Influence of dTMP on the Phenotypic Appearance and Intracellular Persistence of Staphylococcus aureus

Bacterial strains and growth conditions. S. aureus T1172 and S67 are clinical thymidine-dependent SCVs cultured from respiratory specimens from CF patients. Both of these isolates have deletions in thyA; T1172 has an internal 12-bp deletion including the codon for Cys-201, which is implicated in the active site of the thymidylate synthase (3), and S67 harbors a 3-bp deletion (ΔN83). Isolate S114 is a spontaneous revertant of S67 displaying an NCV phenotype with a wild-type thyA sequence. In addition, S. aureus SH1000 was used in this study. This strain is a derivative of S. aureus 8325-4, in which the original small deletion in rsbU has been repaired. The rsbU gene encodes a positive regulator of σ^B activity and consequently influences the expression of many virulence-associated loci (10). A thyA knockout mutant of S. aureus SH1000 (SH1000thyA; SCV phenotype) and thyA-complemented strain SH1000thyA (NCV phenotype) were used as defined SCV and NCV control strains (3).

Bacteria were grown aerobically on sheep blood agar (heipha, Eppelheim, Germany), Mueller-Hinton agar (heipha), and yeast extract-tryptone (YT) agar (5 g/liter yeast extract, 8 g/liter tryptone, 5 g/liter NaCl, 1.5% [wt/vol] agar). Antibiotics for cultivation of the S. aureus SH1000 mutants were used at the following final concentrations: chloramphenicol (Sigma-Aldrich, Munich, Germany), 30 μg/ml; and erythromycin (Sigma-Aldrich), 10 μg/ml.

Influence of dTMP on the phenotypic appearence of S. aureus. (i) Thymidylate synthase inhibition assay.An assay to determine the inhibition of thymidylate synthase was performed by placing a disk impregnated with 150 μg 5-fluorouracil (Sigma-Aldrich) on an agar plate that had been inoculated with an S. aureus strain.

(ii) Nucleoside uptake inhibition assay.An assay to determine the inhibition of thymidine uptake was performed by placing a disk impregnated with 150 μg uridine (Sigma-Aldrich) on an agar plate that had been inoculated with the appropriate S. aureus strain.

(iii) Dihydrofolate reductase and dihydropteroate synthase inhibition assay.An assay to determine the inhibition of dihydrofolate reductase and dihydropteroate synthase was performed by placing an SXT Etest strip (AB Biodisk, Solna, Sweden) on either YT agar or Mueller-Hinton agar that had been inoculated with S. aureus SH1000 (NCV).

(iv) Thymidine auxotrophism.Bacteria unable to grow on Mueller-Hinton agar were analyzed to determine their dependence on thymidine. A strain was considered auxotrophic if a zone of growth was detected around a disk impregnated with 15 μg thymidine (Sigma-Aldrich) after 24 h of incubation at 37°C.

(v) dTMP utilization assay.A disk impregnated with 150 μg dTMP (Sigma-Aldrich) was placed on either Mueller-Hinton agar or sheep blood agar that had been inoculated with S. aureus T1172 (SCV). To prove that an enzyme secreted by proliferating S. aureus enables conversion of dTMP to thymidine, a disk impregnated with 15 μl of a sterile-filtered S. aureus supernatant was placed on a Mueller-Hinton agar plate that had been inoculated with S. aureus T1172. For this purpose, S. aureus SH1000 (NCV) was incubated overnight in Mueller-Hinton broth and centrifuged, and the supernatant was sterile filtered by use of a 0.45-μm-pore size filter. To prove that the observed phenomena were due to a protein, the dTMP utilization assay was repeated with a supernatant exposed to proteinase K (Carl Roth GmbH, Karlsruhe, Germany) at a final concentration of 1 mg/ml for 30 min at room temperature.

(vi) Determination of protein concentration and extraction of nucleotides.Bacteria were grown in 500 ml brain heart infusion broth (Becton, Dickinson and Company, Sparks, MD) to the exponential growth phase and subsequently harvested by centrifugation at 3,000 × g for 20 min at 4°C. For determination of the protein concentration, 50 μl of a bacterial suspension was placed in 100 μl of buffer 1 containing 10 mM Tris-HCl and 85 μg/ml lysostaphin (Sigma-Aldrich) (pH 7.5). After incubation for 30 min at 37°C, 100 μl of buffer 2 containing 10 mM Tris-HCl and 245 μg/ml phenylmethanesulfonyl fluoride (Sigma-Aldrich) (pH 7.5) was added and incubated for 10 min at room temperature. Protein concentrations were then determined by the BCA protein assay (Pierce, Rockford, IL) as recommended by the manufacturer.

Extraction of nucleotides was performed as described by Müller et al. (17), with some modifications. The bacterial suspension described above was centrifuged at 4,000 × g for 20 min at room temperature. Pellets were washed twice in distilled water and resuspended in 2 mM imidazole buffer (pH 7.0). Then 1.5 ml was transferred to 1.125 ml of 1 M phosphoric acid buffer containing 6.6 mg/ml EDTA (Sigma-Aldrich). For nucleotide extraction, bacteria were incubated at 4°C for 50 min, sonicated for 2 min, and centrifuged for 15 min at 16,000 × g. The pH of the supernatant was adjusted to pH 7.7 with 0.72 M KOH containing 0.16 M KHCO₃, and the supernatant was centrifuged again. The supernatant was immediately stored at −20°C for high-performance liquid chromatography (HPLC).

(vii) HPLC analysis.Samples were separated by reversed-phase chromatography using a 5-μm GROM-SIL 120 ODS-5 ST column (GROM, Herrenberg, Germany). The solvent system used comprised solvent A (0.2 M triethylammonium acetate [Sigma-Aldrich] [pH 6.6]) and solvent B (0.2 M triethylammonium acetate [pH 6.6] and acetonitrile [Sigma-Aldrich], 95/5). The following gradient was used: 0 to 10 min, 4% solvent B; 10 to 35 min, 4 to 100% solvent B; 35 to 50 min, 100% solvent B; and 50 to 52 min, 100 to 4% solvent B. Chromatography was carried out at a flow rate of 0.8 ml/min at 30°C. Nucleotides were monitored by determining the absorbance at 260 nm by using a PDA-100 photodiode array detector (Dionex, Idstein, Germany).

Intracellular persistence. (i) Cell culture.The adenovirus-immortalized epithelial IB3-1 cell line (ATCC, Wesel, Germany) was derived from a CF patient and is compound heterozygous for the ΔF508 mutation and a nonsense mutation, W1282X. This cell line was maintained in Dulbecco's modified Eagle medium (DMEM/F-12) (Gibco, Paisley, United Kingdom) supplemented with 10% fetal bovine serum (PAA, Lölbe, Germany) and an antibiotic-antimycotic solution containing (final concentrations) 200 U penicillin G/ml (Gibco), 200 μg streptomycin sulfate/ml (Gibco), and 2.5 μg amphotericin B/ml (Sigma-Aldrich).

(ii) Intracellular persistence assays.Intracellular persistence assays were performed as described previously (11), with some modifications. For invasion experiments, epithelial cells were seeded onto DMEM/F-12 without any supplements and allowed to grow at 37°C in the presence of 5% CO₂ for 7 days until a confluent monolayer (about 5 × 10⁶ cells/tissue culture flask) was obtained. Cells in a cell culture flask treated in the same way were suspended by trypsinization and enumerated using a counting chamber before each invasion experiment to determine the bacterial inoculum. Invasion experiments were performed by adding 2 ml of DMEM/F-12 containing S. aureus at a multiplicity of infection (MOI) of 50:1 to each tissue culture flask (BD Biosciences, Erembodegem, Belgium), followed by incubation for 3.5 h at 37°C in the presence of 5% CO₂. The cells were then washed two times with phosphate-buffered saline (PBS) (PAA). After this, the cells were incubated either with 2 ml of DMEM/F-12 containing lysostaphin, trimethoprim, and sulfamethoxazole (ratiopharm, Ulm, Germany) at final concentrations of 10, 33, and 167 μg/ml, respectively, for 40 min or 24, 48, or 72 h or with DMEM/F-12 containing lysostaphin at a final concentration of 10 μg/ml. The solutions in the appropriate tissue culture flasks were renewed after 24, 48, and 72 h. After the appropriate incubation time, cells were washed four times with PBS. The absence of extracellular bacteria in the supernatant was proven by determination of the number of CFU on sheep blood agar. The tissue culture flasks were incubated with 4.5 ml PBS and 0.5 ml 0.5% trypsin-EDTA (Gibco) at 37°C in the presence of 5% CO₂. After detachment of the IB3-1 cells, the solutions were transferred to 5 ml of DMEM/F-12, and the cells were counted using a counting chamber. Then 166 μl of 0.4% trypan blue (Gibco) was added to 1-ml portions of the cell suspensions to verify the integrity of the human cells by light optical microscopy. Portions (9 ml) of the cell suspensions were centrifuged two times at 3,000 × g for 10 min at 4°C, transferred to 1 ml of distilled H₂O for 25 min at room temperature to release intracellular bacteria, and then plated on sheep blood agar for determination of the number of CFU.

(iii) Immunofluorescence staining and laser scanning microscopy.To determine the intracellular location of S. aureus in IB3-1 cells used in the intracellular persistence assay, laser scanning microscopy was performed. Invasion of epithelial cells was induced as described above, with some modifications. Two hundred microliters of DMEM/F-12 containing S. aureus SH1000 at an MOI of 50:1 was added to each well of a chamber slide (Nunc, Rochester, NY), which was followed by incubation for 3.5 h at 37°C in the presence of 5% CO₂. Cells were washed two times with PBS, and subsequently DMEM/F-12 containing 10 μg lysostaphin/ml was added and the preparations were incubated for 40 min at 37°C.

(a) Staining with SYTO9, propidium iodide, and Alexa Fluor 633 phalloidin.Cells were washed three times with sodium chloride. Then 0.6 μl of a dye mixture consisting of equal volumes of SYTO9 (Invitrogen, Carlsbad, CA), which stained dead bacteria, and propidium iodide (Invitrogen), which stained living bacteria, was added to 200 μl of a physiological sodium chloride solution. This mixture was added to a well, and cells were incubated at room temperature in the dark for 15 min. The cells were washed two times with PBS, and the sample was fixed in a 3.7% formaldehyde solution in PBS for 10 min at room temperature. The cells were washed two times with PBS. Then the adherent IB3-1 cells were incubated with 0.1% Triton X-100 (AppliChem, Cheshire, CT) in PBS for 5 min and washed two times with PBS. To stain F-actin of IB3-1 cells, 5 μl of Alexa Fluor 633 phalloidin (Invitrogen) and 200 μl of PBS were added to each well. To reduce nonspecific background staining, 1% bovine serum albumin (BSA) (PAA) was added to the staining solution, and cells were incubated for 20 min at room temperature, washed two times, and finally covered with coverslips with DAKO fluorescent mounting medium (DAKO Diagnostika GmbH, Hamburg, Germany).

(b) Staining with propidium iodide, Alexa Fluor 633 phalloidin, S. aureus antibody, and Alexa Fluor 488 goat anti-rabbit immunoglobulin G.For propidium iodide staining, formaldehyde fixation, and Triton X-100 incubation, cells were treated as described above. After two washes with PBS, cells were incubated with 1% BSA in PBS for 30 min to block nonspecific binding of the antibodies. Cells were incubated in PBS containing 400 μg/ml of diluted antistaphylococcal rabbit antibody (Abcam, Cambridge, MA) and 1% BSA for 1 h at room temperature. The solution was decanted, and the cells were washed two times in PBS for 5 min. IB3-1 cells were incubated with 5 μg/ml of the Alexa Fluor 488 goat anti-rabbit secondary antibody in 1% BSA in PBS in the dark for 1 h at room temperature. After three 5-min washes, Alexa Fluor 633 was added, and each sample was covered with a coverslip with DAKO fluorescent mounting medium as described above.

(c) Imaging.Human cells and intracellular S. aureus were analyzed with a Zeiss LSM 510 confocal scanning microscope (Zeiss, Göttingen, Germany). For triple labeling Alexa Fluor 633-labeled IB3-1 cells were visualized using 633-nm monochromatic light with a dichroic beam splitter (488, 543, and 633 nm) and a 650-nm emission longpass filter. For detection of living SYTO9-labeled S. aureus and dead propidium iodide-labeled S. aureus, 488- and 543-nm monochromatic light, respectively, and 505- to 530-nm and 580- to 615-nm emission bandpass filters, respectively, were used. For the secondary antibody Alexa Fluor 488 goat anti-rabbit immunoglobulin G-labeled S. aureus, 488-nm monochromatic light, and a 505- to 530-nm emission bandpass filter were used. Confocal images were obtained at a resolution of 1024 × 1024 pixels and a magnification of ×630 in multitracking mode to prevent cross talk between confocal channels.

Statistics.The statistical analysis was performed with the unpaired Student t test. P values of <0.05 were considered statistically significant.

Influence of dTMP on the phenotypic appearance of S. aureus.Inhibition assays were used to analyze the metabolic pathways that are associated with the phenotypic appearance of thymidine-dependent S. aureus SCVs. The influence of 5-fluorouracil, acting mainly as an inhibitor of thymidylate synthase (19), on the phenotype of S. aureus is shown in Fig. 1. S. aureus SH1000, an NCV, exhibited a zone of growth reduction around the disk impregnated with 5-fluorouracil on sheep blood agar (Fig. 1A). A thin bacterial lawn of small colonies grew around the disk, indicating that the strain utilized thymidine provided by the sheep blood agar to establish an alternative pathway for dTMP synthesis. In contrast, when S. aureus SH1000 was cultivated on Mueller-Hinton agar that did not contain thymidine, a clear-cut zone of inhibition was observed around the impregnated disk (Fig. 1B). The thymidine dependence of thymidylate synthase-inhibited S. aureus SH1000, in turn, was proven by growth of S. aureus SH1000 around a disk impregnated with thymidine within the zone of inhibition of 5-fluorouracil on Mueller-Hinton agar (Fig. 1C). S. aureus T1172 did not grow on Mueller-Hinton agar because this strain is a thymidine-dependent SCV. The growth on thymidine-providing sheep blood agar of S. aureus T1172 was not affected by 5-fluorouracil (Fig. 1D), because thymidylate synthase has no function in this strain (3).

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FIG. 1.

Influence of 5-fluorouracil and uridine on the S. aureus phenotype. S. aureus SH1000 (NCV) was grown on sheep blood agar (SB) (A and F) and Mueller-Hinton agar (MH) (B and C), S. aureus T1172 (SCV) was grown on sheep blood agar (D and E), S. aureus SH1000thyA was grown on sheep blood agar (G), and thyA-complemented SH1000thyA was grown on sheep blood agar (H). Strains were exposed to 5-fluorouracil (f), thymidine (t), and uridine (u).

The influence of uridine, an inhibitor of nucleoside transport (18, 33), on the phenotype of S. aureus is shown in Fig. 1E and 1F. The growth of S. aureus SH1000 was not influenced by this inhibitor either when the strain was cultivated on Mueller-Hinton agar or when it was cultivated on blood agar (data not shown), indicating that the thymidylate synthase pathway alone is sufficient to provide the bacterial cell with dTMP. However, S. aureus T1172, which is dependent on an extracellular thymidine supply, did not grow in the presence of uridine, as indicated by the zone of inhibition around the impregnated disk (Fig. 1E). The combined effects of inhibited thymidylate synthase and an inhibited nucleoside transporter are shown in Fig. 1F. S. aureus SH1000 could not grow on blood agar when it was exposed to both 5-fluorouracil and uridine, as indicated by the inhibition zone between the impregnated disks (Fig. 1F). The results observed with S. aureus T1172 (SCV) and SH1000 (NCV) were confirmed with defined SCV and NCV strains, strain SH1000thyA and thyA-complemented strain SH1000thyA, respectively. The growth of SH1000thyA was not impaired by 5-fluorouracil but was inhibited by uridine (Fig. 1G), whereas the growth of the isogenic thyA-complemented strain SH1000thyA was impaired by 5-fluorouracil and inhibited by exposure to both 5-fluorouracil and uridine (Fig. 1H).

The influence of trimethoprim and sulfamethoxazole, inhibitors of dihydropteroate synthase and dihydrofolate reductase, respectively, is shown in Fig. 2. S. aureus SH1000 inoculated onto YT agar exhibited an elliptic zone in which growth was reduced along an SXT Etest strip (Fig. 2A). Tiny colonies were present close to the Etest strip because YT agar provides thymidine and consequently facilitates small-colony growth (Fig. 2B). In contrast, when S. aureus SH1000 was cultivated on Mueller-Hinton agar that did not provide thymidine, a clear-cut zone of inhibition was observed around the Etest strip. In turn, the thymidine dependence of S. aureus SH1000 exposed to SXT on Mueller-Hinton agar was proven by growth of tiny SCVs around a disk impregnated with thymidine (Fig. 2C and D).

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FIG. 2.

Influence of SXT on the S. aureus phenotype. S. aureus SH1000 was inoculated onto YT agar (A and B) and Mueller-Hinton agar (C and D) in presence of a co-trimoxazole Etest strip (A to D) and thymidine (C and D).

There was no direct uptake of extracellular dTMP in S. aureus as thymidine-dependent S. aureus T1172 was not able to grow in the presence of dTMP on Mueller-Hinton agar (Fig. 3A). However, growth of S. aureus T1172 on sheep blood agar was enhanced by extracellular dTMP and blocked by uridine (Fig. 3B and C), indicating that an extracellular enzyme produced by proliferating bacteria dephosphorylated dTMP to thymidine.

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FIG. 3.

Utilization of dTMP. S. aureus T1172 was inoculated onto Mueller-Hinton agar (A and D) and sheep blood agar (B and C) in the presence of dTMP (dt), S. aureus SH1000 supernatant (s), and uridine (u).

To further prove that such an extracellular enzyme is produced by proliferating S. aureus, a disk impregnated with sterile-filtered supernatant of S. aureus SH1000 was placed on Mueller-Hinton agar, which was inoculated with S. aureus T1172. Growth of S. aureus T1172 was then observed in presence of a dTMP-impregnated disk (Fig. 3D). No growth was observed when proteinase K was added to the supernatant (data not shown).

All experiments described above were repeated with the clinical strain S. aureus S67 (SCV) and the corresponding revertant strain S114 (NCV), which had the same phenotypic characteristics as S. aureus T1172 (SCV) and SH1000 (NCV).

To verify the impact of dTMP on the phenotypic appearance of S. aureus, HPLC was performed to determine the intracellular levels of dTMP in S. aureus SCVs and NCVs. S. aureus T1172, S67, S114, and SH1000 (SH1000 with and without addition of SXT) were cultured in brain heart infusion broth, nucleotides were extracted, and the levels of intracellular dTMP, dUMP, and dGMP were determined by HPLC, as described in Materials and Methods. The levels of both dUMP and dTMP were related to either bacterial protein or dGMP concentrations as independent reference parameters. While dUMP accumulated in both clinical and SXT-induced SCVs, dTMP was not detectable in these strains. In contrast, dTMP was detectable in S. aureus NCVs, whereas dUMP was not detectable (Table 1).

Intracellular persistence.In view of the impact of SXT on the phenotypic appearance of S. aureus, an intracellular persistence assay was performed. Monolayers of IB3-1 epithelial CF cells were incubated with S. aureus SH1000 at an MOI of 50:1 in cell culture flasks for 3.5 h to enable intracellular uptake. Cell culture medium supplemented with lysostaphin was then added to inhibit extracellular growth of bacteria. In order to assess the impact of dTMP synthesis inhibitors on intracellular persistence, experiments were performed with and without SXT added to the cell culture medium. The numbers of intracellular bacteria were determined after 40 min and 24, 48, and 72 h of incubation as described in Materials and Methods. After 40 min of incubation approximately 10 bacteria per epithelial cell were observed (Fig. 4). The average number of intracellular S. aureus SH1000 cells was found to decrease steadily over the 72-h time period to 0.03 bacterium per epithelial cell when cells were not exposed to SXT. S. aureus SH1000 exposed to SXT, however, showed significantly greater intracellular persistence after 48 and 72 h of incubation than unexposed S. aureus SH1000. Similar significantly greater intracellular persistence in IB3-1 cells after 72 h was found for S. aureus SCV S67 compared to the isogenic NCV revertant S. aureus S114 (data not shown). To verify the intracellular location of S. aureus, laser scanning microscopy was performed. After 3.5 h of incubation with S. aureus SH1000 and elimination of extracellular bacteria by lysostaphin exposure and washing, IB3-1 epithelial CF cells were stained with different markers as described in Materials and Methods. Staining with propidium iodide, which accumulates in dead bacteria, and SYTO9, which marks living bacteria, revealed that more than 99.9% of the bacteria were alive within F-actin-stained IB3-1 cells (data not shown). In addition, S. aureus-specific immunofluorescence staining confirmed the intracellular location of S. aureus (Fig. 5).

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FIG. 4.

Intracellular persistence of S. aureus SH1000 in CF cells: numbers of intracellular bacteria after incubation with and without SXT. The numbers of CFU were determined after 40 min and 24, 48, and 72 h. Statistically significant differences (P < 0.05) are indicated by asterisks. The bars indicate the means and the error bars indicate the standard deviations of four experiments.

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FIG. 5.

Immunofluorescence staining and laser scanning microscopy of intracellular S. aureus SH1000. Infected IB3-1 CF cells were stained with F-actin-affine Alexa Fluor 633 phalloidin (blue). S. aureus was stained with antistaphylococcal rabbit antibody and Alexa Fluor 488 goat anti-rabbit immunoglobulin G (green).