Detection and characterization of the S. typhimurium HilA protein
© Rodriguez et al; licensee BioMed Central Ltd. 2002
Received: 9 September 2002
Accepted: 23 October 2002
Published: 23 October 2002
Virulence genes on Salmonella pathogenicity island 1 (SPI1) are coordinately regulated by HilA, a member of the OmpR/ToxR family of transcription factors. Although a great deal is known about the complex regulation of hilA gene expression, very little is known about the HilA protein.
In order to detect and localize the HilA protein in S. typhimurium, we raised polyclonal antiserum against purified His-tagged HilA. This allowed us to study the effect of environmental conditions on the production of HilA. We also used the antiserum to examine the fractionation properties and SDS-PAGE mobility of native HilA. Our results indicate that S. typhimurium initiates translation of HilA from the first AUG codon in the hilA open-reading frame (ORF), producing a soluble 553 amino acid (63 kDa) protein product.
Materials and methods are now available to study the environmental regulation of the HilA protein in S. typhimurium. Our results also indicate that future in vitro studies of the interaction between HilA and DNA should utilize soluble preparations of HilA. Previous analyses used preparations of HilA in which the protein fractionated with the membrane, greatly limiting the types of experiments that could be conducted.
Salmonella pathogenicity island 1 (SPI1), a 40 kb region of the Salmonella typhimurium chromosome, encodes a type III secretion system that is important for virulence [1, 2]. SPI1 genes that encode components of the type III secretion apparatus are directly activated by HilA [3, 4]. HilA is a member of the OmpR/ToxR family of transcription regulators based on homology of its N-terminal domain to the conserved OmpR/ToxR DNA binding and transcription activation domain . Members of this family typically bind degenerate direct repeats as a dimer [6, 7]. Although HilA also appears to activate gene expression by binding to direct repeats, other features of HilA deviate from those of well-characterized family members [5, 7, 8]. Whereas OmpR and PhoB are regulated by phosphorylation of an N-terminal phosphoryl-acceptor domain, HilA lacks homology to such a domain. Although ToxR and CadC also lack a phosphoryl-acceptor domain, HilA lacks a predicted membrane spanning domain which is characteristic of these two family members.
HilA is thought to coordinate the regulation of SPI1 genes in response to environmental and regulatory factors. Previous studies have concluded that the regulation of SPI1 genes is primarily controlled by changes in hilA transcription [9–11]. It has been assumed that there is a direct correlation between hilA transcription, production of the HilA protein and HilA-activated gene expression. Here, we test this hypothesis by examining the effects of environmental conditions on HilA protein levels in S. typhimurium.
Results and Discussion
Induction and identification of HilA in S. typhimurium
Effect of a shift to aerobic growth conditions on SPI1 gene expression and HilA protein levels in S. typhimurium
Western blots show that high levels of HilA are present in the pre-shifted cells as well as in the bacteria shifted to the low aeration condition (Fig. 3C). However, HilA levels decrease in bacteria shifted to the high aeration condition, which correlates with the reduced transcription of hilA and prgH. These results suggest that repression of hilA transcription by oxygen leads to a decrease in HilA protein production, which in turn reduces the expression of HilA-activated genes, such as prgH.
SDS-PAGE mobility of native HilA vs. engineered forms of HilA
Sub-cellular fractionation of native HilA vs. HilA-Myc-His
The activity of many OmpR/ToxR family members is modulated by environmental conditions . HilA has a unique C-terminal domain, which could mediate its modulation by environmental or regulatory factors. Our current results support the idea that the regulation of SPI1 gene expression by oxygen is primarily mediated by regulating hilA transcription. However, it is possible that other regulatory signals control SPI1 gene expression by affecting HilA post-translationally. By developing materials and methods to detect HilA protein in S. typhimurium, we can now investigate whether HilA activity is modulated. We may find conditions in which HilA protein levels are high, but expression of HilA-activated SPI1 genes is extremely low, suggesting that HilA activity is down-modulated by these conditions. Alternatively, we may find a condition which down-modulates HilA activity by triggering its degradation. S. typhimurium encounters many different intracellular and extracellular environments during infection. The complex regulation of SPI1 virulence genes may be important to activate SPI1 gene expression during infection, but may also be important to down-regulate SPI1 gene expression when production of the type III secretion system would induce a bacteriocidal host response [20, 21]. Our current work on the HilA protein is an important first step towards future studies aimed at discovering non-transcriptional mechanisms that regulate expression of SPI1 virulence genes.
S. typhimurium strains and growth conditions
S. typhimurium strains used are all derivatives of SL1344. EE658 (hilA080:: Tn5lacZY), CL87 (iagB87::lacZY), EE656 (prgH020:: Tn5lacZY) and EE668 (hilA339::kan prgH020:: Tn5lacZY) are described in [10, 11, 22]. LM558 (hilA339::kan ΔaraBAD22 invF12-5:: Tn5lacZY) carrying pCR53 and pCH112 were used to express engineered versions of HilA from an arabinose-inducible promoter. The hilA ORF contains two possible AUG start codons that are separated by 22 codons. pCR53 expresses a 553 amino acid (63 kDa) derivative of HilA, which starts at the first AUG codon and ends at the natural stop codon of the chromosomal hilA ORF. pCH112 expresses a 575 amino acid (65.5 kDa) derivative of HilA, which starts at the first AUG codon and ends with an extra 22 amino acid C-terminal Myc-His-tag . S. typhimurium cultures were grown in 16 × 150 mm tubes at 37°C in Luria-Bertani (LB) medium composed of 0.5% Bacto-yeast extract, 1% Bacto-tryptone, and 1% NaCl.
To induce hilA expression and the production of HilA protein, S. typhimurium were grown under high osmolarity, low aeration conditions. Bacteria were first grown to saturation in 3 ml LB, by rolling tubes at an angle overnight. The overnight culture was then diluted 1:100 in fresh LB and 10 ml aliquots were shaken at 150 rpm in upright tubes. Aliquots were harvested at various times post-inoculation for analysis. To analyze SPI1 gene expression and HilA protein levels inS. typhimurium shifted from inducing conditions to high aeration vs. low aeration conditions, bacteria were grown in 10 ml aliquots as described above. At 3.5 hours post-inoculation, the induced culture was diluted 1:10 in fresh LB. The shift to high aeration conditions was achieved by rolling tubes containing 2 ml aliquots at an angle. The shift to low aeration conditions was achieved by shaking 10 ml aliquots in upright tubes at 150 rpm. β-Galactosidase assays were performed and Miller Units were calculated as described .
To compare native HilA to engineered HilA derivatives, SL1344 or LM558, carrying pCR53 or pCH112, were grown as described above in high osmolarity, low aeration conditions, except that the media for the LM558 strains was supplemented with 0.2% arabinose and 200 μg/ml ampicillin. Cells were harvested by centrifugation after 4 hours and boiled in 1 × SDS loading buffer.
HilA protein purification and production of polyclonal antisera
E. coli strain VV445 (BL21/pET15b-hilA) produces a C-terminal His-tagged derivative of HilA, HilA-His, that was purified and used for antibody production. pET15b-hilA was generated by cloning a DNA fragment containing 98% of the predicted hilA ORF, from the second AUG to the Bam HI site, into pET15b (Novagen, Inc.). 100 mL of VV445 culture was grown in LB to OD600 0.4–0.6 at 37°C, induced by the addition of 0.5 mM IPTG, and cultured for an additional 1.5 hours at 30°C. Cells were harvested by centrifugation at 5000 × g and resuspended in 10 mL 50 mM Tris-HCl pH 8, 2 mM EDTA, 0.1 mg/ml lysozyme, 0.1% TritonX-100. After a 20 minute incubation, the cells were lysed by sonication and centrifuged at 12000 rpm in an SS34 rotor for 20 minutes. The membrane fraction contained HilA-His and was resuspended in 4 mL of 1 × binding buffer (1 × BB: 20 mM Tris-HCl pH 7.9, 500 mM NaCl, 0.5% SDS, 10% glycerol). After incubation for 10 minutes at 30°C, the solution was passed through a 0.45 μm low protein binding disk filter and loaded onto a 2 mL bed volume of His-bind resin at room temperature. The resin was washed extensively with 1 × BB and then with 1 × BB + 20 mM imidazole. Protein was eluted with 1 × BB + 1 M imidazole and analyzed by SDS-PAGE and Coomassie staining. A 2 mg/mL solution of the purified HilA-His protein was used to inoculate a rabbit (Cocalico Biologicals, Inc.). The initial injection contained 100 μg HilA-His and Freund's complete adjuvant, and boosters contained 50 μg HilA-His and Freund's incomplete adjuvant.
Subcellular fractionation and protein analysis
To examine the subcellular localization of native HilA in S. typhimurium, strain EE656 was grown in inducing, high osmolarity, low aeration conditions as described above. The S. typhimurium cells were harvested by centrifugation for analysis at 3 hours post-inoculation. To examine the subcellular localization of HilA-Myc-His in E. coli, an overnight culture of DH5α/pCH112 was diluted 1:1000 in fresh LB, shaken in a flask for 2.5 hours, and then supplemented with 0.2% arabinose. The E. coli cells were harvested by centrifugation 1 hour and 2 hours after the addition of arabinose. Bacterial cells were lysed by sonication in lysis buffer (20 mM HEPES pH 7.0, 1 mM DTT, 1 mM EDTA, 20% glycerol, 100 mM NaCl). Unbroken cells were removed by low-speed centrifugations and the cleared cell lysates were ultracentrifuged in a TLA100.3 rotor for 2 hours at 45 K rpm 4°C to separate the membrane and soluble fractions.
Samples were boiled for 10 minutes in 1 × SDS loading buffer and were electrophoresed on 5% stacking/7.5% resolving SDS-PAGE gels at 25 mA for 5.5–6 hours until a 45 kDa pre-stained protein standard was running off the bottom of the gel. This protocol was necessary to clearly separate the hilA-specific 63 kDa protein from the 65.5 kDa reactive protein that was present in both hilA- and hilA+ strains. Proteins were transferred from the gel to a PVDF membrane in a semi-dry blotter at 150 mA for 2 hours. HilA was detected following Western analysis using a 1:5000 dilution of polyclonal antiserum in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 1% BSA, followed by secondary detection using a goat anti-rabbit horseradish peroxidase conjugate diluted 1:10,000 in TBST. Whole cell and subcellular samples were normalized relative to bacterial number as estimated from OD600 measurements of the original cultures. Normalization was verified by running samples on SDS-PAGE and examining protein levels by Coomassie staining.
This work was supported by NIH grants, T32 AI07410 and R01 AI33444, and the John Taplin Funds for Discovery. We thank Vivek Bajaj for constructing strain VV445 and Priscilla McDowell for assistance with preliminary studies. We thank Sumita Jain and Jim Day for helpful comments about the manuscript.
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