The Response Regulator PhoP Is Important for Survival under Conditions of Macrophage-Induced Stress and Virulence in Yersinia pestis

Bacterial strains, plasmids, growth conditions, and chemicals.Bacterial strains and plasmids used in this study are listed in Table 1. Y. pestiswas routinely cultured aerobically at 28°C in blood agar base (BAB) broth, on BAB agar (30), on Congo red agar (including 25 g of heart infusion broth/liter) (33), or onYersinia selective agar (Oxoid, Basingstoke, United Kingdom). Defined media were prepared as described by Straley and Bowmer (39). Escherichia coli strains were cultured and stored as described by Sambrook et al. (34). All chemicals were purchased from Sigma-Aldrich (Poole, United Kingdom). Ampicillin and tetracycline were used at final concentrations of 55 and 20 μg/ml, respectively. Unless otherwise stated, plasmid and genomic extractions, restriction enzyme digestions, DNA ligations, and transformations into E. coli were performed by standard procedures (34) using enzymes provided by Promega Ltd. (Southampton, United Kingdom) or Boehringer Mannheim (Lewes, United Kingdom).

PCR and cloning procedures.Oligonucleotide primers used for PCR are summarized in Table 2. TheY. pestis, Yersinia pseudotuberculosis, andYersinia enterocolitica phoP gene fragments were amplified using PCR with degenerate oligonucleotide primers (PCRDOP) (42) with primers P3 and P4, based on known PhoP sequence data for E. coli and Salmonella serovar Typhimurium (27). The PCR was performed with 40 cycles of 1 min at 94°C, 1 min at 40°C, and 1 min at 72°C. The 262-bp products were digested with PstI and HindIII, ligated into similarly digested pUC19, and transformed in E. coli XL2-Blue MRF′ cells (Stratagene Europe, Amsterdam, The Netherlands). The cloned fragments were sequenced using the dideoxynucleotide chain termination method with an Applied Biosystems (Warrington, United Kingdom) PRISM sequencing kit, and the data were compared with other PhoP sequences using BLASTX software (1).

To determine the entire phoP sequence, the cloned Y. pseudotuberculosis phoP gene fragment was used with Southern blotting to identify a 4-kbp DraI fragment and a 1.5-kbpSspI fragment from digests of Y. pseudotuberculosis chromosomal DNA. The fragments were isolated from a SeaPlaque GTG agarose gel (Flowgen, Sittingbourne, United Kingdom), ligated into pUC19/SmaI/BAP (Amersham Pharmacia Biotech, St. Albans, United Kingdom), and transformed into E. coli XL2-Blue MRF′ cells to generate plasmids pDA5 and pSA2, respectively. The nucleotide sequences of these cloned fragments allowed the design of primers P41 and P42, which were used to amplify a 1.5-kb product containing the entire Y. pestis or Y. pseudotuberculosis phoP genes. Both PCR products were purified through S-300HR (Amersham Pharmacia Biotech) and nucleotide sequenced. The complete phoP gene sequences from Y. pestisand Y. pseudotuberculosis were determined from sequence data from three independent PCR reactions.

A 31-bp deletion and unique BglII site were engineered into the phoP gene in plasmid pSA2, using inverse-PCR mutagenesis (IPCRM) (10, 43), with primers P9 and P10. Fifty picograms of alkali-denatured pSA2 was added to a PCR mixture containing primers P9 and P10 and subjected to PCR with 40 cycles of 1 min at 94°C, 1 min at 50°C, and 4 min at 72°C. PCR products were digested withBglII, self ligated, and transformed into E. coliXL2-Blue MRF′ to generate plasmid pSAI1. Primers P27 and P24, which flank the deletion site, were used to screen for mutants by PCR, using 1 μl of boiled cell supernatant added to a standard reaction mixture, which was subjected to 40 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C.

Construction of a Y. pestis phoP mutant.Plasmid DNA from pSAI1 was digested with XbaI and PvuII, and the excised fragment containing the mutated Y. pseudotuberculosis phoP gene was ligated with pCVD442, which had been previously digested with XbaI and SmaI. This ligation was electroporated into E. coli CC118 λpir cells to form pSAI2. pSAI2 was introduced into Y. pestis strain GB by conjugation in a three-way mating (25), using a Y. pestis GB wild-type recipient, E. coli CC118 λpir pSAI2, and E. coli S17 λpir pNJ5000 (18). Equal volumes (50 μl) of the three strains were mixed, and 100 μl was spotted onto an L agar plate. After incubation (28°C for 8 h), the bacteria were recovered, resuspended in 1 ml of Luria-Bertani (LB) broth, washed twice in LB broth, and cultured on Yersiniaselective agar containing ampicillin at 28°C. Individual ampicillin-resistant Y. pestis colonies were inoculated into BAB broth, grown overnight at 28°C, and plated onto BAB agar or BAB agar supplemented with 5% (wt/vol) sucrose (9). Sucrose-tolerant revertants occurred at a rate of 1 in 3 × 10⁴, and these colonies were screened using PCR with primers P27 and P24, followed by digestion of the PCR product withBglII. An ampicillin-sensitive Y. pestis phoPdouble-crossover mutant, termed SAI2.2, was identified and used for further studies.

Survival following environmental stresses and exposure to defensins.The effect of low pH, high osmolarity, and oxidative stress on Y. pestis was determined as described by Badger and Miller (3). Y. pestis wild-type andphoP mutant strains were grown overnight at 28°C, and the cells were pelleted and resuspended appropriately. For oxidative stress experiments, cells were incubated for 1 h at 28°C in 15 mM H₂O₂ in deionized water. For high-osmolarity stress, cells were incubated in 2.4 M NaCl in deionized water for 1 h at 28°C. For low-pH stress, cells were incubated in LB broth, adjusted to pH 3 with HCl, for 5 min at ambient temperature (22°C). Control bacteria were incubated in sterile phosphate-buffered saline (PBS) (pH 7.4). After exposure, the bacteria were pelleted at 12,000 × g for 5 min at 22°C, resuspended in 0.9% (wt/vol) NaCl, and enumerated after growth at 28°C for 48 h on Congo red agar. All survival experiments were repeated in triplicate.

Survival in J774 macrophage cell cultures. Y. pestiscells from 1-ml volumes of an 18-h culture grown at 28°C were diluted with 9 ml of PBS, centrifuged at 10,000 × g for 10 min at 22°C, and resuspended in 10 ml of PBS. The number of viable cells was determined after culturing dilutions of the cell suspensions on Congo red agar. J774 macrophage cells were seeded at a density of 5 × 10⁵ ml⁻¹ in Dulbecco's modified essential medium (Sigma-Aldrich) into 24-well tissue culture dishes and cultured until confluent. The tissue culture medium was removed, 150 μl (10⁶ cells) of the bacterial suspension in PBS was added, and the cells were incubated at 37°C for 30 min. The suspension above the cell monolayer was removed, and the cells were washed three times with PBS. One milliliter of 2% L15 medium (Sigma-Aldrich) containing 10 μg of gentamicin/ml was added, and the cells were incubated for 30 min at 37°C. The cells were washed twice with PBS, and 1 ml of 2% L15 medium containing 2 μg of gentamicin/ml was added to the cells. The cells were incubated at 37°C, and at various time points the growth medium was removed, the cells were washed with PBS, and 200 μl of 0.1% sodium deoxycholate was added to the cells, which were lysed by aspiration. The lysate was diluted in PBS, and the number of viable cells was determined after growth at 28°C for 48 h on Congo red agar. Duplicate samples were taken at all time points, and the assay was repeated four times.

2D gel electrophoresis.Protein profiles of Y. pestis wild-type and phoP mutant strains were compared by two-dimensional (2D) gel electrophoresis. Bacteria were grown for 6 h at 28°C or 37°C in BAB broth. Cells were washed once with PBS and then boiled for 10 min in a 0.1% (wt/vol) sodium dodecyl sulfate (SDS) solution. Cellular debris was removed by centrifugation, and then the proteins were concentrated using Amicon Centriplus concentrators (Millipore, Watford, United Kingdom) and stored at −20°C. Culture supernatants were treated with protease inhibitors (Complete; Roche Diagnostics Ltd., Lewes, United Kingdom), 9 mM urea, 65 mM dithiothreitol, and 2% (vol/vol) Triton X-100 to obtain completely denatured and reduced proteins. Proteins were separated using 2D gel electrophoresis as described by Gorg et al. (17). Immobiline dry strips pH 4-7 linear (18 cm; Amersham Pharmacia Biotech) were rehydrated with each protein sample (30 to 40 μg) as recommended by the manufacturers. Isoelectric focusing was carried out for 44,900 V · h at 20°C. After equilibration, horizontal SDS electrophoresis was carried out using Excel Gel SDS, with a gradient of 12 to 14% T (Amersham Pharmacia Biotech) at 15°C. The separated proteins were then visualized by a silver staining method (21). Molecular weight standards (Bio-Rad, Hemel Hempstead, United Kingdom) were also applied to the gel.

Determination of virulence in mice.The median lethal doses (MLD) of the Y. pestis wild-type and phoP mutant were assessed by subcutaneous injection of groups of five female 6-week-old BALB/c mice (Charles River Laboratories, Margate, United Kingdom) with serial dilutions of exponential-phase broth cultures grown at 28°C (33). Humane endpoints were strictly observed, and animals deemed incapable of survival were humanely killed by cervical dislocation. Times to humane death (typically 3 to 5 days) were recorded, and the MLD of the phoP mutant was determined by the method of Reed and Muench (32).

Nucleotide sequence accession numbers.The nucleotide sequences of the Y. pestis and Y. pseudotuberculosis phoP genes have been submitted to the EMBL database under accession numbers Y08758 and X66587 , respectively.