INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Rickettsia typhi, the causative agent of murine typhus, is one of the most widely distributed arthropod-borne diseases. This intracellular bacterium is transmitted to humans by infected fleas (1). Upon introduction to the human host, R. typhi infects endothelial cells and replicates in the cell cytoplasm. Ultimately, the cell bursts open, releasing the rickettsial progeny. The lysis of endothelial cells causes widespread vasculitis and is the basis of pathology for this rickettsial disease. Studies regarding the specific interactions involved in rickettsial entry and exit of the host cell have been hindered by lack of an efficient genetic manipulation system for rickettsiae. Only recently has transformation of rickettsiae been accomplished. By using electroporation and homologous recombination, it is now possible to introduce mutated rickettsial genes into the rickettsial cell (reference 9 and unpublished data). However, there are still many obstacles to overcome before transformation of rickettsiae is optimized. To date, no plasmids capable of autonomous replication within rickettsiae have been identified, nor have suitable selectable markers been found. Selectable markers such as antibiotic resistance to tetracycline or chloramphenicol would greatly simplify isolation of rickettsial transformants; however, their use is not advisable due to the importance of these antibiotics in treatment of rickettsial diseases.

In a previous study, a gene conferring resistance to the antibiotic rifampin (rpoB) was used to demonstrate transformation of R. prowazekii, the etiologic agent of epidemic typhus (9). Rifampin is a transcriptional inhibitor which acts by binding to the B subunit of RNA polymerase (RpoB). Certain amino acid substitutions within RpoB ablates rifampin binding and thereby confer resistance to this antibiotic (5-8, 11). Rachek et al. demonstrated transformation of R. prowazekii by electroporating the bacteria with a plasmid encoding a fragment of the rickettsial rpoB gene containing a G-to-A point mutation at position 1727 which results in rifampin resistance, along with two silent mutations as additional markers (9). Sequencing of the rpoB genes of the rifampin-resistant rickettsiae confirmed the presence of the engineered mutations in transformed rickettsiae but also detected untransformed rickettsiae with additional rpoB mutations which occurred spontaneously. In a similar study, we also obtained spontaneous rifampin-resistant mutations in rpoB of R. typhi (11). In both cases, the presence of spontaneous rifampin-resistant mutants confounded attempts to obtain pure transformed populations of rickettsiae.

We chose GFP_UV (a green fluorescent protein variant optimized for fluorescence under UV light) expression as a marker for R. typhi transformation experiments. The present study demonstrates the successful transformation of rickettsiae with a GFP_UV fusion construct and the isolation of transformed rickettsiae by fluorescence-activated cell sorting (FACS). This is the first report of transformation of rickettsiae with a nonrickettsial (GFP_UV) gene and demonstrates the utility of fluorometric identification of rickettsial transformants.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

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 with R. 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.

P. mirabilis internalization assay. Vero monolayers were inoculated with approximately 200 µl of overnight P. 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 a ClaI site incorporated to facilitate subcloning into the rpoB 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 in Escherichia 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 the rpoB primers noted above. The plasmid was then purified from a positive colony with a Wizard miniprep kit (Promega), digested with ClaI (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 rpoB and 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.

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 , 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 with ClaI-GFP_UV reverse and an rpoB-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. typhi RpoB-GFP_UV expression and isolation of transformants. Vero infected with mock-transformed R. typhi or rpoB-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.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

In this study, we used GFP_UV as a fluorescent marker in the transformation of R. typhi. Since transformation of rickettsiae has been accomplished only by homologous recombination (1), we created a GFP_UV fusion construct by ligating the GFP_UV coding region to the 3' terminus of the rpoB gene of R. typhi (Fig. 1, top). We selected the 3' terminus of rpoB since studies of E. coli have not shown the C terminus to be a functional domain. In-frame ligation of GFP_UV with rpoB was confirmed by sequence analysis, and the 1.7-kb fragment encoding the fusion protein was amplified by PCR for use in the transformation of R. typhi by electroporation. Electroporated R. typhi was subsequently allowed to infect Vero cells, and after 8 days infected Vero cultures were assayed first for the presence of rickettsiae and second for transformation of rickettsiae. Over 80% of the cells were infected with rickettsiae as revealed by Diff-Quik staining. Successful transformation of R. typhi with the rpoB-GFP_UV amplicon was initially confirmed by PCR. Using primers specific for GFPuv (ClaI-GFP_UV forward and ClaI-GFPuv reverse), the 730-bp GFP_UV amplicon was detected in Vero cells infected with transformed R. typhi but not in uninfected Vero cells or in Vero cells infected with untransformed R. typhi (data not shown). To confirm integration of the construct into the rickettsial genome, we performed a second PCR using ClaI-GFP_UV reverse and an rpoB-specific forward primer (nt 3097 to 3113; CTT TGG CAG ATT ACA GT) which is located 5' of the construct incorporation site. The 1.08-kb rpoB-GFP_UV amplicon was detected in Vero cells infected with transformed rickettsiae but not in uninfected Vero cells or Vero cells infected with untransformed R. typhi (data not shown). We confirmed the identity of the amplicon by RFLP using ClaI digestion (Fig. 1, bottom). We attempted to visually detect R. typhi RpoB-GFP_UV expression by placing infected culture flasks on a UV light box, but fluorescence was not visible by the naked eye. We also attempted to confirm RpoB-GFP_UV expression by viewing cultures microscopically. Low-level fluorescence was seen, but it was not significantly brighter than background fluorescence; therefore, we used FACS analysis to quantitatively assess fluorescence of the transformants.

View larger version (83K): [in this window] [in a new window]   FIG. 1.   (Top) Construction of the rpoB-GFPUV fusion. The coding region of GFPUV was amplified from pGFPuv with primers containing a ClaI site to facilitate ligation to a ClaI site near the 3' terminus of R. typhi rpoB. In-frame ligation was confirmed by sequence analysis. (Bottom) Integration of the rpoB-GFPUV construct into the genome of transformed R. typhi, 8 days posttransformation, as confirmed by PCR of infected Vero cells and ClaI digestion of the amplicons. The 1.08-kb amplicon was obtained by using primers specific for rpoB nt 3097 to 3113 and ClaI-GFPUV reverse. Lane 1, 100-bp marker (Gibco); lane 2, Vero cells infected with transformed R. typhi (electroporated with the rpoB-GFPUV construct); lane 3, ClaI digestion of the amplicon yielding fragments of 375 and 730 bp.

As a positive control for GFP_UV fluorescence at 488 nm, we compared the fluorescence of rickettsial RpoB-GFP_UV expression to that of GFP_UV expression in another gram-negative intracellular bacterium. For this investigation, we used a hemolysin-negative P. mirabilis strain (WPM111) transformed with pGFPuv. P. mirabilis expressing GFP_UV was clearly visible as pale green colonies when grown on LB plates, and colonies fluoresced brightly when viewed on a UV light box. FACS analysis of Vero cells infected with GFP_UV-expressing P. mirabilis revealed that 41% of the Vero cells were infected with GFP_UV-expressing bacteria (Fig. 2). P. mirabilis-infected Vero cells had a mean fluorescence of 18.7, compared to 1.8 for uninfected Vero cells.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   FACS analysis of pGFPuv expression by P. mirabilis after infection of Vero cells with P. mirabilis (dashed line) and with P. mirabilis expressing GFPUV (solid line). y axis, relative cell counts; x axis, FL1-H detection of relative fluorescence intensity.

Samples of the infected Vero cells were analyzed by FACS to determine the proportion of fluorescent cells and stained with Diff-Quick to determine the infection rate. Flow cytometry analysis of R. typhi-infected Vero cells revealed that approximately 10% of the Vero cell culture was infected with RpoB-GFP_UV-expressing rickettsiae by day 8 postinfection (Fig. 3A). Vero cells infected with transformed R. typhi had a mean fluorescence of 9.9 (Fig. 3) and Vero cells infected with untransformed R. typhi had a mean fluorescence of 4.2 (Fig. 3A). Although over 80% of Vero cells sampled after 6 weeks of continuous culture contained R. typhi, FACS analysis revealed that only 1% of the cells were infected with R. typhi expressing functional fusion protein. The presence of fluorescent rickettsiae after 6 weeks of continuous culture demonstrates the viability of the transformants. Both FACS and PCR analyses revealed that the transformed rickettsiae expressing the RpoB-GFP_UV fusion protein are present and that both proteins in the fusion construct are functional. However, while the expression of RpoB-GFP_UV does not significantly alter rickettsial infectivity, the number of the transformed rickettsiae was reduced 10-fold after the continuous culture in Vero cells. There are several possibilities to explain the decrease in the relative abundance of transformants during prolonged in vitro culture. It is possible that the RpoB-GFP_UV fusion protein in transformed rickettsiae either inhibits rickettsial growth or interferes with the transcriptional function of RpoB, and as a result the transformants replicate more slowly than untransformed rickettsiae. It is also possible that some of the transformed rickettsiae no longer had a functional GFP_UV fusion construct. The nonfluorescent transformants would not be detected in this assay. In the present study, we treated the R. typhi-infected Vero cells with paraformaldehyde prior to FACS analysis in order to prevent contamination of our FACS facility and did not attempt to purify the transformed rickettsiae after separation of transformed and untransformed populations. In future studies, individual transformed clones can be isolated via limiting dilution and plaque purification after the separation of live rickettsiae by FACS.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Detection of rpoB-GFPUV expression by FACS (488 nm) 8 days after infection. (A) Vero cells infected with untransformed R. typhi (dashed line) and with rpoB-GFPUV-transformed R. typhi (solid line). (B) Selected population of Vero cells infected with rpoB-GFPUV-transformed R. typhi. y axis, relative cell counts; xñ

In summary, our results not only confirm the recent report of Rachek et al. (9) that it is possible to transform rickettsiae but also demonstrate that nonrickettsial DNA flanked by rickettsial DNA can be incorporated into the rickettsial chromosome. Using GFP_UV expression as a fluorescent marker allowed the separation of transformed from untransformed R. typhi by FACS analysis 8 days postinfection. This is the first report of transformation of rickettsiae with a nonrickettsial gene. We have also demonstrated the utility of GFP as a marker for rickettsial transformation studies. The GFP variant used in this study, GFP_UV, is optimized for fluorescence under UV light. We chose this GFP variant to facilitate identification of Vero cells infected with transformed rickettsiae by macroscopic analysis on a UV light box. We are continuing to experiment with other GFP_UV fusion constructs and rickettsial plating techniques to facilitate the identification of transformants macroscopically. In addition, we are creating several fusion constructs by using GFP variants optimized for excitation at 480 nm in order to enhance selection of transformants by FACS analysis.