Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium
Article first published online: 30 JUN 2005
Volume 57, Issue 3, pages 691–705, August 2005
How to Cite
Ellermeier, C. D., Ellermeier, J. R. and Slauch, J. M. (2005), HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium. Molecular Microbiology, 57: 691–705. doi: 10.1111/j.1365-2958.2005.04737.x
- Issue published online: 30 JUN 2005
- Article first published online: 30 JUN 2005
- Accepted 19 May, 2005.
Salmonella enterica serovar Typhimurium invades intestinal epithelial cells using a type three secretion system (TTSS) encoded on Salmonella Pathogenicity Island 1 (SPI1). The SPI1 TTSS injects effector proteins into the cytosol of host cells where they promote actin rearrangement and engulfment of the bacteria. We previously identified RtsA, an AraC-like protein similar to the known HilC and HilD regulatory proteins. Like HilC and HilD, RtsA activates expression of SPI1 genes by binding upstream of the master regulatory gene hilA to induce its expression. HilA activates the SPI1 TTSS structural genes. Here we present evidence that hilA expression, and hence the SPI1 TTSS, is controlled by a feedforward regulatory loop. We demonstrate that HilC, HilD and RtsA are each capable of independently inducing expression of the hilC, hilD and rtsA genes, and that each can independently activate hilA. Using competition assays in vivo, we show that each of the hilA regulators contribute to SPI1 induction in the intestine. Of the three, HilD has a predominant role, but apparently does not act alone either in vivo or in vitro to sufficiently activate SPI1. The two-component regulatory systems, SirA/BarA and OmpR/EnvZ, function through HilD, thus inducing hilC, rtsA and hilA. However, the two-component systems are not responsible for environmental regulation of SPI1. Rather, we show that ‘SPI1 inducing conditions’ cause independent activation of the rtsA, hilC and hilD genes in the absence of known regulators. Our model of SPI1 regulation provides a framework for future studies aimed at understanding this complicated regulatory network.
Salmonella serovars cause a variety of diseases ranging from mild gastroenteritis to life threatening systemic infections. During the course of infection, Salmonella enterica serovar Typhimurium invades intestinal epithelial cells using a type three secretion system (TTSS) encoded by Salmonella Pathogenicity Island 1 (SPI1). The SPI1 TTSS forms a needle-like structure that injects effector proteins directly into the cytosol of host cells (Kimbrough and Miller, 2000, 2002; Sukhan et al., 2001). A number of functions have been attributed to the SPI1 TTSS and its effector proteins including: actin rearrangement that promotes invasion (Zhou and Galan, 2001), Salmonella-induced necrosis of macrophages (Monack et al., 1996, 2001; Hersh et al., 1999; Brennan and Cookson, 2000), enteropathogenesis (Wallis and Galyov, 2000), and secretion of a pathogen-elicited epithelial chemoattractant (PEEC) that promotes transepithelial migration of PMN's (McCormick et al., 1998; Lee et al., 2000).
Expression of the SPI1 TTSS is controlled in response to a specific combination of environmental signals that presumably act as cues that the bacteria are in the appropriate anatomic location (Bajaj et al., 1996; Schechter et al., 1999). HilA, a member of the OmpR/ToxR family of transcriptional regulators, is encoded on SPI1 and directly activates the TTSS structural genes located on SPI1 (Lee et al., 1992; Bajaj et al., 1995; Ahmer et al., 1999; Lostroh and Lee, 2001). HilA activates the SPI1-encoded prg/org and inv/spa operons by binding just upstream of the −35 sequences of PprgH and PinvF (Lostroh and Lee, 2001). The inv/spa transcript reads through the sic/sip operon (Darwin and Miller, 1999; Eichelberg and Galan, 1999). Activation of PinvF leads to production of InvF, a member of the AraC family of transcriptional regulators (Kaniga et al., 1994). InvF, in an apparent complex with the chaperone protein SicA, then induces expression of genes both within SPI1 (the sic/sip operon encoding the translocon and chaperones) and encoded on various bacteriophage and islands elsewhere in the chromosome (e.g. sopE on SopEφ and sopB on SPI5; Darwin and Miller, 2000, 2001).
Production of HilA serves as an integration point for a variety of environmental signals and regulatory elements that impinge on SPI1 induction (Lostroh and Lee, 2001; Lucas and Lee, 2001). In the laboratory, expression of hilA is activated in ‘SPI1 inducing conditions’ (high osmolarity and low oxygen), which are thought to mimic the conditions found in the small intestine (Lee et al., 1992; Bajaj et al., 1996). Three homologous proteins, RtsA, HilC and HilD, which belong to the AraC/XylS family of transcriptional regulators, bind to the DNA immediately upstream of hilA leading to induction of hilA expression (Schechter and Lee, 2001; Olekhnovich and Kadner, 2002; Ellermeier and Slauch, 2003). HilC and HilD are encoded within SPI1, whereas RtsA is encoded in an operon at 93.9 centisomes that also encodes RtsB, which negatively regulates flhDC and hence the flagellar regulon (Ellermeier and Slauch, 2003). Null mutations in hilD decrease expression of hilA∼10-fold under SPI1 inducing conditions, whereas hilC or rtsA mutations decrease expression of hilA approximately twofold (Eichelberg et al., 1999; Schechter et al., 1999; Schechter and Lee, 2001; Ellermeier and Slauch, 2003). These proteins also act independently of HilA to activate expression of the invF operon, albeit at significantly lower levels than HilA-dependent activation (Eichelberg et al., 1999; Rakeman et al., 1999; Akbar et al., 2003; Ellermeier and Slauch, 2003). The three regulators, RtsA in particular, also activate, independent of HilA and InvF, the effector gene slrP (Ellermeier and Slauch, 2003) and dsbA, encoding the periplasmic disulphide bond isomerase required for the function of SPI1 and other TTSSs (Ellermeier and Slauch, 2004).
In addition to these direct regulators, genetic studies have identified a plethora of regulatory proteins encoded outside of SPI1 that contribute to regulation of hilA. These include several two-component regulatory systems. PhoPQ, a significant regulator of virulence and physiology (Groisman, 2001), represses expression of the SPI1 TTSS (Behlau and Miller, 1993; Pegues et al., 1995) in response to low magnesium concentrations and low pH (Chamnongpol et al., 2003). This presumably ensures that SPI1 is inactive during systemic stages of infection. The PhoBR two-component regulatory system also represses expression of hilA in response to its normal signal, low extracellular Pi (Lucas et al., 2000; Lucas and Lee, 2001). Mutations that disrupt the osmoregulatory two-component system, OmpR/EnvZ reduce expression of hilA but do not apparently alter the osmoregulation of hilA (Lucas and Lee, 2001).
SirA/BarA is a two component regulatory system that is required for maximal expression of hilA; sirA mutations decrease expression of hilA approximately 10-fold (Johnston et al., 1996; Ahmer et al., 1999; Rakeman et al., 1999; Lucas and Lee, 2001). Mutations in sirA also decrease expression of hilD and hilC approximately 1.5- to twofold (Lucas and Lee, 2001). Although it has been proposed that SirA directly controls expression of both hilA and hilC (Teplitski et al., 2003), it was also determined that HilC was not required for SirA to control hilA expression (Lucas and Lee, 2001). BarA/SirA have also been implicated in controlling SPI1 gene expression in response to bile (Prouty and Gunn, 2000).
In Escherichia coli, the SirA/BarA homologues, UvrY/BarA, are involved in a regulatory loop with csrA, csrB and csrC (Suzuki et al., 2002; Weilbacher et al., 2003), which function as both positive and negative regulators of gene expression. CsrA is a 61 amino acid protein that can directly bind mRNA transcripts affecting their stability (Romeo, 1998). The 360 nucleotide RNA csrB and the 245 nucleotide RNA csrC each antagonize the activity of CsrA by binding multiple CsrA molecules (Romeo, 1998; Weilbacher et al., 2003). The transcription of csrB and csrC is regulated by UvrY (SirA) (Suzuki et al., 2002). The csr system is clearly implicated in SPI1 regulation (Altier et al., 2000a,b; Lawhon et al., 2002). Indeed, overproduction of csrB suppresses the decrease in hilA expression conferred by loss of BarA (Altier et al., 2000b), suggesting that SirA/BarA function through the csr system to control SPI1 gene expression.
A null mutation in hilE increases expression of hilA under both SPI1 inducing and SPI1 repressing conditions (Fahlen et al., 2000; Baxter et al., 2003). Using two-hybrid analysis it was determined that HilE interacts with HilD suggesting that HilE represses expression of hilA by inhibiting the activity of HilD (Baxter et al., 2003). Transcription of the hilE gene is activated by FimZY, the regulators of Type I fimbriae in serovar Typhimurium (Baxter and Jones, 2005). It is not known if HilE also controls the activity of either RtsA or HilC. It has been reported that PhoPQ and PhoBR, in addition to FimZY, control hilA expression by acting through HilE (Baxter and Jones, 2003).
A number of additional regulatory genes impinge on SPI1 expression. Mutations in ams, hha and a previously unidentified PhoP-activated gene (pag) lead to increased hilA expression (Fahlen et al., 2000, 2001). Deletion of the sirB locus decreases hilC (and presumably hilA) expression (Rakeman et al., 1999). Several studies have identified the class 3 flagellar protein FliZ as an activator of hilA expression (Eichelberg and Galan, 2000; Lucas et al., 2000; Iyoda et al., 2001). It is also known that a number of mutations in the SPI2 TTS apparatus decrease expression of hilA (Deiwick et al., 1998). The nucleoid proteins HU and Fis are also required for hilA expression (Wilson et al., 2001; Schechter et al., 2003). In contrast, H-NS seems to be involved in repressing the hilA promoter (Schechter et al., 2003). Indeed, it has been proposed that the molecular role of HilD and HilC is to derepress hilA (Schechter et al., 1999, 2003; Schechter and Lee, 2001), likely by counteracting H-NS, although there is debate on this point (Boddicker et al., 2003; Olekhnovich and Kadner, 2004). Recent studies have shown that HilC and HilD are degraded by the Lon protease, resulting in decreased production of HilA, presumably allowing downregulation of the system after invasion of host cells (Takaya et al., 2002, 2005; Boddicker and Jones, 2004). The signal molecule ppGpp is also critical for SPI1 expression and invasion. Transcription of hilC, hilD and hilA was significantly reduced in a strain background incapable of producing ppGpp (Pizarro-Cerda and Tedin, 2004; Song et al., 2004). With the exceptions of Lon and some of the nucleoid proteins, the molecular mechanism by which these factors influence hilA expression and the physiological relevance of these effects remain unclear.
Although numerous models have been proposed to explain particular aspects of SPI1 regulation, the large number of regulators affecting HilA production has made it difficult to comprehend the system as a whole. Yet, if we are to understand this complex regulatory circuit, we need a model that can be tested and built on by various investigators. Based on our genetic analyses, and taking into account the published results outlined above, we present a model in which expression of hilA is controlled by the combined action of HilC, HilD and RtsA, each of which can independently bind upstream of hilA to induce its expression (Schechter and Lee, 2001; Olekhnovich and Kadner, 2002; Ellermeier and Slauch, 2003). We demonstrate that HilC, HilD and RtsA are each capable of inducing expression of hilC, hilD and rtsA. Thus these regulators form a self-reinforcing feed forward regulatory loop and increased expression or activity of one regulator leads to an increase in expression of the other regulators (Fig. 1). Of these three, HilD has a predominant role, but apparently cannot act alone either in vitro or in vivo to sufficiently activate SPI1. The two-component regulatory systems, SirA/BarA and OmpR/EnvZ function through HilD, thus inducing hilC, rtsA, hilA and invF. However, we show that the environmental signals used to induce SPI1 in vitro directly affect transcription of hilC, hilD and rtsA, independent of any known regulator. Future studies should be focused on determining where additional factors affecting SPI1 regulation fit into this regulatory circuit and how environmental regulation is mediated.
HilC, HilD and RtsA can independently induce expression of hilC, hilD and rtsA
We have shown that RtsA (Ellermeier and Slauch, 2003), like HilC and HilD (Schechter and Lee, 2001; Olekhnovich and Kadner, 2002), induces expression of hilA by binding to the HilA promoter region. It has been suggested that HilC and HilD may be capable of inducing expression of one another as well as inducing their own expression (Lucas and Lee, 2001; Olekhnovich and Kadner, 2002). We had previously demonstrated that RtsA increased expression of hilC and hilD (Ellermeier and Slauch, 2003). However, in that experiment, the other regulator, HilD or HilC, respectively, was still present (Ellermeier and Slauch, 2003). We wanted to determine if HilC, HilD and RtsA could induce expression of hilC, hilD, rtsA and hilA in the absence of the other regulators. To test this, we introduced plasmids pBAD (vector control), pRtsA, pHilD (pLS118) and pHilC (pLS119) into strains containing hilC–lac, hilD–lac, rtsA–lac, or hilA–lac fusions and in which the chromosomal hilC, hilD and rtsA genes were all deleted. The respective lac fusions are single copy chromosomal fusions constructed from deletion mutations of the regulator of interest (Ellermeier et al., 2002). The data in Fig. 2 demonstrate that RtsA, HilC and HilD are able to induce expression of hilA in the absence of the other regulators. Production of HilC induced expression of hilA∼120-fold, while RtsA and HilD induced expression of hilA 30- to 40-fold, similar to previously reported values (Eichelberg et al., 1999; Rakeman et al., 1999; Schechter et al., 1999; Schechter and Lee, 2001; Ellermeier and Slauch, 2003). It is not clear if the absolute levels of induction conferred by the various plasmids reflect real differences in the three regulators. It is equally likely that these differences are due to differential expression of the plasmid-borne genes. RtsA, HilC and HilD also induced expression of hilC, hilD and rtsA (Fig. 2). For example, RtsA and HilD induced expression of rtsA approximately four- to fivefold while HilC induced expression of this gene ∼30-fold. RtsA, HilC and HilD induced expression of hilC approximately three- to fourfold and hilD∼10- to 12-fold. These data show that HilC, HilD and RtsA are each capable of independently inducing expression of hilC, hilD and rtsA, consistent with our model that HilC, HilD and RtsA constitute a feed forward regulatory loop (Fig. 1).
To further characterize this regulatory loop we tested the effect of hilC, hilD and rtsA null mutations on expression of the three regulatory genes (Fig. 3). We observed that the loss of HilC and HilD decreased expression of rtsA; loss of HilD had the greater effect. Similarly, loss of RtsA and HilD significantly affected hilC transcription with the largest effect seen in the hilD mutant (Fig. 3). In contrast, loss of RtsA and HilC did not significantly alter hilD expression, even though RtsA and HilC can clearly activate hilD (see Discussion).
RtsA, HilC and HilD each contribute to activating hilA in vitro
The proposed model predicts that HilD, HilC and RtsA each have a role in activating hilA. To test this prediction, we introduced hilC, hilD and rtsA single, double and triple mutations into a hilA–lac fusion strain and monitored β-galactosidase activity in cells grown under SPI1 inducing conditions. The data in Fig. 3 show that loss of any one of the three regulators decreased hilA transcription; the hilD mutation had the greatest impact, followed in intensity by hilC, then rtsA. Double mutations further decreased hilA expression. Loss of both RtsA and HilC had an effect equivalent to loss of HilD. Indeed, deletion of hilD and either hilC or rtsA led to a level of expression essentially equivalent to loss of all three regulators.
RtsA, HilC and HilD each contribute to invasion in vivo
Previous published data suggested that a complete deletion of SPI1 had no effect on invasion of serovar Typhimurium in the host small intestine, yet an insertion in hilA (which is encoded on SPI1) did significantly decrease the ability of the bacterium to gain access to systemic sites of infection (Murray and Lee, 2000). These results were counterintuitive, especially considering that the SPI1 deletion used included a deletion of the sit operon, which encodes a manganese transporter known to be required for full virulence in oral infections (Janakiraman and Slauch, 2000; Kehres et al., 2002). Therefore, we wanted to explicitly test SPI1 and hilA deletions to determine the effect on virulence. We constructed two strains: one containing a deletion of SPI1 that leaves the sit operon intact and a second containing a deletion of hilA. We tested the ΔSPI1 strain and the ΔhilA strain against wild-type serovar Typhimurium using oral competition assays in which we recover bacteria from both the small intestine and the spleen. Decreased recovery of a strain from the spleen is presumed to reflect a defect in intestinal invasion, assuming that the mutations do not confer a virulence defect when the bacteria are growing systemically (see below). Strains carrying either a ΔSPI1 or a ΔhilA mutation were significantly attenuated in both the small intestines and spleens of orally infected animals (Table 1). We also performed direct competition assays between the ΔSPI1 strain and the ΔhilA strain. As HilA appears to be the integration point for all regulators of the SPI1 TTSS, we expected that these two strains should compete evenly. This is indeed what was seen in vivo (Table 1). To show that any difference in virulence is due to an effect on invasion rather than systemic growth, we tested the ΔSPI1 and ΔhilA strains against wild type in intraperitoneal (i.p.) competition assays. In both instances the mutant competed evenly with wild type (Table 2). In addition, in vitro competition assays prove that the ΔSPI1, the ΔhilA, or the ΔhilCΔrtsAΔhilD strains are unaffected in their growth in laboratory medium under SPI1 inducing conditions (data not shown). Note that both the ΔSPI1 and ΔhilA strains are capable of reaching systemic sites despite that lack of a functional SPI1 system, as previously shown (Vazquez-Torres et al., 1999). However, the simplest interpretation of our data is that loss of SPI1 leads to an invasion defect and that this defect is mimicked by loss of HilA, ultimately responsible for transcriptional activation of the SPI1 TTS apparatus genes. These data also support the use of oral competition assays for studying the roles of SPI1 TTS or its regulators in vivo.
|Strain A||Strain B||Spleen||Small intestine||No. mice|
|Name||Relevant genotype||Name||Relevant genotype||CI||P||CI||P|
|Strain A||Strain B||Spleen||No. mice|
|Name||Relevant genotype||Name||Relevant genotype||CI||P|
The in vitro data suggest that HilC, HilD and RtsA act in concert to induce expression of hilA. To see if the apparent redundancy seen in vitro is manifested in vivo, we tested various hilC, hilD and rtsA single, double and triple mutants in oral competition assays, monitoring bacteria recovered from the spleen as an indication of invasion abilities. The data from these experiments are presented in Table 3. Strains carrying ΔrtsA or ΔhilC mutations are not significantly attenuated as compared with wild type, while a ΔhilD strain is attenuated about sixfold. This is consistent with in vitro data that suggest HilD is the most potent hilA activator and that loss of hilC or rtsA individually has a less dramatic effect on hilA expression. However, loss of both HilC and RtsA conferred a phenotype equivalent to the ΔhilD mutation. Indeed, the ΔhilCΔrtsAΔhilD strain was attenuated compared with a ΔhilD strain, suggesting that in the absence of HilD, the remaining regulators RtsA and HilC can significantly contribute to invasion. In contrast, the ΔhilCΔrtsAΔhilD strain was not significantly different than a ΔhilCΔrtsA double mutant in its ability to invade, suggesting that HilD alone is unable to activate expression of hilA to a level needed for invasion. Loss of all three regulators confers a phenotype that is not significantly different than that conferred by loss of HilA. These data indicate that all three regulators contribute to activation of the SPI1 TTSS. Moreover, the activation in vivo apparently mimics the results observed in vitro.
|Strain A||Strain B||Spleen||No. mice||Oral dose (cfu)|
|Name||Relevant genotype||Name||Relevant genotype||CI||P|
The SirA/BarA two-component regulatory system functions through hilD
The SirA/BarA two-component regulatory system affects expression of hilA, hilC and hilD (Lucas and Lee, 2001). We wanted to confirm a role of SirA in expression of hilA, hilC and hilD as well as determine if SirA also regulates expression of rtsAB. We introduced a sirA null mutation (Ahmer et al., 1999) into the various fusion strains and monitored β-galactosidase activity after growth under SPI1 inducing conditions. Figure 4 shows that loss of SirA decreased expression of hilC and hilD approximately two- and threefold, respectively, while decreasing expression of hilA∼6.5-fold. These results are similar to those obtained by others (Johnston et al., 1996; Lucas and Lee, 2001). Figure 4 also shows that a sirA mutation decreased expression of the rtsA–lac fusion approximately 6.5-fold. Thus SirA controls expression of the three hilA regulators and hilA.
Our model (Fig. 1) suggests that SirA could control expression of hilC, hilD and rtsA by either independently activating each gene or by regulating the expression of a single regulator. Increased expression of this regulator would then induce expression of the others. Previous attempts have been made to determine how SirA controls expression of hilA (Lucas and Lee, 2001; Teplitski et al., 2003). However, these experiments were often limited by the fact that loss of the regulatory proteins decreased expression of hilA such that further decreases in transcription were difficult to determine accurately (Lucas and Lee, 2001). To avoid this caveat we utilized strains producing SirA from an arabinose inducible promoter (Ahmer et al., 1999). Overproduction of SirA was predicted to increase expression of the regulatory genes. We introduced plasmids pBAD (vector control) or pSirA into rtsA–lac, hilC–lac and hilD–lac fusion strains. We then introduced deletions of rtsA, hilC, hilD or hilDC and assayed the β-galactosidase activity of the strains after growth in Luria–Bertani (LB) containing arabinose. As shown in Fig. 5A, production of SirA from the plasmid increased expression of rtsA and hilC approximately twofold while expression of hilD was induced approximately 1.5-fold. The loss of HilC decreased the overall expression of rtsA but did not affect the ability of SirA to induce expression of the rtsA–lac, hilC–lac, or hilD–lac fusions. (The lac fusions are constructed from null mutations in the regulator of interest; Ellermeier et al., 2002). Thus SirA does not require HilC to regulate expression of the three hilA regulators. In contrast, the presence of a hilD mutation completely blocked SirA induction of rtsA and hilC (Fig. 5A). A hilDC double mutant further decreased expression of rtsAB and this expression was similarly unaffected by SirA. This suggests that SirA regulates expression of rtsAB and hilC via HilD. SirA apparently controls hilD at the level of the mRNA; overproduction of SirA increased hilD expression in a HilD-independent fashion. (The hilD–lac fusion is a hilD null.)
SirA induction of hilA and invF requires HilD
Previous evidence suggested that SirA required the HilC/HilD/RtsA binding sites in the hilA promoter to induce expression of hilA and thus the entire SPI1 TTSS (Schechter et al., 1999). Transcription of invF is primarily regulated by HilA, but HilD, HilC and RtsA are each capable of partially activating invF (Eichelberg et al., 1999; Rakeman et al., 1999; Akbar et al., 2003; Ellermeier and Slauch, 2003). Our data suggest that SirA induces expression of hilC and rtsA via HilD. Therefore, our model predicts that SirA induces expression of hilA (and invF) via HilD (Fig. 1). We introduced deletions of rtsA, hilC, or hilD and determined if SirA produced from a plasmid was still capable of inducing expression of hilA– and invF–lac fusions. As shown in Fig. 5B, loss of RtsA or HilC decreased the level of hilA and invF expression but did not significantly affect induction by SirA. In contrast, the absence of HilD alone blocks the ability of SirA to induce expression of the hilA–lac or invF–lac fusions. This suggests that SirA induction of hilA expression requires the presence of HilD. This is consistent with our feedforward regulatory model. Although all three regulators contribute to the induction, the effect is mediated solely through HilD.
HilD is required for EnvZ/OmpR regulation of hilA
It was previously concluded that the EnvZ/OmpR two-component system functioned through hilC to regulate expression of invasion genes (Lucas and Lee, 2001). This conclusion was based on the observation that an envZ null mutation had a greater effect on the expression of a hilC–lac fusion than it did on a hilD–lac fusion. However, these investigators also suggested that the EnvZ-mediated regulation required HilD (Lucas and Lee, 2001). The feedforward loop model affects these interpretations. Therefore, to determine where the EnvZ/OmpR system feeds into the regulatory scheme, we analysed the effects of hilC, hilD and rtsA deletions alone, and in various combinations, on expression of a hilA–lac fusion in strain backgrounds containing wild-type ompR or the ompR1009::Tn10dTc allele. The data (Fig. 6) show that EnvZ/OmpR regulation of hilA–lac is abolished in the presence of a hilD deletion. In all instances where a hilD deletion was present, there was no difference in hilA expression between the wild type and ompR1009::Tn10dTc strains. Deletion of rtsA or hilC alone decreased the absolute expression of hilA–lac, but did not affect the relative level of expression in the wild-type ompR versus the ompR1009::Tn10dTc strains. Thus, neither RtsA nor HilC are required for OmpR-mediated regulation. However, in the rtsA hilC double mutant, although OmpR regulation was still apparent, there was an overall decrease in the fold-effect of the ompR1009::Tn10dTc on hilA expression. This is consistent with the feedforward loop model; when neither RtsA nor HilC are present, regulation of hilA is dampened, even though the primary signal is mediated through HilD. We conclude that the EnvZ/OmpR two-component system regulates hilA expression via HilD and that HilC and RtsA act to amplify the effect.
Environmental regulation is mediated by unknown factors affecting transcription of the hilD, hilC and rtsA promoters
In the laboratory, the SPI1 system is maximally induced in high salt, low oxygen conditions (Bajaj et al., 1996). Conversely, growth in a low salt, high oxygen medium is usually used as the repressive condition. We have found that oxygen levels have the most profound effects of hilA transcription and that the best repressing conditions are high salt, high oxygen (data not shown). It is also clear that expression of the rtsA, hilC and hilD genes is induced under SPI1 inducing conditions (Lucas and Lee, 2001; Ellermeier and Slauch, 2003), suggesting that environmental regulation is mediated by factors that act upstream of these proteins in the regulatory circuit. The data above suggest that the SirA/BarA and EnvZ/OmpR two-component systems function through HilD. We wanted to test if the so-called SPI1 inducing conditions also fed into the system through HilD. Therefore, we examined the expression of hilA, rtsA, hilC and hilD transcriptional fusions in the presence and absence of RtsA, HilC, and/or HilD (Fig. 7). As expected, hilA transcription was largely dependent on the three regulators, although there might be a slight effect of oxygen on the promoter. However, transcription of rtsA, hilC and hilD was induced in response to low oxygen independent of RtsA, HilC, or HilD. Consistent with the feedforward loop model, the absolute level of transcription of hilC and particularly rtsA was reduced in the absence of the other regulators. However, these data suggest that at least oxygen regulation is mediated at the level of transcription of all three regulators by an unknown mechanism.
We propose a model for SPI1 regulation in which the three AraC-like regulators HilD, HilC and RtsA act in a feedforward loop to activate expression of the hilA gene. We show that HilD, HilC and RtsA can each independently activate expression of the hilD, hilC, rtsAB and hilA genes. Previous data show that these three regulators also activate, to a lesser extent, the inv/spa operon and hence production of InvF (Eichelberg et al., 1999; Rakeman et al., 1999; Akbar et al., 2003; Ellermeier and Slauch, 2003). Each can independently bind upstream of hilA to induce its expression (Schechter and Lee, 2001; Olekhnovich and Kadner, 2002; Ellermeier and Slauch, 2003). However, HilD, HilC and RtsA normally act in concert to activate hilA. This is evidenced by the effects of single, double and triple mutations in the hilD, hilC and rtsA genes on both in vitro expression of a hilA–lac fusion and in vivo invasion of the intestine after oral infection of mice. Of the three regulators, HilD apparently has a dominant role. However, HilD does not work alone under normal conditions: in the absence of HilC and RtsA, loss of HilD has little effect on transcription of hilA or invasion of the intestine.
HilC and HilD bind to overlapping sites in the hilA promoter (Olekhnovich and Kadner, 2002). Recent data suggest that RtsA binds to the same sequence, although the sequence requirements differ subtly between the three regulators (Ellermeier and Slauch, 2003; I.N. Olekhnovich and R.J. Kadner, unpubl.). This complicates the model by raising the possibility that the three regulators compete for binding to the same site. However, our most striking results show that even though HilD appears to be the best hilA activator of the three in vitro, when working alone it cannot induce hilA enough to stimulate invasion. Coupled with the data suggesting that HilC and RtsA are together sufficient to stimulate invasion at some level, these results lead us to propose that at least two of the three regulators need to be present to activate transcription. It follows that mixed dimers could be the active entities. The affinity of the three regulators for various binding sites almost certainly differs. If there are also subtle differences in the ability of the three regulators to activate transcription, then mixed dimers could constitute a range of activating moieties with different efficiencies. Recent data suggest that HilC and HilD are differentially degraded by Lon (Takaya et al., 2005), suggesting that the stability of the two could differ under certain conditions. Moreover, AraC-like regulators often interact with a small molecule, like arabinose, to activate transcription. It is possible that RtsA, HilC and HilD each respond to an independent signal to activate SPI1, but what these signals are is not clear. If the three are also binding to overlapping sites at the rtsA, hilC and hilD promoters, then predicting the level of hilA transcription under a given condition becomes even more difficult.
A simple interpretation of the model would suggest that it acts as a switch; an increase in the expression or activity of one regulator leads to an increase in expression of the other regulators (Mangan and Alon, 2003), turning the entire system on. However, at least at the population level, we know that the system is not fully on under ‘SPI1 inducing’ conditions because overexpression of one of the regulators can significantly induce the system further (Ellermeier and Slauch, 2003). Likewise, loss of OmpR, for example, does not shut the system off, but rather attenuates expression about twofold (Fig. 6). Thus, the system has intermediate levels rather than two simple states. This could reflect a population dynamic. In other words, the system could act as a switch at the single cell level, while we are monitoring the average over the population (Batchelor et al., 2004). Alternatively, either the factors outlined above or feed back control of the system (Darwin and Miller, 2000, 2001; Ellermeier and Slauch, 2004) could instil continuous control (Batchelor et al., 2004).
As stated above, HilD seems to play a predominant role in the system. Loss of HilD has a quantitatively greater effect of hilA transcription. In addition, loss of HilC and RtsA does not significantly decrease hilD transcription, although the two can clearly activate hilD independently. One could argue that HilD is the most active of the three regulators, but this could be misleading. Indeed, in some of our experiments, HilC seems to be the most efficient activator of the three (Fig. 2). However, given that the external regulators of the system, e.g. SirA/BarA and OmpR/EnvZ, seem to function through HilD, loss of HilD could have more profound effect than loss of RtsA or HilC due to the lack of these external activation signals rather than some inherent difference in efficiency of HilD versus HilC or RtsA. Likewise, these external regulators could largely be responsible for hilD transcription, negating the effects of HilC and RtsA.
Regulation of the SPI1 TTSS and Salmonella invasion has been intensively studied, leading to identification of a bewildering number of regulatory proteins that play major or minor roles in the process. We show that the SirA/BarA two-component regulatory system is required for maximal expression of hilA, hilC, hilD and rtsA. In support of our model, we demonstrate that SirA induction of hilC, rtsA, hilA and invF requires the presence of HilD. This genetic analysis is apparently inconsistent with previous gel shift data suggesting that SirA binds both the hilC and hilA, but not the hilD, promoters (Teplitski et al., 2003). However, the data in Fig. 5 show that SirA, even overproduced, can activate neither hilC nor hilA in the absence of HilD. Indeed, the effects of SirA are likely to be indirect, mediated by csrB and csrC acting through CsrA (see Introduction). Although SirA functions through HilD, it has only a modest effect on expression of hilD (Fig. 4; Lucas and Lee, 2001). Is the two- to threefold decrease in hilD expression caused by a sirA mutation enough to account for the nearly 10-fold decrease in hilA transcription? It is possible, given the nature of the feedforward regulatory loop, that moderate effects on HilD production are amplified. Decreases in hilD expression could have a dramatic effect on hilA expression because expression of the two other hilA activators, hilC and rtsA, is also decreased. Moreover, because HilD autoregulates and the hilD–lac fusion we are using is a hilD null, SirA may have a greater effect on production of HilD than what is apparent in our assays. If SirA is acting through the csr system (Suzuki et al., 2002) and thereby affecting the stability of the hilD mRNA, this could also explain a greater effect on the native transcript versus the fusion.
OmpR also exerts its regulatory effect on hilA by working through HilD. In future studies, we will determine if OmpR works by regulating the transcription of hilD, or by some other mechanism. Genetic studies have identified a number of hilA regulators (see Introduction). It is clear that many of these regulators control expression of hilC and hilD (Lucas and Lee, 2001). It will be critical to determine if they also regulate expression of rtsA and if these regulators function via a single regulator, much like SirA and OmpR. Understanding how these regulators fit into the circuit to control expression of hilA is crucial to understanding the global regulation of the SPI1 TTSS.
Jones and colleagues have identified HilE as a negative regulator of hilA transcription (Fahlen et al., 2000; Baxter et al., 2003; Baxter and Jones, 2005). Based on two-hybrid analysis suggesting a direct interaction, they propose that HilE directly inhibits the activity of HilD (Baxter et al., 2003). They have also provided data suggesting that the two-component systems PhoPQ and PhoBR act through HilE to regulate SPI1 (Baxter and Jones, 2003). It is not clear if HilE is involved in SirA or OmpR regulation. However, our data suggest that at least part of the effect of SirA is mediated at the level of hilD transcription or mRNA stability (Fig. 4). This is inconsistent with post-transcriptional regulation by HilE. Thus, the various two-component systems apparently work by different mechanisms.
The data presented here and by Jones and colleagues suggest that all of the two-component systems that impinge on hilA regulation function through HilD. However, we show that the environmental conditions that are considered ‘SPI1 inducing’ act, at least partially, at the level of hilD, hilC and rtsA transcription, in the absence of the three AraC-like regulators. Thus, these environmental conditions are apparently not mediated by these known two-component regulators. It seems clear that SPI1 is activated in response to particular environmental signals that are integrated at the level of hilA transcription. Given the data presented here and despite the many years of study, we have no idea how the presumed primary environmental signals are sensed. Thus, understanding the transcriptional regulation of hilC, hilD and rtsA becomes key.
Media, reagents and enzymatic assays
Luria–Bertani media was used in all experiments for growth of bacteria and SOC was used for the recovery of transformants (Maloy et al., 1996). Bacterial strains were routinely grown at 37°C except for strains containing the temperature sensitive plasmids, pCP20 or pKD46, which were grown at 30°C. Antibiotics were used at the following concentrations: 50 µg ml−1 ampicillin (Ap); 20 µg ml−1 chloramphenicol (Cm); 50 µg ml−1 kanamycin (Km). Primers were purchased from IDT. β-Galactosidase assays were performed using a microtiter plate assay as previously described (Slauch and Silhavy, 1991) on strains grown under the indicated conditions. β-Galactosidase activity units are defined as (µmol of ONP formed min−1) × 106/(OD600 × ml of cell suspension) and are reported as mean ± standard deviation where n = 4. Cultures used to measure β-galactosidase activity were initially inoculated into LB (0.5% NaCl), grown for 8–12 h, then subcultured 1/100 and grown under one of the following conditions: (i) statically overnight in 3 ml of LB with 1% NaCl in a 13 × 100 mm tube, referred to as either Low Oxygen or SPI1 inducing; (ii) on a platform shaker at 225 r.p.m. in 2 ml of LB with 1% NaCl in a 18 × 150 mm tube to an OD600 of 0.8, referred to as High Oxygen; (iii) shaking at 225 r.p.m. in LB no salt/Ap/0.2%l-arabinose and grown to an OD600 of ∼0.6.
Bacterial strains and plasmids are described in Table 4. All S. enterica serovar Typhimurium strains created for this study are isogenic derivatives of strain 14028 [American Type Culture Collection (ATCC)] and were constructed using P22 HT105/1 int-201 (P22)-mediated transduction (Maloy et al., 1996). Deletion of various genes and concomitant insertion of an antibiotic resistance cassette was carried out using Lambda Red-mediated recombination (Datsenko and Wanner, 2000; Yu et al., 2000) as described in (Ellermeier et al., 2002). The end-points of each deletion are indicated in Table 4. In all cases, the appropriate insertion of the antibiotic resistance marker was checked by P22 linkage to known markers and/or polymerase chain reaction analysis. In each case, the constructs resulting from this procedure were moved into a clean wild-type background (14028) by P22 transduction. Antibiotic resistance cassettes were removed using the temperature sensitive plasmid pCP20 carrying the FLP recombinase (Cherepanov and Wackernagel, 1995). In some instances, the insertion mutations were converted to transcriptional lac+ fusions using an FLP/FRT-mediated site-specific recombination method as previously described (Ellermeier et al., 2002).
|Strain||Genotypea||Deletion end-pointsb||Source or referencec|
|14028||Wild-type serovar Typhimurium||ATCC|
|BA746||sirA3::Cm||(Ahmer et al., 1999)|
|JS107||zjg8101::Kn||(Mann and Slauch, 1997)|
|JS248||ΔrtsA5||4561755–4560884||(Ellermeier and Slauch, 2003)|
|JS251||ΔhilA112::Cm||3019885–3021480||(Ellermeier and Slauch, 2003)|
|JS252||ΔhilC113::Cm||3012135–3012976||(Ellermeier and Slauch, 2003)|
|JS253||ΔhilD114::Cm||3017865–3018730||(Ellermeier and Slauch, 2003)|
|JS256||ΔhilC-D2915::Cm||3012135–3018730||(Ellermeier and Slauch, 2003)|
|JS279||φ(hilA-lac+)112||(Ellermeier and Slauch, 2003)|
|JS282||φ(invF-lac+)100||(Ellermeier and Slauch, 2003)|
|JS324||φ(rtsA-lac+)5||(Ellermeier and Slauch, 2003)|
|JS481||Δ(invH-avrA)2916::Cm (called ΔSPI1-2916::Cm)||3009858–3044879|
|JS482||ompR1009::Tn10dTc||(Gibson et al., 1987)|
|JS520||ompR1009::Tn10dTc ΔhilD114::Cm φ(hilA-lac+)112|
|JS521||ompR1009::Tn10dTc ΔhilC113::Cm φ(hilA-lac+)112|
|JS522||ompR1009::Tn10dTc ΔhilC-D2915::Cm φ(hilA-lac+)112|
|JS524||ompR1009::Tn10dTc ΔrtsA5ΔhilD114::Cm φ(hilA-lac+)112|
|JS525||ompR1009::Tn10dTc ΔrtsA5ΔhilC113::Cm φ(hilA-lac+)112|
|JS526||ompR1009::Tn10dTc ΔrtsA5ΔhilC-D2915::Cm φ(hilA-lac+)112|
|Plasmid||Relevant characteristics||Cloned end-pointsb||Source or referencec|
|KD46||bla PBADgam bet exo pSC101 oriTS||(Datsenko and Wanner, 2000)|
|pCP20||bla cat cI857 λPRflp pSC101 oriTS||(Cherepanov and Wackernagel, 1995)|
|pKD3||bla FRT cat FRT PS1 PS2 oriR6K||(Datsenko and Wanner, 2000)|
|pCE36||ahp FRT lacZY+ this oriR6K||(Ellermeier et al., 2002)|
|pCE37||ahp FRT lacZY+ this oriR6K||(Ellermeier et al., 2002)|
|pBAD30||bla araC PBAD pACYC184 ori||(Guzman et al., 1995)|
|pBA204||bla araC PBAD pACYC184 ori sirA +||(Ahmer et al., 1999)|
|pRtsA||bla PBADλAttB1 rtsA+λAttB2 pACYC184 ori||4561766–4560885||(Ellermeier and Slauch, 2003)|
|pLS118||bla PBADhilD-myc-His pACYC184 ori||(Schechter and Lee, 2001)|
|pLS119||bla PBADhilC-myc-His pACYC184 ori||(Schechter and Lee, 2001)|
BALB/c mice (Harlan) of 5- to 7-week-old were inoculated either orally or i.p. with 0.2 ml of a bacterial suspension diluted to either 5 × 107 cells per millilitre or 5 × 108 cells per millilitre in sterile 0.1 M sodium phosphate buffer, pH 8.0 (oral) or to 5 × 103 cells per millilitre in sterile 0.85% NaCl (i.p). After 4–5 days, the mice were sacrificed by CO2 asphyxiation and the small intestine (oral) and spleen (oral and i.p) were harvested. These organs were homogenized, and serial dilutions were plated on appropriate media to determine the colony-forming unit (cfu) per organ. The relative percentage of each strain recovered was determined by replica plating to the appropriate antibiotic containing medium. In all competition assays, the inoculum consisted of a 1:1 mix of two bacterial strains. The actual cfu and relative percentage represented by each strain was determined by direct playing of the inoculum. The competitive index (CI) was calculated as (% strain A recovered/% strain B recovered)/(% strain A inoculated/% strain B inoculated). Each mutant strain was reconstructed at least once to ensure that the virulence phenotype is the result of the designated mutation(s). The Student's t-test was used to determine if the output ratio is significantly different than the input ratio.
We would like to thank Igor Olekhnovich and Bob Kadner for allowing us to discuss unpublished data, and Brian Ahmer for kindly providing the sirA null allele and plasmid. This work was supported by Grant 00–25 from the Roy J. Carver Charitable Trust and by Public Health Service Grant AI63230.
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