Green Fluorescent Protein as a Marker inRickettsia typhi Transformation

Host cells.Vero cells were cultured in EMEM (Eagle’s minimum essential medium with glutamine; Biofluids, Rockville, Md.) and 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, Ga.) at 37°C and 5% CO₂. The Vero cells were grown to complete monolayer in 25-cm² or 150-cm² flasks (Nunc, Inc., Naperville, Ill.) prior to infection. After infection withR. typhi, the cells were cultured in EMEM with 4% FBS at 34°C and 5% CO₂. At various time points postinfection, Vero cells were assessed for the level of rickettsial infection by staining with Diff-Quik (Dade International Inc., Miami, Fla.). Briefly, a portion of cells were detached from the culture flask by using a cell scraper (Nunc), and 1.5 ml of medium from the flask was spun at 1,000 × g for 5 min, and the supernatant was discarded. The pellet was gently resuspended in SPG buffer (0.218 M sucrose, 0.0038 M KH₂PO₄, 0.0072 M K₂HPO₄, 0.0049 M l-glutamate [pH 7.2]), placed in a Cytospin chamber slide (Wescor, Logan, Utah), and spun at 2,000 × g for 10 min in a Cytopro centrifuge (Wescor). Slides were air dried for 10 min and then processed by Diff-Quik staining as directed by the manufacturer. Slides were viewed at a magnification of ×100 under oil immersion.

Bacterial strains. R. typhi Wilmington (10⁸ PFU/ml) was stored frozen at −80°C. A 200-μl aliquot was quickly thawed in a 37°C water bath and used to infect 10 150-cm² flasks of Vero cell monolayers. Infected cells were grown at 34°C with 5% CO₂ for 8 days (approximately 40 bacteria per cell). Vero cell cultures infected with R. typhi were harvested by centrifugation at 13,000 × g for 10 min. The supernatant was discarded, and the pellet (containing infected Vero cells and released rickettsiae) was resuspended in 10 ml of sterile electroporation buffer (EB; H₂O, 10% glycerol; 0.2-mm-pore-size Millipore [Bedford, Mass.] filter). The suspension was spun at 1,500 × g for 10 min to pellet the cellular debris, and the supernatant (containing predominantly R. typhi) was transferred to a new tube and spun at 13,000 × g for 10 min. The pellet was washed three times in EB, finally resuspended in 500 μl of fresh EB, and kept on ice for use in the transformation assay.

As an added control for assessing bacterial GFP_UVexpression in Vero cells, we chose a hemolysin-negative mutant ofProteus mirabilis, strain WPM111 (3). This bacterium can invade mammalian cells but does not lyse the cells (3). We previously transformed this strain with pGFPuv, which encodes a GFP variant optimized for expression in prokaryotes and fluorescence with UV light (Clontech, Palo Alto, Calif.) (2, 4, 10). P. mirabilis cultures were grown overnight on Luria-Bertani (LB) agar plates at 37°C. Expression of GFP_UV was visible by viewing culture plates on a UV light box (Transilluminator UVP; San Gabriel, Calif.).

P. mirabilis internalization assay.Vero monolayers were inoculated with approximately 200 μl of overnightP. mirabilis culture diluted in 5 ml of EMEM. Flasks were placed on a rocker at room temperature for 2 h and then at 37°C for an additional hour. Medium was discarded from the flasks, and the monolayers were rinsed with phosphate-buffered saline (PBS) to remove any extracellular P. mirabilis. Cultures were harvested by adding 5 ml of trypsin-EDTA (Gibco BRL, Gaithersburg, Md.) to each flask. After 3 min, trypsin was discarded, and the cells were washed with 5 ml of PBS and transferred to a 15-ml centrifuge tube (Fisher, Pittsburgh, Pa.). Samples were spun at 1,000 × g for 5 min, the medium was discarded, and the pellet was resuspended in 5 ml of cold PBS. Infected cells were washed twice more in PBS, then resuspended in 1.5 ml of cold 1.0% paraformaldehyde (Sigma, St. Louis, Mo.), and incubated at room temperature for 90 min. Cells were spun at 1,000 × g for 5 min; the pellet was washed twice in PBS and resuspended to a final volume of 1 ml of PBS. Cells were analyzed the same day on a flow cytometer (FACSort; Becton Dickinson, San Jose, Calif.).

rpoB-GFP_UV fusion construction.The GFP_UV gene was amplified from pGFPuv by PCR. The primers used for amplification (ClaI-GFP_UV forward [5′ CAT CGA TAG AAA AAA TGA GTA AAG GA 3′] and reverse [5′ GAT CGA TTC ATT ATT TGT AGA GCT C 3′]) each had aClaI site incorporated to facilitate subcloning into therpoB gene. PCRs were done with 0.5 μg of pGFPuv, 1 μM each ClaI-GFP_UV forward and reverse primers, and 47 μl of Supermix (Gibco BRL), overlaid with 50 μl of mineral oil. Unless otherwise noted, temperature conditions for all PCRs were as follows: 94°C for 1 min, 37°C for 1 min, and 72°C for 1 min for 30 cycles. The final cycle had an additional incubation step at 72°C for 10 min. All PCRs were performed on a Perkin-Elmer DNA thermocycler (Perkin-Elmer Cetus, Norwalk, Conn.). The amplicon was subcloned into a TA cloning vector (Invitrogen, Carlsbad, Calif.) for propagation inEscherichia coli 505 (Dcm⁻ Dam⁻). Plasmid was purified from culture by using a Wizard miniprep kit (Promega, Madison, Wis.) and was digested with ClaI (Gibco BRL) at 37°C for 1 h to excise the GFP_UV fragment. The digestion products were analyzed on a 1.0% agarose gel and subsequently purified from the gel by using GeneClean (Bio 101, Vista, Calif.). The purified GFP_UV-ClaI fragment was ligated into a ClaI site located at nucleotide (nt) 3472 in the C′ terminus of the rickettsial rpoB gene, which was prepared in the following manner. The last 1 kb of R. typhi rpoB was PCR amplified from R. typhi genomic DNA with primers rpoB forward (nt 3122 to 3138; 5′ CAA ATG TAA TGA ATG AA 3′) and rpoB reverse (nt 4105–4089; 5′ AGC TTT ACG TTG AGA CA 3′). The amplicon was subcloned into the TA cloning vector and propagated in E. coli One Shot (Invitrogen). Positive colonies were screened by PCR using GeneReleaser (BioVentures, Inc., Murfreesboro, Tenn.) and therpoB primers noted above. The plasmid was then purified from a positive colony with a Wizard miniprep kit (Promega), digested withClaI (Gibco BRL) 37°C for 1 h, and treated with calf intestinal alkaline phosphatase (1 μl in 1× buffer; New England Biolabs, Beverly, Mass.) at 37°C for 1 h. The linearized plasmid was gel purified by using GeneClean (Bio 101). For ligation with GFP_UV, approximately 0.1 μg of linearized rpoBand 0.3 μg of the GFP_UV-ClaI fragment were mixed with 1 μl of T4 DNA ligase (Gibco BRL) in 1× buffer. The ligation reaction mixture was incubated at 16°C for 4 h, and 2 μl was used to transform E. coli (Invitrogen). Transformants were grown on LB-ampicillin (100 μg of ampicillin per ml) plate, and positive colonies were screened by GeneReleaser (BioVentures), using primers for ClaI-GFP_UV as described above. Positive colonies were assessed for in-frame ligation of GFP_UV with rpoB by sequencing using dye terminator method with an Applied Biosystems (Foster City, Calif.) model 373 automated sequencer. The DNA sequences were analyzed with the Sequence Editor 675 SeqEdTM software package (Applied Biosystems). Forward and reverse sequences were aligned and compared for fidelity.

The cloned rpoB-GFP_UV region (1.7 kb) was amplified by PCR using rpoB forward and reverse primers as described above. The amplicon was purified from the PCR mixture by phenol-chloroform-isoamyl alcohol extraction followed by ethanol precipitation. The resulting pellet was resuspended in H₂O and was stored on ice until used in the electroporation of R. typhi.

Transformation of rickettsiae.For the transformation procedure, 100 μl of R. typhi in EB was placed in a 0.2-cm-gap cuvette (BTX Inc., San Diego, Calif.) and kept on ice for 1 min; 1 to 5 μl of rpoB-GFP_UV DNA (total of 3 μg) was added to the cuvette (controls without DNA received equal amounts of water). The samples were electroporated at 2.5 kV and 200 Ω, with a time constant of approximately 5 ms (nonelectroporated controls remain on ice). Immediately, 1 ml of prechilled SPG buffer was added, and the electroporated rickettsiae were allowed to infect Vero cells grown in 25-cm² flasks (Nunc). Vero cells were then incubated at room temperature for 30 min. After addition of 5 ml of medium (EMEM, 4.0% FBS), each flask was incubated at 34°C with 5% CO₂. Controls included Vero cell monolayers incubated with the following: 100 ml of SPG, 100 μl of nonelectroporated rickettsiae, 100 μl of electroporated rickettsiae (no DNA added), 100 μl of rickettsiae with 3 μg of DNA added (no electroporation), and 100 μl of electroporated rickettsiae with 3 μg of DNA. After 8 days, all samples were assayed by PCR for detection of GFP_UV, using the ClaI-GFP_UV forward and reverse primers described above. To confirm integration of the construct into the rickettsial genome, a second PCR was performed withClaI-GFP_UV reverse and anrpoB-specific forward primer (nt 3097 to 3113; CTT TGG CAG ATT ACA GT) which is located 5′ of the construct incorporation site. PCR templates were prepared by harvesting infected cells from flasks and boiling them in sterile H₂O for 5 min. Five microliters of the boiled mixture was used as the template for PCR. Temperature conditions for PCR were as follows: 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min for 35 cycles. All PCR products were analyzed on 1.0% agarose gels. Samples positive by PCR were further analyzed by restriction fragment length polymorphism (RFLP) to confirm identities of the amplicons. Ten microliters of each PCR mixture was added to 10 μl of water, 2.2 μl of 10× buffer, and 5 U of ClaI. Cultures that were positive by PCR and RFLP were transferred to fresh Vero cell monolayers. Flasks of uninfected or infected Vero cells were placed on a UV light box for visualization of GFP_UV expression by R. typhi.

Flow cytometry for detection of R. typhiRpoB-GFP_UV expression and isolation of transformants.Vero infected with mock-transformed R. typhi orrpoB-GFP_UV-transformed R. typhi were harvested 8 days postinfection by the addition of 5 ml of trypsin-EDTA (Gibco BRL) to the 150-cm² culture flasks. After 3 min, trypsin was discarded, and the cells were washed with 10 ml of EMEM and transferred to a 15-ml centrifuge tube (Fisher). Samples were spun at 1,000 × g for 5 min, the medium was discarded, and the pellet was resuspended in 10 ml of cold PBS. Infected cells were washed twice more in PBS, resuspended in 1 ml of cold 1.0% paraformaldehyde (Sigma), and incubated at room temperature for 1.5 h. The cells were spun at 1,000 × g for 5 min, and the pellet was washed twice in PBS and resuspended in a final volume of 600 μl of PBS. Cells were analyzed the same day on a flow cytometer, and GFP_UV fluorescence was detected by excitation at 488 nm and emission at 520 nm (FACSort). Cultures which were positive by flow cytometry analysis were maintained for 6 additional weeks by harvesting the transformed R. typhi from a 10-day-old culture and subculturing them to a new monolayer of Vero cells. Infected cultures were subcultured every 7 to 10 days; after 6 weeks, cells were harvested and analyzed by flow cytometry as described above.