ABSTRACT
The expression of genes encoding proteins secreted by the SPI1 (Salmonella pathogenicity island) type III secretion apparatus is known to require the transcriptional activators SirA and HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of theinv-spa gene cluster, is predicted to encode an AraC-type transcriptional activator and is required for invasion into cultured epithelial cells. However, the genes which are regulated by InvF have not been identified. In this work, an in-frame deletion ininvF was constructed and tested for the expression of Φ(sigD-lacZYA),sipC::Tn5lacZY, and a plasmid-encoded Φ(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion ofSalmonella typhimurium into cultured eucaryotic cells.sicA (Salmonella invasion chaperone) is the first gene of a putative operon encoding the Sip/Ssp (Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of thesigD, sicA, and sipC fusions. This is the first demonstration that there is a functional promoter in the intergenic sequence between spaS and sicA. In addition, several proteins were either absent from or found in reduced amounts in the culture supernatants of the invF mutant. Therefore, invF is required for the optimal expression of several genes encoding SPI1-secreted proteins. Genetic evidence is also presented suggesting there is HilA-dependent readthrough transcription from the invF promoter at least through sipC.
The expression of genes required for the invasion of eucaryotic cells is stimulated by environmental cues including osmolarity (12), pH (4), and growth state and oxygen tension (10, 27, 38). Several regulators of SPI1 (Salmonella pathogenicity island 1) gene expression have been identified, but it is not known how they recognize environmental signals which affect gene expression. A pho-24mutant produces constitutively active PhoP, a response regulator which directly or indirectly represses the expression of SPI1 genes (4, 6, 15, 21). One of these genes, hilA, encodes an activator of SPI1 gene expression (3, 28). HilA is believed to directly activate expression from the invF andprgH promoters (3), although this has not been established biochemically. invF is predicted to encode an AraC-type transcriptional activator (for a review, see reference13), but it is not known which genes it activates (23). invF is not autoregulated, nor does it activate the expression of invH, a gene which encodes an outer membrane lipoprotein component of the SPI1 type III secretion apparatus and which is divergently transcribed from invF(2, 8, 23). Nonetheless, invF is required for efficient invasion of cultured epithelial cells (23), suggesting that it is important for the expression of other genes required for invasion. This hypothesis was tested by examining the effect of an invF mutation on the expression of genes known to be required for invasion.
MATERIALS AND METHODS
Bacterial strains and plasmids.Bacterial strains and plasmids used in this study are described in Table1. Electroporation of plasmids into bacteria was carried out as previously described (36). Plasmids that were manipulated in Escherichia coli were passaged through a restriction-minus (hsd) Salmonella typhimurium LT2 strain (LB5000) (37) prior to electroporation into S. typhimurium SL1344 (19). P22 HT int lysates were harvested and used for transductions as previously described (30).
Bacterial strains and plasmids used in this work
pDL7-2 (generously provided by Catherine Lee) is a pLAFR2-based clone (11) containing the inv-spa andsicAsipB genes (32). Two subclones were made by digesting pDL7-2 with EcoRI and cloning an ∼8-kb fragment containing invHFGEABC′ and a 6.7-kb fragment containing theinvIJ spaOPQRS sicA sequence into pWKS130 (40), resulting in pHD7 and pHD8, respectively. pHD7 was digested withPstI to subclone a 1.7-kb fragment containinginvF, creating pHD9-1. This fragment includes about 250 bp of sequence upstream of the invF start codon and is transcribed in the same direction as the lacZ promoter in pWKS130. The same PstI fragment was cloned into the medium-copy-number vector pHG329 (39), forming pHD10-1.
To construct pHD14, a 2.5-kb SalI-BamHI fragment containing spaQRS was cloned from pHD8 into pMAK705 (16). A unique BglII site was used to clone a ∼2-kb streptomycin-spectinomycin resistance cassette from pSmUC intospaS, resulting in pHD14.
To construct the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible sigDE clone pHH37, a 3.2-kbEcoRI-BamHI fragment from pHH20 (20) was cloned into pVLT33 (9). Induction of sigDEexpression was done by the addition of IPTG to a final concentration of 100 μM in overnight cultures.
pHD3 (invF-lacZYA) and pHD11 (sicA-lacZYA) were made by PCR amplification (Pfu polymerase; Stratagene) of putative promoter sequences and cloning into pRW50 (29). For pHD3, an invH primer with an EcoRI linker (invH-1 [5′-GGAATTCCGGCGCCATGTTTTTACACAACCGTCAGAAC-3′]) and aninvF primer with a BamHI linker (invF-1 [5′-CGGGATCCCGGCAGCTTTTGCGCGGAACACGTCTGTATAAACC-3′]) (Gibco BRL) were used to amplify ∼460 bp betweeninvF and invH from pDL7-2. The resulting fragment was cloned into the BamHI and EcoRI sites of pRW50. For pHD11, the primers spaS-EcoRI-3 (5′-GGAATTCCGCGGAGAAGGTTGGCGTACCTG-3′) andsicA-BamHI-1 (5′-CGGGATCCCGGCGTGGCGCCTTCACTAACGGCATCC-3′) were used to amplify the intergenic sequence (137 bp) between spaS andsicA along with 192 bp of the 3′ end of spaS and 76 bp of sicA. This amplified product was cloned into pRW50 as described above, and both strands were sequenced with the same primers used for amplification to confirm the sequence.
To construct the sigD chromosomal lacZYAreporter, plasmid pFUSE was used (5). pFUSE contains an R6K origin of replication and cannot replicate in SL1344. A 1-kbXbaI fragment from pHH15 (20) containing 0.4 kb of sequence upstream of sigD and 0.6 kb of sigDcoding sequence was cloned into the unique XbaI site of pFUSE. Ligations were transformed into E. coliS17-1λpir, and clones were screened by restriction digestion with PstI and EcoRI for inserts in the correct orientation relative to lacZYA. One clone was selected and named pHD5. pHD5 was transferred by conjugation into SVM252 (14028s sirA::Tn10dTc) (20, 22), where it integrated into sigD, leaving an intactsigDE+ copy in addition to asigD-lacZYA operon fusion. ThesirA::Tn10dTc strain was used as a recipient in order to provide a selectable marker (tetracycline resistance) for S. typhimurium. Correct integration of pHD5 was confirmed in several exconjugants by transduction linkage analysis using SVM167 (sigE::Tn10dTc) (86% linkage). The Φ(sigD-lacZYA) fusion was then transduced into the appropriate strains.
Growth conditions. S. typhimurium and E. coli strains were grown in Luria-Bertani (LB) broth (Difco) at 37°C with aeration on a roller drum or without aeration in standing cultures, as indicated. Antibiotics were used at the following final concentrations: chloramphenicol, 25 μg/ml; kanamycin, 100 μg/ml; streptomycin, 100 μg/ml; spectinomycin, 100 μg/ml; and tetracycline, 15 or 30 μg/ml for single-copy or multicopy tetracycline resistance, respectively. For the detection of β-galactosidase activity, solid medium (LB agar) was supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-Gal) at 40 μg/ml. IPTG was used at a final concentration of 100 μM.
Tissue culture invasion assays.HEp-2 cells were maintained and passaged as recommended by the American Type Culture Collection. For invasion assays, 2 × 105 cells/ml were seeded into Falcon 24-well tissue culture plates (Becton Dickinson, Lincoln Park, N.J.) to obtain about 90% confluent monolayers on the following day. For bacterial cultures, single colonies were inoculated into 2 ml of LB broth and grown for 18 h without shaking at 37°C. Aliquots of 5 μl (107 to 108 CFU) were used per well of tissue culture cells. The invasion assay was performed as previously described (20).
Construction of spaS disruption mutants.To construct the spaS::ΩStr/Sp mutant SMV514, a 2.5-kb SalI-BamHI fragment containingspaQRS was cloned into pMAK705, creating pHD12. ABamHI streptomycin-spectinomycin resistance cassette from pSmUC was cloned into the unique BglII site within the 5′ region of spaS. This plasmid, pHD14, was used to exchange the disrupted spaS allele with the wild-type allele on the chromosome as previously described (16).
To confirm the disruptions on the chromosome, Southern analysis was performed (36). Labeling of a DNA probe and detection of disrupted sequences were done with enhanced chemiluminescence (ECL). Southern blotting detection reagents (Amersham Pharmacia Biotech). To confirm the spaS disruption, chromosomal DNA was digested with BamHI and probed with a spaS PCR-amplified product. As predicted, the spaS probe hybridized to a 7-kb fragment of SL1344 chromosomal DNA and a 9-kb fragment in thespaS-disrupted strains (data not shown). OnespaS::ΩStr/Sp mutant was chosen and called SVM514.
Construction of the invF in-frame deletion mutant.To make an in-frame deletion in invF, pHD10-1 was digested with ClaI and SacII, the 5′ and 3′ single-stranded overhangs were removed with mung bean nuclease (New England Biolabs), and the blunt ends were ligated with T4 DNA ligase (New England Biolabs). Mung bean nuclease can sometimes remove double-stranded DNA in addition to single-stranded DNA; therefore, several clones were sequenced to identify clones with an in-frame deletion in invF. One clone, pHD13, contained an in frame deletion of 465 bp. A 1.25-kb PstI fragment from pHD13 was subcloned into pMAK705, producing pHD15, which was then used to exchange the deletion onto the chromosome as previously described (16).
To screen for the invF deletion on the wild-type chromosome, bacteria with resolved plasmids were pooled in phosphate-buffered saline, subcultured into LB broth, and infected with P22 HTint containing pHH21 (sigD-lacZYA reporter plasmid, medium copy number) (20). If invF were required for the expression of sigD as predicted, the expression of Φ(sigD-lacZYA) would be reduced in an ΔinvF strain. Transductants were grown on MacConkey lactose agar supplemented with ampicillin. Four of several hundred transductants that formed less red or white colonies were purified and subsequently cured of the reporter plasmid by growing bacteria in LB broth for 5 days, subculturing 1:500 on each day. Individual colonies were screened for ampicillin sensitivity on LB agar containing ampicillin. One mutant was chosen and called SVM579. TheinvF mutation in this strain was complemented byinvF in a low-copy-number vector (pHD9-1), suggesting that the phenotype of this mutant (see Results) was due to the deletion ininvF and not another mutation elsewhere on the chromosome. The same technique was used to introduce the invF deletion onto the chromosome of BJ68 (sipC::Tn5lacZY) (34), creating SVM725. The ΔinvF resulted in reducedlacZY expression from thesipC::Tn5lacZY fusion when tested on LB agar plates supplemented with X-Gal.
To confirm the presence of the deletion on the chromosome and the absence of any gross chromosomal rearrangements, Southern analysis was performed (36). Chromosomal DNA was digested withPstI and probed with the 1.7-kb PstI fragment from pHD10-1. The probe hybridized to a 1.7-kb PstI fragment of chromosomal DNA from SL1344 and a 1.2- to 1.3-kb fragment of SVM579 and SVM725 as predicted (data not shown).
To construct the ΔinvF spaS::ΩStr/SpsipC::Tn5lacZY triple mutant, SVM754, the spaS::ΩStr/Sp mutation in SVM514 was transduced into SVM579 (ΔinvF). Nine purified transductants were screened by Southern hybridization to confirm that the invF deletion had not been lost upon transduction due to its spaS-linked location on the chromosome (data not shown). One mutant, SVM733 (ΔinvF spaS::ΩStr/Sp), was used to make a P22 HT int lysate. ThespaS::ΩStr/Sp mutation from SVM733 was transduced into SVM725 (ΔinvF sipC::Tn5lacZY). Tetracycline-, spectinomycin-, and streptomycin-resistant transductants were purified and checked for P22 sensitivity.
Analysis of culture supernatants.Cultures were grown in 5 to 10 ml of LB broth with antibiotics for 18 h without aeration, and equivalent units of optical density at 600 nm were harvested as previously described (20). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (26) on 7.5% gels for silver stain analysis (7) and 5% gels for immunoblot (Western) analysis (ECL Western blotting detection system; Amersham Pharmacia Biotech). It is notable that in these gels (30% acrylamide to 1.6% bisacrylamide), some proteins migrated more slowly than through gels poured with 0.8% acrylamide (data not shown). For immunoblots, proteins were transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore).
Antibodies.To make antibodies to SigD, the first 575 bp ofsigD were cloned into the expression vector pMAL-c1 (New England Biolabs). Expression and affinity purification of the maltose binding protein (MBP)-SigD′ fusion protein expressed from this plasmid were performed as described by the manufacturer. Protein eluted from the amylose column was separated on a 7.5% SDS-polyacrylamide gel, and the fusion protein was excised from the gel. Lyophilized gel slices containing MBP-SigD′ were used to raise rabbit antibodies to SigD (Covance Research Services, Denver, Pa.). The anti-SigD antibodies were preadsorbed with an acetone powder of E. coli DH5α expressing MBP (17) to reduce cross-reactivity of the antibody to proteins other than SigD. The preadsorbed antiserum was used at a 1:1,000 dilution. Anti-rabbit horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma (St. Louis, Mo.).
Enzyme assays.β-Galactosidase assays were performed and values were calculated as previously described (31). Cultures were grown in 5 ml of LB with appropriate antibiotics for 18 h at 37°C in 13- by 100-mm screw-capped tubes. Samples of 1.5 ml were harvested, washed once in 0.88% NaCl, and resuspended in 1 ml of working buffer; 100 μl of each cell suspension was used per assay.
Sequence analysis.Sequencing was performed by using the BigDye Terminator Cycle Sequencing Ready Reaction system (PE Applied Biosystems, Foster City, Calif.). Reactions were analyzed by the Washington University Nucleic Acid Chemistry Laboratory (St. Louis, Mo.). Sequence analyses (homologies, mapping, etc.) were performed using the Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc.).
RESULTS
InvF is essential for the expression of effector genes. invF is the first open reading frame of a large gene cluster encoding components of the SPI1 type III secretion apparatus (23). It has been proposed that InvF activates the expression of genes required for invasion (23), but this has not been tested. Therefore, a strain with an in-frame deletion of 465 bp in invF, SVM579, was constructed and tested for expression of the Φ(sigD-lacZYA) (chromosomal),sipC::Tn5lacZY, and Φ(sicA-lacZYA) (episomal) reporters. sigD, which is unlinked to SPI1, encodes a protein secreted by the SPI1 type III secretion apparatus and is required for efficient invasion into cultured epithelial cells (20). The SigD homologue inS. dublin, SopB (Salmonella outer protein), has been implicated as a factor important for causing enteritis in a calf model of infection (14). Contrary to our previous report (20), sigD expression was found to behilA dependent (1). Therefore, strain VV302 (ΔhilA-523) was resent to us and retested for the expression of Φ(sigD-lacZYA) in pHH21. In addition to the episomal sigD-lacZYA fusion, a chromosomal reporter fusion, Φ(sigD-lacZYA)/sigDE+, was constructed and tested in the wild-type and VV302 strains. The expression of both Φ(sigD-lacZYA) fusions was significantly reduced in the hilA background (data not shown). Thus, although the hilA deletion in VV302 in our previous work was confirmed by Southern analysis and by tissue culture invasion assays (20), it appeared that the original strain of VV302 we received had acquired a suppressing mutation which allowed for hilA-independent expression of Φ(sigD-lacZYA).
sicA is predicted to encode a chaperone for one or more of the secreted proteins encoded in SPI1 (sip/ssps) and is believed to be cotranscribed with these genes (18, 21, 24, 25). The expression of Φ(sigD-lacZYA) and Φ(sicA-lacZYA) in SVM579 was found to be much lower than in the wild-type strain SL1344 (Table 2). The regulation defect due to the invF deletion was complemented by pHD9-1 (invF+), demonstrating that the regulation phenotype was due to ΔinvF. Expression of the Φ(sicA-lacZYA) and Φ(sigD-lacZYA) reporters was not complemented by philA(hilA+) (Table 2), suggesting that the regulatory effect of InvF on the sigD and thesicA promoters is downstream of HilA.
Complementation of the ΔinvF mutant for regulation of gene expressiona
The expression of a sipC::Tn5lacZYchromosomal reporter fusion from strain BJ68 (34) was also tested in the ΔinvF background. The expression ofsipC::Tn5lacZY was significantly reduced in the ΔinvF mutant (Table 2). This regulatory defect could be complemented by pHD9-1 (invF+). Interestingly, hilA provided in multicopy could also increase the expression ofsipC::Tn5lacZY, suggesting thatsipC expression could be activated from either a HilA- or an InvF-dependent promoter (Table 2). The HilA-dependent expression ofsipC::Tn5lacZY in the ΔinvF mutant could be virtually eliminated by a polar disruption in spaS, a gene upstream of sicA andsipC. In contrast, the InvF-dependent expression ofsipC::Tn5lacZY was not affected by the polar disruption in spaS. These results suggest the production of a readthrough transcript beginning upstream ofspaS that is HilA dependent and a second transcript beginning downstream of spaS (probably the sicApromoter) that is InvF dependent (Table 2).
Expression from the invF promoter was measured from pHD3 (invF-lacZYA) in SVM579 to determine if invF was autoregulated. As previously reported for a different invFmutant (23), the in-frame deletion in invF did not significantly affect expression of the invF promoter (64 (wild type) versus 53 [ΔinvF] U of β-galactosidase activity).
InvF is not sufficient to activate the expression of Φ(sicA-lacZYA) or Φ(sigD-lacZYA) inE. coli.Previous work demonstrated that an Φ(invF-lacZYA) reporter (pVV562) could be activated byhilA (in pVV214) expressed in E. coli(3). To determine if HilA and/or InvF directly activatessigD or sicA expression, the effect of providinghilA or invF in trans on the expression of Φ(sigD-lacZYA) in pHH21 (20) and of Φ(sicA-lacZYA) in pHD11 was tested in E. coli. Although philA and pVV214 (data not shown) were able to activate Φ(invF-lacZYA) (pHD3) in E. coli, neither pVV214 nor philA could activate Φ(sicA-lacZYA) or Φ(sigD-lacZYA), respectively, in E. coli (Table3). Expression of these fusions was also tested in the presence of invF. Like hilA,invF, with or without hilA, was unable to activate the expression of either fusion in E. coli (Table3). Therefore, invF may require additional factors or signals that are absent from E. coli for the activation of the sigD and the sicA promoters.
Expression of Φ(invF-lacZYA), Φ(sigD-lacZYA), and Φ(sicA-lacZYA) in E. colia
InvF is required for efficient invasion into cultured epithelial cells.Invasion into cultured epithelial cells was also quantitated for the SVM579 mutant and wild-type S. typhimurium. Invasion of HEp-2 cells by the invFdeletion strain was reduced significantly (Table4), but not to the extent previously reported (23). It is possible that the invFmutation tested by Kaniga et al. (23) was having a polar effect on the expression of the inv genes and thus had a stronger phenotype. Moreover, invasion by the ΔinvF mutant was not as reduced as invasion by a secretion-defective invA(SB154) or spaS (SVM514) mutant (Table 4). Invasion by SVM579 into HEp-2 cells was fully complemented to wild-type levels by pHD9-1. Interestingly, invasion was partially restored by philA (Table 4).
Complementation of the ΔinvF mutant for invasion of HEp-2 cells
Analysis of secreted proteins from the invFmutant.Proteins that are secreted by the SPI1 type III machinery are believed to be the effectors which stimulate the uptake of bacteria by eucaryotic host cells (18, 20, 21, 24, 25). Because the expression of genes encoding several of these proteins was reduced in the invF mutant, the secreted protein profile of SVM579 was analyzed by SDS-PAGE and silver staining. Compared to the wild-type strain, several proteins were missing from the culture supernatants of the invF mutant, including SigD (confirmed by immunoblotting with anti-SigD antibodies [data not shown]) and a smaller protein migrating at about 36 kDa (Fig. 1, lanes 2 and 5) (it is notable that most of the known proteins, including SipA, SipB, and SigD, did not migrate according to their predicted molecular weights in these gels [see Materials and Methods]). Unexpectedly, Sip/SspA was clearly observed in culture supernatants of the invF mutant, although in slightly lower amounts, compared to the wild-type supernatant proteins. Sip/SspA and the other SPI1 secreted proteins were clearly absent from the supernatants of thespaS mutant SVM514 (Fig. 1, lane 1). pHD9-1 restored the secretion of SigD and the ∼36-kDa protein into culture supernatants of the invF mutant and increased Sip/Ssp secretion to wild-type levels (Fig. 1, lane 6).
Supernatant proteins from wild-type and ΔinvF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7,S. typhimurium SL1344 containing pWSK130, pHD9 (invF+), and philA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by immunoblot analysis. Proteins were prepared and analyzed as described in Materials and Methods.
Unlike pHD9-1, philA did not restore SigD or the ∼36-kDa protein into the culture supernatants of the ΔinvF mutant. However, philA did increase the amounts of several other proteins including the Sip/Ssps to culture supernatants of the ΔinvF mutant (Fig. 1, lane 7). Because philAcould increase the expression ofsipC::Tn5lacZY in the ΔinvF mutant, it is not surprising that the Sip/Ssp proteins in culture supernatants are increased accordingly (Fig. 1, lane 7). This result may explain why philA partially restores invasion of an invF mutant into tissue culture cells.
To determine if InvF was required for the secretion of SigD in addition to the regulation of sigDE expression, an inducible clone ofsigDE, pHH37 (Ptac-sigDE), was transformed into SVM579. If invF were required for secretion, SigD would not appear in culture supernatants even after the induction of sigDE expression with IPTG. The secretion of SigD was clearly restored to the supernatants of the invFmutant containing pHH37, demonstrating that InvF is not required for the secretion of proteins by the SPI1 type III secretion system (Fig.1, lane 9).
DISCUSSION
invF was identified by sequence analysis of the SPI1 region and was predicted to encode an AraC-type transcriptional activator (23). However, InvF-dependent genes were not identified. In this study, invF was shown to be required for the expression of Φ(sigD-lacZYA) and Φ(sicA-lacZYA) fusions. An in-frame invFdeletion mutant was fully complemented for the expression of these reporters by invF cloned into a low-copy-number vector but was not complemented by hilA, a central activator of SPI1 gene expression (3). In contrast, although asipC::Tn5lacZY reporter also requiredinvF for optimal expression, hilA provided in multicopy was also able to increase sipC expression in the ΔinvF mutant.
Several of the genes encoding secreted proteins, specifically thesip/ssp genes, are immediately downstream of theinv-spa gene cluster (18, 21, 24, 25). Because the sip/ssp locus is only 137 bp downstream of thespa genes, it was possible that sip/sspexpression could be activated from the invF promoter ∼12 kb upstream of sicA; no transcriptional terminator is evident in this intergenic region. This observation is supported by the analysis of secreted proteins from the invF mutant which revealed the presence, arguably in lesser amounts, of several of the Sip/Ssp proteins. Moreover, these proteins could be restored to wild-type levels in the invF mutant by providinghilA on a low-copy-number plasmid. hilA could also partially complement the invasion defect of the invFmutant. Most importantly, the expression ofsipC::Tn5lacZY could be activated in aninvF mutant with the addition of hilA in multicopy, and this expression could be eliminated by a polar disruption in spaS. These results suggest that readthrough expression of the sip/ssp genes could be activated from theinvF promoter by HilA.
To determine if a promoter was present immediately upstream of thesip/ssp gene cluster, an episomal, Φ(sicA-lacZYA) reporter fusion in a low-copy-number vector was made. Expression of this fusion was dramatically reduced in theinvF mutant. The regulation defect was complemented byinvF but not hilA. This result demonstrates for the first time the presence of an InvF-dependent promoter immediately upstream of sicA. From these results taken together with the invasion assays, the β-galactosidase assays, and the SDS-PAGE data, it appears that expression of the sip/ssp genes can be driven in part from the HilA-dependent invF promoter in addition to an InvF-dependent promoter immediately upstream ofsicA (Fig. 2).
Model for the regulation of invasion/virulence gene expression in S. typhimurium. The direction of transcription for each gene cluster is indicated by closed arrows; open arrows represent putative transcripts of the inv-spa andsip/ssp genes. Question marks indicate either unidentified regulatory factors or unclear relationships between the designated regulator and the noted promoter.
The expression of a Φ(sigD-lacZYA) fusion in theinvF deletion mutant was also dramatically reduced. SigD was absent from culture supernatants of the ΔinvF mutant and restored by pHD9-1 (invF+). In contrast to the Sip/Ssp proteins, SigD was not restored to culture supernatants when philA was placed in the invF mutant. In addition to SigD, a ∼36-kDa protein was absent from invF mutant culture supernatants. This protein was also restored by the presence ofinvF but not hilA on a low-copy-number plasmid. Therefore, unlike the sip/ssp genes, sigD (and possibly the ∼36-kDa protein) is not likely to be directly dependent on hilA for expression. It is notable that unlike the case for hilA, providing invF in multicopy does not result in the hypersecretion of proteins found in the culture supernatants of the wild-type strain (Fig. 1, lane 3 versus lane 4). Although InvF activates the expression of genes encoding secreted proteins, it probably does not increase the transcription of the apparatus genes required for the secretion of these proteins. This may explain why the hypersecretion of proteins does not occur despite the hyperexpression of the effector genes observed when invF is present in multicopy.
It is clear that the regulation of SPI1 gene expression is complicated and multifactorial (Fig. 2). hilA expression is dependent, directly or indirectly, on SirA, a protein which is known to be conserved in several of the Enterobacteriaceae (22, 33, 35). HilA, a member of the OmpR/ToxR family of regulators, in turn activates the expression of genes encoding the type III secretion apparatus (3). This effect is predicted to be direct becausehilA expressed in E. coli can activate the expression of either Φ(invF-lacZYA) or Φ(prgH-lacZYA) (3). This work provides the first demonstration that the AraC-type transcriptional activator InvF is required for the expression of genes encoding proteins secreted by the type III secretion system. Further analysis will be necessary to determine if InvF itself binds to sequences upstream of sicAand sigD or if it activates the expression of another gene required for their expression. Because invF andhilA cannot activate the expression of Φ(sicA-lacZYA) or Φ(sigD-lacZYA) in E. coli, it seems unlikely that InvF alone is sufficient for activation. Perhaps InvF, like AraC, requires a cofactor (in the case of AraC, arabinose) (13) which induces a conformational change in InvF allowing it to bind to the appropriate promoters. Future studies will elucidate if and how InvF directly interacts with the promoters of genes encoding secreted effectors.
ACKNOWLEDGMENTS
We thank Andrew Darwin for critically reviewing the manuscript. We also thank Brad Jones and Catherine Lee for strains indispensable for this work. We especially thank C. Lee for helpful and open discussions about this project.
This work was supported by National Institutes of Health grant AI01230 to V.L.M.
FOOTNOTES
- Received 17 March 1999.
- Accepted 10 June 1999.
- Copyright © 1999 American Society for Microbiology