characterized. By dissecting the molecular mechanism of CRP-cAMP-mediated SPI-1 repression in exponentially growing Salmonella, we here reveal novel mechanisms by which sRNAs target mRNAs. Our data point towards an unusual post-transcriptional stimulation of the hilD mRNA by Spot 42. Different from other trans-acting sRNA characterized, Spot 42-mediated activation occurs in the 3’ UTR of the hilD mRNA, adding to a growing appreciation of mRNA 3’ ends as sites for post-transcriptional control in bacteria.
  To characterize the role of the metabolic sensor CRP-cAMP in SPI-1 expression, we monitored transcription of the main regulator HilA in wild-type and Δcrp derivative strains grown in LB at 37°C. The expression pattern was determined in mid-logarithmic cultures (OD600nm 0. 4, non-permissive conditions for SPI-1 expression) and at early stationary phase (OD600nm 2. 0, permissive conditions for SPI-1 expression) (Fig 1A). Consistent with previous reports, a growth phase dependent profile in SPI-1 expression was observed [6]. In the wild-type strain, hilA expression levels were 8-fold higher at early stationary phase when compared to mid-logarithmic cultures. Remarkably, we also observed a growth-phase dependent effect of the Δcrp mutation. In agreement with previous work [18], the Δcrp mutation reduces hilA transcription in early stationary phase. In mid-logarithmic cultures, however, the Δcrp mutation caused an upregulation of hilA expression (4-fold, as compared to the wild-type). Using a chromosomally encoded FLAG-tagged HilA variant, these transcriptional profiles were corroborated on the protein level. More HilA protein accumulated in the Δcrp mutant in mid-logarithmic cultures and less in early stationary cultures, relative to wild-type levels (Fig 1B). CRP is active upon binding of the cofactor cAMP [14]. Therefore, lack of either CRP or cAMP should have a similar effect on SPI-1 expression. HilA levels were monitored in a Δcya mutant strain, which is deficient in the synthesis of cAMP (Fig 1C). As expected, Δcya mutation caused an almost 4-fold increase in HilA levels in mid-logarithmic phase cells. Chemical complementation was performed by monitoring HilA abundance after addition of cAMP (Fig 1C). An 8-fold decrease in HilA levels was observed when cAMP was added to cultures of the Δcya strain. Interestingly, when cAMP was added to a culture of a cya+ (i. e. wild-type) strain, a 2-fold drop in HilA levels was observed. These results may indicate that the intracellular cAMP levels were not saturating all CRP molecules. Consequently, external addition of the cofactor to wild-type cultures would lead to an increase in the number of CRP-cAMP complexes, causing further repression of HilA expression. To further corroborate the involvement of cAMP in the control of HilA expression, the intracellular levels of cAMP were lowered by ectopically over-expressing CpdA in Salmonella, a putative cAMP phosphodiesterase [30]. Over-expression of CpdA, confirmed by immunodetection (S1 Fig), caused a 4-fold increase in HilA expression in the wild-type background. This clearly depended on cAMP turnover, since CpdA over-expression had no effect in a Δcya strain (Fig 1D). HilA regulates the transcriptional expression of most SPI-1 genes, including those required for the synthesis of a type III secretion system (T3SS) and several effector proteins that are translocated to the host cell during Salmonella infection [2]. A ΔhilA mutation impairs secretion of SPI-1 effector proteins [31]. Comparative studies of the secreted protein profile between wild-type and ΔhilA strains were performed to identify protein bands corresponding to SPI-1 effectors (S2 Fig). Major protein bands exclusively detected in extracts of the wild-type strain were identified by LC-MS/MS as the SPI-1-encoded proteins SipA and SipC. The secretome of wild-type, Δcrp and Δcya derivative strains was characterized in LB cultures grown to mid-logarithmic and early stationary phase (Fig 2A). Consistent with previous reports [16], CRP-cAMP-deficient cells in early stationary phase showed a lower amount of those secreted proteins. Yet, CRP-cAMP-deficient Salmonella hyper-secreted SPI-1 effector proteins in mid-logarithmic phase cultures. The Δcrp-dependent overproduction in mid-logarithmic phase of the larger protein, the effector protein SipA, was confirmed by using a SipA-3xFLAG variant (Fig 2B). HilA has also been reported to regulate the expression of SopE, an effector protein that is encoded outside the SPI-1 locus but it is secreted by the SPI-1 encoded T3SS [32]. SopE levels were monitored in secreted protein extracts of wild-type and Δcrp mutant strains (Fig 2A). Again, the Δcrp strain secreted more SopE protein in the mid-logarithmic phase and less in early stationary phase, as compared to wild-type. The fact that CRP-cAMP-mediated repression of hilA expression has a concomitant effect on the expression and secretion of SPI-1 effector proteins highlights the biological relevance of CRP-cAMP in the control of Salmonella virulence under non-permissive conditions. In support of this notion, a Δcrp mutant grown to mid-logarithmic phase prior to infection, invaded HeLa cells more efficiently (>10-fold) than the wild-type (Fig 2C). In contrast, the wild-type strain showed a higher rate (>4. 5-fold) than the Δcrp derivative when cultures were grown to early stationary phase prior to infection. Three AraC-like transcriptional activators, HilD, HilC and RtsA, are directly involved in hilA activation [8]. To determine at which level CRP-cAMP controls SPI-1 through HilA, the expression of hilD, hilC and rtsA mRNA was monitored. RNA was extracted from mid-logarithmic cultures (OD600nm 0. 4) of both wild-type and Δcrp derivative strains and the relative amounts of mRNA of all three AraC-like regulators were determined by qRT-PCR. As shown in Fig 3A, in the Δcrp mutant higher transcripts levels of hilD, hilC and rtsA were detected than in the wild-type, indicating that the effect of CRP-cAMP on SPI-1 expression occurs upstream of HilA. The hilA transcript was also monitored by qRT-PCR as a control; as expected, it too over-accumulated in the Δcrp strain (S3 Fig). HilD, HilC and RtsA form a feed-forward regulatory loop to stimulate hilA expression. In order to elucidate the direct target of CRP-mediated regulation of SPI-1, mutants of each of the three regulators in a crp proficient and deficient background were generated and hilA expression monitored in mid-logarithmic cultures (Fig 3B). In the crp+ strains, hilA expression was very low in all genetic backgrounds, further validating the silenced hilA expression during logarithmic growth. The hilA derepression in the absence of CRP was altered in the different mutants. Remarkably, in the absence of HilC and HilD the derepression of hilA transcription was greatly reduced. The expression of hilC and hilD was further studied using transcriptional lacZ fusions. As expected, both hilC and hilD were deregulated in a Δcrp mutant background (Fig 3C). Particularly, HilD seems to be required for the observed upregulation of hilC in the Δcrp background, whereas hilD induction does not require HilC. Taken together, these results suggest that HilD is the direct target of CRP-cAMP-mediated regulation of SPI-1 expression. Northern blot detection further corroborates an increase in the levels of hilD mRNA in the Δcrp strain as compared to wild-type in mid-logarithmic phase (Fig 3D). The hilD mRNA possess an unusually long (310 nt) 3’UTR that has an overall negative effect on hilD expression [11]. If the 3’UTR is deleted, the hilD mRNA accumulates and the SPI-1 genes are induced concomitantly [11]. Of note, the above-described effect of CRP-cAMP on hilD transcription (Fig 3C) was elucidated using a hilD-lacZ fusion at position +1,235 (relative to the transcription start site), containing the hilD coding sequence and the full-length 3’UTR. To determine whether the hilD 3’UTR is important in CRP-mediated regulation, a proximal fusion at position +76 was constructed. Remarkably, the Δcrp mutation had no effect on this proximal fusion (Fig 4A). This indicates that either CRP-cAMP does not regulate hilD expression at the level of transcription initiation or that the HilD protein is required for the induction of transcription initiation in a crp-deficient strain. To discriminate between these two possibilities, a hilD-lacZ fusion at position +965 was constructed, carrying the whole hilD coding sequence but lacking the hilD 3’UTR. As shown in Fig 4A, the Δcrp mutation did not lead to a significant induction even when the full coding sequence was included (hilD965-lacZ), as compared to a 6-fold induction detected using the hilD1235-lacZ fusion which includes both the hilD coding sequence and its 3’UTR. Although we cannot fully rule out a potential effect of CRP on hilD transcription, the different behavior of the hilD965-lacZ and hilD1235-lacZ reporters clearly points towards the 3’UTR being crucial for CRP-mediated post-transcriptional regulation of hilD. The relevance of the 3’UTR in CRP-mediated regulation of HilD expression was supported by i) a Δcrp-dependent increase in the levels of HilD-3xFLAG protein was only detected when the hilD-3xFLAG mRNA contained the 3’UTR (Fig 4B) and ii) similarly, the Δcrp-dependent increase in SipA levels was only detected in strains carrying a hilD allele with the 3’UTR (Fig 4C). Additionally, the transcriptional expression of the SPI-1 gene sipC can be monitored as a proxy for HilD activity in the cell, since sipC upregulation in a crp mutant strain requires the presence of HilD (S4 Fig). Consistently, sipC-lacZ was upregulated in a Δcrp mutant background only when the hilD allele carried its native 3’UTR (Fig 4D). Our data also demonstrate that, according to the role attributed to the 3’UTR in the expression of hilD mRNA [11], there was an increase in the levels of HilD-3xFLAG, SipA-3xFLAG and sipC-lacZ when the 3’UTR was removed as compared to the parental strains carrying the native hilD mRNA containing the 3’UTR. Based on the fact that CRP-cAMP is a transcriptional factor, it is surprising that the CRP-mediated regulation of HilD occurs at the post-transcriptional (and not the transcriptional) level, requiring the hilD 3’UTR. In other words, the data shown suggest that CRP-cAMP modulates hilD expression by an indirect mechanism. In line with previous reports [11,33], we found that the Δcrp-dependent activation of hilD expression, as judged by the hilD1235-lacZ fusion (containing the 3’UTR), was impaired in the absence of the sRNA chaperone Hfq (Fig 5A). Similarly, the drastic increase (16-fold) in sipC expression caused by the deletion of crp was abolished in the absence of Hfq (Fig 5B). We thus hypothesized that Hfq-dependent sRNA may be involved in the CRP-mediated regulation of hilD. In search for candidate sRNAs in Salmonella, we focused on Spot 42 (encoded by the spf gene) which is transcriptionally controlled by CRP-cAMP in the closely related species, E. coli [34]. Work by the Storz and Valentin-Hansen laboratories had established this sRNA to be a general repressor of sugar-related mRNAs during CRP-mediated catabolite repression [23]. In Salmonella, Spot 42 is highly abundant, with maximal expression in mid-logarithmic phase and reduced upon entry into stationary phase [35]. We tested by Northern blot whether Spot 42 is under CRP-cAMP control also in Salmonella (Fig 5C). In the mid-logarithmic growth phase a 12-fold upregulation of Spot 42 sRNA was detected in the CRP-deficient compared to the wild-type strain. This pattern was further validated using a chromosomal spf-lacZ fusion (S5 Fig). Additionally, spf expression was assessed at early stationary phase. Interestingly, the spf induction detected in the Δcrp derivative strain in mid-logarithmic phase was no longer observed in early stationary phase (S5 Fig), reflecting the divergent effects observed for CRP on SPI-1 expression in exponential versus early stationary phase. To establish whether Spot 42 is involved in the CRP-cAMP-mediated regulation of hilD, expression studies in strains either deficient in Spot 42 or over-expressing the sRNA were performed. In the absence of CRP, a partial but significant drop in the upregulation of hilD in the Spot 42-deficient background (Δspf) was detected (Fig 5D). In contrast, ectopically expressing Spot 42 stimulated hilD expression. This suggests that Spot 42 is indeed involved in CRP-mediated repression of hilD. Importantly, over-expression of Spot 42 caused a 3-fold increase in hilD expression only when the 3’UTR was present, which implicates the hilD 3’UTR as a previously unknown target of this sRNA (Fig 5E). Consistently, a 2. 5-fold increase in HilD-3xFLAG levels was detected upon the over-expression of Spot 42 (S6 Fig). In agreement with previous data (Fig 5A, [29,35,36]), the positive effect of Spot 42 on hilD requires the chaperone Hfq (Fig 5E). As it has been shown before [29], Hfq binds to both Spot 42 and the hilD 3’UTR (S7 Fig). In addition, the major endoribonuclease RNase E has been suggested to play a role in 3’UTR mediated silencing of hilD expression [11]. Accordingly, hilD induction upon over-expression of Spot 42 was partially lost in the rne537 background encoding an RNase E with a truncated C-terminal domain that is defective in degradosome assembly [37] (Fig 5E). These results suggest that both Hfq and RNase E are involved in the Spot 42-mediated effect on hilD expression. The involvement of Spot 42 in the control of SPI-1 gene expression was further assessed by examining the transcriptional activity of a sipC-lacZ reporter. Transcription was monitored in either strains carrying the native hilD (+UTR) or strains from which the hilD 3’UTR had been removed (-UTR). As shown in Fig 5F, there was a 5-fold induction of sipC-lacZ upon over-expression of Spot 42 in the +UTR background, whereas sipC transcription was unaffected when Spot 42 was over-expressed in a background lacking the hilD 3’UTR (-UTR). To confirm that the hilD 3’UTR is targeted by Spot 42, the hilD 3’UTR was cloned downstream of the gfp coding sequence expressed from a constitutive promoter. Expression of this genetic reporter was monitored in either the presence or absence of Spot 42. Co-expression of the sRNA led to a nearly two-fold increase in fluorescence, suggesting that Spot 42 targets the hilD 3’UTR regardless of the genomic location of the latter (S8 Fig). Overall, these results led us to conclude that Spot 42 sRNA activates hilD expression (either directly or indirectly) in a manner that requires the presence of the hilD 3’UTR. Spot 42 from E. coli and Salmonella share 98% sequence identity. Three unstructured regions (denoted I, II and III, Fig 6A) of Spot 42 have been identified in E. coli to participate in gene regulation through base-pairing interactions [24]. To dissect the mechanism of action of Spot 42 on hilD expression, we determined if specific regions within the sRNA were essential for regulation. The software IntaRNA [38], developed to search for putative interaction sites between two given RNA molecules, predicted an interaction between unstructured region III of Spot 42 and positions 1,129–1,138 of the hilD mRNA (i. e. a region within the 3’UTR). To test whether this unstructured region III of Spot 42 is required for the regulation of SPI-1 genes, two Spot 42 mutant variants were generated, spf-mut1 and spf-mut2 (Fig 6B). Over-expression of these Spot 42 derivatives was performed in strains carrying a deletion of the endogenous spf gene, and their effect on hilD expression was monitored by determination of i) hilD1235-lacZ expression, ii) hilD mRNA levels by qRT-PCR, and iii) sipC-lacZ expression as a readout for HilD activity. In accordance with our previous results, over-expression of wild-type Spot 42 (Spot 42WT) upregulated hilD1235-lacZ and sipC-lacZ expression. Likewise, relative hilD mRNA levels were elevated upon Spot 42WT over-expression (Fig 6C). Conversely, over-expression of neither Spot 42mut1 (spf-mut1) nor Spot 42mut2 (spf-mut2) induced hilD1235-lacZ expression, hilD mRNA levels or sipC expression, demonstrating that mutations in region III disrupt the stimulatory effect of Spot 42 on hilD expression. (Fig 6C). The substitutions introduced to generate spf-mut1 (GUA-CAU) and spf-mut2 (GGA-CAC) have previously been described in E. coli, where those substitutions were shown to retain Spot 42 steady-state levels [37,38]. Similarly, Northern blots showed that these mutations did not dramatically affect Spot 42 stability in Salmonella either (Fig 6D), arguing that the reduced capability of the Spot 42 mutant variants to induce SPI-1 was not due to lowered sRNA levels. Taken together, the results indicate that the unstructured region III of Spot 42 is required for the regulation of hilD expression. The in silico prediction suggests that region III of Spot 42 interacts within the hilD 3’UTR, between positions 1,129 and 1,138 of the hilD mRNA. Accordingly, we generated two chromosomal compensatory mutations in the hilD 3’UTR that restore the base pairing of Spot 42mut1 or Spot 42mut2 with the putative target sequence within hilD. The mutant alleles were designated hilD 3’UTRmut1 and hilD 3’UTRmut2, respectively (Fig 6B). sipC expression was used as a readout for HilD activity. Over-expression of Spot 42WT in both hilD 3’UTRmut1 and hilD 3’UTRmut2 genetic backgrounds induced expression of sipC-lacZ, indicating that substitution of those residues within the hilD 3’UTR did not impair the positive effect of Spot 42 on SPI-1 expression. Additionally, over-expression of either Spot 42mut1 or Spot 42mut2 in both hilD 3’UTRmut1 and hilD 3’UTRmut2 backgrounds did not reestablish the ability to induce sipC expression (S9 Fig). Despite unstructured region III of Spot 42 being responsible for hilD activation, these results suggest that the in silico predicted interaction site—positions 1,129–1,138 within hilD mRNA—is not the actual target site or, at least, not the unique interaction site with Spot 42. More complex interaction mechanisms cannot be ruled out such as multiple interactions sites of Spot 42 within the hilD 3’UTR. Biochemical approaches were used to confirm the physical interaction between Spot 42 and the hilD 3’UTR. The ability of Spot 42 to bind to the hilD 3’UTR-derived fragments was assessed by electrophoretic mobility shift assays (EMSAs). EMSAs of radiolabeled full-length hilD 3’UTR incubated with increasing concentrations of Spot 42 confirmed a direct interaction between the two RNA species (Fig 7A). Consistent with our in vivo data, the unstructured region III of Spot 42 is required for the interaction with the hilD 3’UTR, since the binding affinity of the mutant version of Spot 42 (spf-mut2) was markedly reduced. Next, the hilD 3’UTR was divided into two halves, UTRL and UTRR (Fig 7B). The UTRL fragment spans positions +927 to +1114 of the hilD mRNA (roughly the first half of the hilD 3’UTR), while UTRR covers the second half of it (position +1090 to +1275) and includes the putative interaction site with the unstructured region III of Spot 42 (Fig 6B) as well as two Hfq binding sites as inferred from CLIP-seq [29]. EMSAs with radiolabelled Spot 42 and increasing concentrations of either one of the two UTR fragments were conducted. A concentration dependent upshift of Spot 42 was only observed upon addition of the UTRR fragment with an apparent Kd of 80 nM but not upon addition of the UTRL fragment (Fig 7C), indicating that Spot 42 interacts with the second half of hilD 3’UTR. Again, Spot 42-UTRR interaction was specific as the affinity of the UTRR fragment to the mutant version of Spot 42 (spf-mut2) was markedly reduced (Fig 7D). Further supporting this notion, in the reverse experiment increasing concentrations of Spot 42 did not lead to a band shift of the UTRL but only of the UTRR fragment (S10 Fig). Our results indicate that the loss of hilD activation by Spot 42mut2 in vivo is due to the inability of this mutant sRNA version to directly interact with the hilD mRNA, specifically with the second half of its 3’UTR. Overall, this makes Spot 42 the first Hfq-associated sRNA that potentially activates a trans-encoded target gene via its 3’UTR.
  During the infection process, Salmonella relies on the expression of genes encoded on SPI-1 for epithelial cell invasion. Although SPI-1 is therefore crucial for Salmonella infection, it has a retarding effect on the growth rate, presumably as a consequence of the energetically high costs to produce the SPI-1 T3SS [3]. Accordingly, the expression of SPI-1 genes is tightly regulated [1]. Most relevant studies have focused on the regulatory pathways dedicated to induce SPI-1 under permissive conditions. However, as SPI-1 expression affects cell fitness, SPI-1 silencing mechanisms under non-permissive conditions, for instance in fast growing cells in the mid-logarithmic phase, are equally important. In this study, we identified CRP-cAMP, a metabolic sensor and global transcription factor [14,39], as a key player in the repression of SPI-1. CRP-cAMP is involved in a post-transcriptional regulatory circuit, controlling the expression of hilD by a mechanism dependent on its 3’UTR. Coordination of metabolism and stress-related functions is crucial for the evolutionary success of bacterial populations. In pathogenic bacteria, the cross regulation between virulence factors, which can be considered within-host stress-related factors, and physiology is crucial for efficient colonization. A sudden shift between the expression of genes involved in active growth and genes involved in adaptation to stress might be required for rapid adaptation to changing conditions during the infection process. Secondary messengers such as cAMP, the intracellular levels of which can be altered by the action of both synthetases (adenylate cyclases) and degrading enzymes (phosphodiesterases), provide a rapid response system that can promote rapid changes in the expression profile. Although cAMP has traditionally been described as a regulator of metabolism, its role in the modulation of virulence-related functions has been extensively studied in several pathogens [40]. In E. coli, CRP-cAMP has been described to repress type 1 fimbriae expression during logarithmic growth [41]. Other secondary messengers, such as ppGpp, have also been reported to participate in the interplay between cell metabolism and virulence control [42]. Post-transcriptional regulation by small non-coding RNA confers to the cell another level for a rapid response to environmental conditions, in fact, a number of sRNAs have been found to play a relevant role in the metabolism-virulence crosstalk [43,44]. In this study we found that CRP-cAMP represses SPI-1 expression by modulating the expression of the regulator HilD (Figs 1–3). The role of HilD is not restricted to SPI-1; rather there is a complex cross-talk between HilD and master regulators of other virulence associated pathways. For example, it has been shown that HilD activates, under certain conditions, SPI-2 expression that is required for survival within macrophages [6,45]. Within macrophage-like cells, SPI-1 genes are downregulated and SPI-2 genes are induced [46]. CRP-cAMP is a regulator tailored to mediate rapid responses to environmental changes and may therefore be relevant for HilD-mediated regulation of virulence in response to the environmental conditions that Salmonella encounters through the infection process. The CRP-mediated regulation of hilD does not occur at the transcriptional initiation level (Fig 4). Rather CRP-cAMP modulates hilD expression at the post-transcriptional level through the long 3’UTR (310 nt) of hilD. Post-transcriptional regulation is an extensively used mechanism to finely regulate virulence in bacterial pathogens [47,48]. The role of 5’UTRs in post-transcriptional gene expression control has been established and it is noteworthy that mRNAs of important SPI-1 regulators, such as invF, hilA and hilE, all carry long 5’UTRs [43,49–51]. In contrast, the involvement of 3’UTRs in post-transcriptional regulation is still poorly understood. Recently, it has been reported in Staphylococcus aureus that one-third of the cellular transcripts carry 3’UTRs longer than 100 nt [52]. In addition to be targets of regulation, 3’UTRs may provide regulators themselves, namely 3’UTR-derived sRNAs [36,53]. The 3’UTR of hilD constitutes a silencing module, since its deletion causes significant hilD upregulation. Although we are yet to elucidate the full molecular mechanism, the observed Hfq dependency suggested that one or several sRNAs are targeting the hilD 3’UTR [11]. Likewise, the CRP-mediated repression of hilD requires both the presence of the hilD 3’UTR and Hfq, indicating that CRP regulates hilD expression in an sRNA-mediated manner (Fig 4 and Fig 5). Spot 42 is an integral member of the CRP-mediated gene expression network in E. coli [23–27] and its expression is repressed by CRP-cAMP in both E. coli and Salmonella ([47], Fig 5). Here we found that Spot 42 is involved in the CRP-mediated regulation of hilD expression, since the derepression of hilD in a Δcrp strain was diminished in the absence of Spot 42 and over-expressing Spot 42 caused a concomitant upregulation of hilD expression. Remarkably, Spot 42-mediated regulation targets the long hilD 3’UTR. The fact that the absence of Spot 42 did not completely abolish the hilD deregulation caused by the Δcrp mutation points at additional factors that could be involved in the described regulation. Although the nature of these factors remains fully elusive, it should be noted that these putative factors seem to also act through the hilD 3’UTR. Further studies will be required to determine whether other sRNAs or proteins plays a role in the CRP-mediated repression of hilD expression. Both genetic and biochemical approaches point towards a direct interaction between Spot 42 and the hilD 3’UTR, involving the unstructured region III of Spot 42 (Figs 6 and 7). Although the exact target sequence within the hilD 3’UTR have not been identified, EMSA experiments indicate that the interaction occurs between Spot 42 and the downstream half of the 3’UTR (last 185 nt). This interaction is strongly diminished when the unstructured region III of Spot 42 is altered by base substitution in three positions previously described to be involved in base-pairing [23]. The recent finding that the transcription elongation factors GreA and GreB target the hilD 3’UTR to regulate hilD at permissive conditions [54] led us to speculate that transcriptional pausing might trigger a specific folding of the hilD 3’UTR important for post-transcriptional regulation. Overall, the regulation through the hilD 3’UTR seem to be complex and presumably several factors target the hilD 3’UTR. Although it has been proposed that intrinsic motifs in the long 3’UTR of hilD might confer susceptibility to degradation in a polynucleotide phosphorylase (PNP) and RNase E dependent manner, no effect in the stability of the hilD mRNA was detected [11]. Consistent with these data, we found no difference in the stability of the hilD mRNA between the wild-type and Δcrp strain in mid-logarithmic phase (S11 Fig). The exact mechanism by which these factors converge to regulate hilD expression should be the focus of future studies. Our results highlight the 3’UTR of hilD as a central hub in SPI-1 regulation and indicate that the whole hilD 3’UTR is required for the post-transcriptional regulation of hilD. To our knowledge, there are no other examples of trans-encoded sRNAs targeting 3’UTRs. Of note, the cis-encoded sRNA GadY, which is encoded on the opposite strand of gadX 3’UTR, seems to positively regulate gadX through interaction with the gadX-gadW intergenic region [55]. Unlike Spot 42 and hilD which are expressed from regions in the chromosome over 1 Mb apart, GadY and gadX physically overlap. Global screens for Hfq-mediated sRNA-mRNA interactions [56,57] suggest, however, that 3’UTR targeting may be more common than currently appreciated. In conclusion, our findings imply a novel mechanism in the complex regulatory network of SPI-1 expression. Under non-permissive conditions, very low transcriptional expression from the hilD gene occurs. Additionally, CRP-cAMP represses the transcription of the sRNA Spot 42, thereby maintaining basal levels of hilD expression. Consequently, despite hilD transcription occurs, only low levels of HilD protein arise (Fig 8 panel I). In contrast, environmental and/or physiological signals may relieve CRP-dependent Spot 42 repression. Upon binding to its 3’UTR in an Hfq-dependent manner, Spot 42 may exerts a positive effect on hilD mRNA, thereby activating HilD protein expression. As HilD auto-activates itself by promoting its own transcription, expression of some copies of HilD protein would likely be sufficient to amplify the final output (Fig 8 panel II). Thus, CRP-cAMP seems to play a relevant role by coordinating post-transcriptional virulence control in Salmonella. Somewhat similar to the described Spot 42-mediated regulation of SPI-1, the sRNA PinT acts as a timer of virulence gene expression in Salmonella, regulating SPI-2 genes through the modulation of CRP-cAMP [58]. Altogether, this highlights the emerging importance of collaborative activities of general transcription factors and sRNAs to precisely adjust the costly expression of major virulence factors to internal and external metabolic cues [44].
  The bacterial strains and plasmids used in this study are listed in S1 Table. Salmonella enterica serovar Typhimurium SL1344 and derivative strains were cultivated either in Luria Bertani broth (tryptone 10 g/l, yeast extract 5 g/l and sodium chloride 10 g/l). When required, the media was supplemented with ampicillin (Amp) 100 μg/ml, kanamycin (Km) 50 μg/ml, chloramphenicol (Cm) 15 μg/ml, or tetracycline (Tc) 15 μg/ml. Induction of genes cloned into pTRc99a was achieved by adding 0. 1 mM IPTG. Liquid cultures (20 ml of LB in 100 ml culture flasks) were inoculated to an OD600nm of 0. 001 and incubated at 37°C with vigorous shaking (200 rpm). An OD600nm of 0. 3–0. 4 was considered mid-logarithmic phase of growth, while an OD600nm of 2. 0 was considered early stationary phase of growth. The cpdA (SL1344_3157) gene was cloned into the IPTG inducible vector pTRc99a [59]. The cpdA coding sequence was PCR amplified by Phusion polymerase (Invitrogen) with the primers cpdA_XbaI_Fw and cpdA_SalI6xHis_rev (S2 Table), subsequently digested with XbaI and SalI and ligated into XbaI/SalI digested pTRc99a. The spf gene encoding Spot 42 sRNA was cloned into pBRplac vector [60]. spf was PCR amplified with the primers spf_AatII_Fw and spf_EcoRI_rev (S2 Table), subsequently digested with AatII and EcoRI and ligated into AatII/EcoRI digested pBRplac. Mutations in Spot 42 (spf-mut1 and spf-mut2) were generated by assembly PCR and subsequent cloning in the pBRplac vector. A gfp-hilD 3’ UTR construct was cloned in the backbone of plasmid pXG1 [61]. The hilD 3’UTR region was fused to gfp by overlapping PCR using chromosomal SV5015 and plasmid pXG1 as templates and the primers gfp_NheI_Fw, gfp_hilD_rev, hilD_gfp_Fw and hilD_XbaI_rev (S2 Table). The purified PCR fragment was subsequently digested with XbaI/NheI and ligated into an XbaI/NheI digested pXG1 vector, resulting in the plasmid pXG1 gfp-3’UTR. Deletion mutants were generated by gene replacement as described by Datsenko and Wanner [62]. Briefly, antibiotic resistance cassettes carrying either KmR or CmR resistance genes were amplified from pKD4 and pKD3, respectively. Primers used include a 40 bp sequence complementary to the region where the insertion was desired. Purified fragments were electroporated into strains carrying pKD46. Positive clones were selected in presence of the required antibiotic. When desired, the antibiotic resistance cassette was removed by expression of the Flp recombinase from the pCP20 plasmid, as described [63]. Deletion mutants, where the antibiotic cassette was removed, were further used for the generation of reporter gene fusions. Transcriptional lacZ fusions were generated as described [64], the remaining FRT-site was used to integrate plasmid pKG136. Epitope tagged proteins were constructed as follows: HilA, HilD and SipA 3xFLAG tagged proteins were generated by a λRed recombinase system as described [65]. When desired, the KmR cassette downstream of the 3xFLAG epitope was removed using the Flp recombinase (see above). In the HilD 3xFLAG construct retaining the KmR cassette, the hilD coding sequence and 3’UTR are split and not co-transcribed. Thus, the KmR cassette was removed, with the hilD 3’UTR now located right downstream of the 3xFLAG epitope. Oligonucleotides used to generate the constructs are listed in S2 Table. All strains were PCR confirmed and integrity of the sequence was checked by DNA sequencing. Chromosomal modifications in the hilD 3’UTR region were generated by scarless mutation [66]. Briefly, a 1 kb fragment containing the hilD 3’UTR sequence was cloned into pGEM vector, desired point mutations were generated via the Quick change method using the hilD-UTR oligonucleotides listed in S2 Table. Subsequently, the vector was digested with SacI/XbaI and ligated into the suicide plasmid pDMS197 [67]. The derivative pDMS197 was propagated in S17-1 lambda pir and used as donor in matings with SV5015 Δspf sipC-lacZ. Trans-conjugants were selected for tetracycline resistance. Selected clones were grown in salt-free nutrient broth supplemented with 5% sucrose. Individual tetracycline-sensitive clones were checked by PCR and subsequent DNA sequencing to select the clones carrying the desired chromosomal mutation. To obtain whole cell and secreted protein extracts, LB cultures were grown at 37°C and processed as previously described [54]. Samples in Laemmli sample buffer were subjected to SDS-PAGE separation. Normalization of the loading samples was performed based on the culture biomass (OD600nm). Coomassie stain was used to visualize protein bands. Protein extracts were subjected to SDS-PAGE separation, transfer to PVDF filter and subsequent immunodetection using monoclonal Anti-FLAG (Sigma), Anti-His (Sigma) or polyclonal Anti-SopE [68] as primary antibodies. Commercial polyclonal anti-mouse (Promega) and anti-rabbit (GE Healthcare) secondary antibodies conjugated to horseradish peroxidase were used. For detection, ECL Prime Western Blotting Detection Reagent (GE Healthcare) served as a substrate. Chemi-luminescence was detected using Chemidoc equipment (Bio-Rad). As a control prior to the immunodetection, all whole cell extract samples were analyzed by SDS-PAGE and Coomassie staining to ensure proper normalization of the loaded amounts. For protein identification, the protein bands from Coomassie stained SDS-PAGE gels were trypsin digested and analyzed by LC-MS/MS by the Proteomic facility from the Scientific Park of Barcelona (PCB). β-galactosidase activity was measured as described previously [69]. Activity determination was performed in technical duplicates for each of three biological replicates. For each strain, samples from three independent LB cultures grown at 37°C to mid-logarithmic phase (OD600nm 0. 4) were processed. RNA was extracted by classical hot phenol method. RNA quality and concentration was assessed by an Agilent Technologies Bioanalyzer 2100. Quantitative reverse transcription-PCR (qRT-PCR) was performed as previously described [54]. The relative amount of target cDNA was normalized using the gapA (GAPDH) gene as an internal control. Oligonucleotides used for qRT-PCR are listed in S2 Table. Electrophoretic separation of total RNA samples was carried out in Tris-Borate-EDTA (TBE) 8% acrylamide gels containing 8. 3 M urea. Samples were prepared by mixing 10 μl of RNA samples with 10 μl of urea dye (2x) loading buffer and incubated for 10 minutes at 65°C, immediately chilled on ice and loaded for electrophoretic separation at 30 mA for 2 hours. RNAs were transferred to Hybond N+ (GE Healthcare) filters by semi-dry TBE based transfer for 2 hours at 400 mA. RNAs were subsequently fixed to the filter by UV crosslinking. Filters were then hybridized with radiolabeled oligos, sequences are given in S2 Table. Images of radioactive filters were obtained with the FLA-5100 imaging system (Fujifilm) and quantification was performed using Image J software. RNA-RNA interactions were detected by Electrophoretic Mobility Shift Assay (EMSA) as described in [70]. First, DNA templates for in vitro T7 RNA transcription were generated by PCR, primers used are listed in S2 Table. RNA was produced in vitro by following the Megascript transcription procedure from Ambion. Then, either the sRNA (Spot 42) or the target RNAs (hilD 3’UTR, UTRR or UTRL) was dephosphorylated and 5’ labeled with [ (α-32P) ATP]. The putatively interacting RNAs were next incubated in structure buffer (Ambion): In a 10 μl final volume, 4 nM of the radiolabeled RNA was incubated with increasing concentrations of the unlabeled RNA (0,56,280,560,1700nM). Samples were incubated at 37°C for 1 hour and subjected to electrophoresis in a native 6% acrylamide gel. For specific RNA detection, acrylamide gels were dried and exposed. Images were obtained as for Northern blots. For single-cell analysis, cell cultures were grown to the desired conditions, pelleted and resuspended in PBS. The bacterial suspensions were then fixed in 4% formaldehyde. The fluorescence of 20,000 bacterial cells was measured by flow cytometry using preset parameters for GFP (excitation wavelength of 484 nm and emission wavelength of 512 nm). Measurements were performed in technical duplicates for each three biological replicates; average was used to compare GFP expression. HeLa human epithelial cells (ATCC CCL2) were cultured in tissue culture medium (Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal calf serum and 1mM glutamine). HeLa cells were seeded the day before the infection in 24-well plates containing 0. 5 ml of DMEM per well and grown at 370°C, 5% CO2. Bacterial cells grown at 37°C to different phases of growth were prepared in DMEM. The bacterial mixture was added to HeLa cells to reach a multiplicity of infection (MOI) of 75 bacteria per eukaryotic cell. 30 minutes post-infection HeLa cells were washed twice with phosphate buffered saline (PBS) and incubated in fresh DMEM medium containing 100 μg/ml gentamicin for 90 minutes. Numbers of viable intracellular bacteria were obtained after lysis of infected cells with 1% Triton X-100, and subsequent plating. Infections were carried out in triplicate. Invasion rate is defined as the intracellular bacteria recovered versus viable bacteria used to infect the HeLa cells (initial inoculum). Invasion rates were normalized to bacterial culture of a wild type strain. GraphPad Prism 5. 0 software was used for data analysis. Two-tailed Student’s t-test were carried out and p-values < 0. 05 were considered significant.
summary: Salmonella infection is one of the major causes of foodborne illness worldwide. During infection, Salmonella expresses a set of virulence genes encoded in discrete regions of the genome. The expression of these genes is tightly regulated, being specific for different stages of the Salmonella infection process. While many regulatory mechanisms that lead to the activation of infection-related gene expression have been described, little is known about silencing mechanisms under conditions when expression is not needed and may rather represent a burden than a benefit for the bacterial fitness. Here, we report a condition-specific silencing mechanism of bacterial virulence. That is, the global transcriptional regulator CRP-cAMP represses, indirectly through a post-transcriptional mechanism, the expression of the major Salmonella virulence regulator HilD. In bacteria, post-transcriptional regulation has so far been mainly focused on 5’ untranslated regions (5’UTR). Remarkably, here we describe a molecular mechanism targeting the 3’untranslated region (3’UTR) of the mRNA of the major regulator of Salmonella virulence by a small non-coding RNA under the transcriptional control of the global regulator CRP-cAMP. Our data highlight the importance of 3’UTR in the regulation of gene expression in bacteria.
keywords: medicine and health sciences
  pathology and laboratory medicine
  molecular probe techniques
  gene regulation
  pathogens
  messenger rna
  rna extraction
  microbiology
  bacterial diseases
  northern blot
  regulator genes
  post-transcriptional gene regulation
  enterobacteriaceae
  gene types
  molecular biology techniques
  gel electrophoresis
  bacteria
  extraction techniques
  bacterial pathogens
  research and analysis methods
  infectious diseases
  electrophoretic techniques
  transcriptional control
  electrophoretic blotting
  medical microbiology
  gene expression
  microbial pathogens
  molecular biology
  salmonella
  biochemistry
  rna
  nucleic acids
  genetics
  biology and life sciences
  organisms
title: CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level