veterinary sciences

Article Fluorescent Protein Expressing buchneri and Rickettsia peacockii for Tracking Symbiont-Tick Cell Interactions

Timothy J. Kurtti *, Nicole Y. Burkhardt, Chan C. Heu and Ulrike G. Munderloh

Department of Entomology, University of Minnesota, 1980 Folwell Avenue, Saint Paul, MN 55108, USA; [email protected] (N.Y.B.); [email protected] (C.C.H.); [email protected] (U.G.M.) * Correspondence: [email protected]; Tel.: +1-612-624-4740

Academic Editor: László Fodor Received: 30 September 2016; Accepted: 14 November 2016; Published: 17 November 2016

Abstract: Rickettsiae of indeterminate pathogenicity are widely associated with ticks. The presence of these endosymbionts can confound a One Health approach to combatting tick-borne diseases. Genomic analyses of symbiotic rickettsiae have revealed that they harbor mutations in gene coding for proteins involved in rickettsial pathogenicity and motility. We have isolated and characterized two rickettsial symbionts—Rickettsia peacockii and R. buchneri—both from ticks using tick cell cultures. To better track these enigmatic rickettsiae in ticks and at the tick-mammal interface we transformed the rickettsiae to express fluorescent proteins using shuttle vectors based on rickettsial plasmids or a transposition system driving insertional mutagenesis. Fluorescent protein expressing R. buchneri and R. peacockii will enable us to elucidate their interactions with tick and mammalian cells, and track their location and movement within individual cells, vector ticks, and host animals.

Keywords: ticks; endosymbionts; Rickettsia buchneri; Rickettsia peacockii; transformation; shuttle vector; Himar 1 transposition; green fluorescent protein; mCherry fluorescent protein; spectinomycin; rifampin

1. Introduction Rickettsiae of indeterminate pathogenicity are often detected in ticks, raising questions about their infectivity for vertebrate hosts. Some are labeled as non-pathogenic beneficial endosymbionts (e.g., Rickettsia buchneri)[1–4] while others are considered potential pathogens (e.g., “Candidatus R. amblyommii”) [5,6]. Rickettsiae are maintained in nature via horizontal (infectious) transmission and/or vertical (transovarial) transmission. The current model specifies that horizontal transmission of rickettsiae during the bloodmeal of an infected arthropod feeding on a vertebrate host favors the evolution of pathogenic rickettsiae while non-pathogenic rickettsiae transmitted transovarially evolve to become mutualists [7]. Major advances are being made in our understanding of virulence mechanisms and pathogenesis of horizontally transmitted rickettsiae [7,8]. In contrast, we have limited understanding of the host parasite interactions and transmission mechanisms of endosymbiotic rickettsiae that are transmitted transovarially. Non-pathogenic rickettsial endosymbionts, such as R. buchneri and Rickettsia peacockii, are transtadially and transovarially transmitted in ticks and are mainly associated with ovarian tissues. Both endosymbionts are non-infectious for vertebrates and mammalian cells [1,3]. Surprisingly, they are closely related to pathogenic rickettsiae that are horizontally transmitted to vertebrate hosts by ticks; R. peacockii is closely related to R. rickettsii [9], and R. buchneri is closely related to Rickettsia monacensis [3,10]. Genomic analyses of these endosymbionts have identified gene mutations that dampen their virulence and ability to cause cytopathic effects [2,11]. Both endosymbionts possess

Vet. Sci. 2016, 3, 34; doi:10.3390/vetsci3040034 www.mdpi.com/journal/vetsci Vet. Sci. 2016, 3, 34 2 of 13 genomes extensively rearranged following introduction of multiple copies of transposons, which led to numerous mutations via recombination between transposon copies and deletion or disruption of several genes important to rickettsial pathogenicity, restricting them to the tick [2,11]. Both species are maintained in nature in ticks via transovarial transmission, and the ovaries of these ticks harbor numerous rickettsiae [1,3,12]. Interstitial cells as well as developing oocytes are colonized and fecundity is not adversely affected [1,13]. Other organs in the tick are rarely infected, supporting the observation that these species are solely transmitted transovarially, but not horizontally. The study of these enigmatic rickettsiae is facilitated by their isolation and propagation in tick cell culture systems, demonstrating that they have retained the ability to invade and replicate in host cells [3,9,10,14]. Tick endosymbionts are closely related to pathogenic rickettsiae that can be horizontally transmitted to vertebrate hosts by ticks [3,10]. Pathogenic rickettsiae cause disseminating infections in both vertebrate hosts and ticks and a reduction in tick fecundity [15–18]. However, strains of pathogenic rickettsial species (e.g., the agent of Rocky Mountain spotted fever) can vary considerably in their infectivity for vertebrates and ticks [15,16]. Avirulent R. rickettsii, e.g., strain Iowa, retain the ability to disseminate and persist in the tick, infect ovaries, and be transmitted transovarially [15,19]. Such avirulent strains need to be clearly delineated from nonpathogenic rickettsial endosymbionts. A goal of our research is to study the interaction between rickettsial endosymbionts and their host cells in vivo and in vitro in order to characterize the cellular processes involved in rickettsial symbiosis with ticks. Recent advances in the genetic transformation of rickettsiae provide us with new research tools for studies on endosymbiotic and other enigmatic rickettsiae [20]. These techniques enable us to genetically manipulate these rickettsiae and track the events involved with rickettsial endosymbiosis (e.g., cellular adherence, entry, and motility). Here we describe the transformation of R. buchneri and R. peacockii to express fluorescent proteins (GFPuv or mKate), using a shuttle vector based on the plasmid pRAM18 of Candidatus Rickettsia amblyommii [21]. Transformed symbionts grew and brightly expressed fluorescent reporter proteins in host cells. Second, we used a transposition system based on a hyperactive Himar1 transposase for driving insertional mutagenesis of mCherry in R. peacockii [22]. The flanking genomic insertions were sequenced to confirm transposition.

2. Materials and Methods

2.1. Tick Cell Lines Cell lines ISE6 and IRE11 from embryos of I. scapularis (ISE 6) [23] and Ixodes ricinus (IRE 11) [10], respectively, were maintained in L15C300 medium [24] supplemented with fetal bovine serum (FBS, 5%), tryptose phosphate broth (TPB, 5%; Difco), and lipoprotein concentrate (LPC, 0.1%; MP Biomedicals). Cultures were incubated at 32 or 34 ◦C.

2.2. Rickettsiae R. buchneri (str ISO7T) (clone B8), isolated from ovaries of an I. scapularis female [3], was grown using IRE11 [10]. Cultures were maintained in ambient air and incubated at 26 or 28 ◦C in L15C300 medium supplemented with FBS (10%), TPB (5%), and LPC (0.1%). Every four weeks, 1 mL (~2 × 106 cells) of infected cells were transferred to 5 mL of uninfected IRE11 cells (~107 cells) in a 25 cm2 flask. R. peacockii (str. Rustic) [9] was maintained in ISE6 using L15C300 medium supplemented with FBS (10%), TPB (5%), LPC (0.1%), HEPES (25 mM), and NaHCO3 (0.25%). Infected cultures were incubated at 32 or 34 ◦C, and 0.1 mL of an infected cell suspension (~2 × 105 cells) was added to a fresh cell layer (5 mL of uninfected ISE6 cells, ~107 cells, in a 25 cm2 flask) every ~3 weeks.

2.3. Plasmid Constructs The plasmid constructs that we used in this study are listed in Table1. Vet. Sci. 2016, 3, 34 3 of 13

Table 1. Plasmid constructs used for the transformation of Rickettsia buchneri and Rickettsia peacockii.

Construct MW (bp) Fluoro-Chrome Antibiotic Selection Symbiont Shuttle Vector Plasmids pRAM18dRGA 10,248 GFPuv Rifampin R. buchneri pRAM18dRGA[MCS] 10,309 GFPuv Rifampin R. buchneri pRAM18dSGA[MCS] 10,736 GFPuv Spectinomycin R. peacockii pRAM18dSGK(23)[MCS] 11,525 GFPuv Spectinomycin R. peacockii pRAM18dSFA[MCS] 10,829 mKate Spectinomycin R. peacockii Himar I Transposase-Transposon Plasmid pCis mCherry-SS HIMAR1 A7 8423 mCherry Spec. Spectinomycin R. peacockii

2.4. Plasmid Transformation of R. buchneri and R. peacockii Shuttle vector pRAM18dRGA [21] was used to transform R. buchneri and R. peacockii. The shuttle vector was developed using pRAM18 originally found in Candidatus Rickettsia amblyommii [21]. The vector carries a selection cassette encoding R. prowazekii arr-2 rifampin resistance gene (rpsLp-arr-2Rp) or a gene for spectinomycin and streptomycin resistance (aadA) and a reporter gene encoding a green (GFPuv) (Becton Dickenson, Palo Alto, CA, USA) [25] or far red (mKate) (DNA 2.0, Newark, CA, USA) [26] fluorescent protein. Rickettsia buchneri was transformed with pRAM18dRGA encoding rifampin resistance and GFPuv fluorescence by electroporation using procedures described previously [21]. Briefly, rickettsiae were released from IRE11 cells using shear created by forcing cells suspended in medium through a 25 G needle attached to a 5 mL syringe. The lysate was filtered through a 1.5 µm pore size filter, washed twice (13.6 rcf × 4 min × 4 C) in 300 mM sucrose, concentrated into 50 µL of 300 mM sucrose with 1 µL (1–2 µg) of pRAM18dRGA, and transferred to a cuvette (Gene Pulser Cuvette, 0.1 cm gap electrode, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cuvettes containing rickettsia-plasmid preparations were held on ice for 15 min and then pulsed once (1.8 kV, 200 ohms, 25 µF, ~5 ms) using a Gene Pulser II electroporation apparatus. Electroporated rickettsiae were transferred to a 2 mL microfuge tube containing IRE11 cells (2 × 106 cells in 1.5 mL), centrifuged at 5000 rcf for 5 min and incubated at room temperature for 30 min. Finally, the R. buchneri-IRE11 suspension was transferred to a vented cap flask (12.5 cm2) in 5 mL L15C300 supplemented as described, and incubated at 26 ◦C in candle jars having an atmosphere of approximately 3% CO2 and 17% O2. After three days, 0.8 µg/mL of Rifampin was added. The medium was changed weekly while maintaining continuous rifampin selection. Cultures were monitored for presence of green fluorescent rickettsiae by epifluorescence microscopy using an inverted Nikon Diaphot fitted with a Sapphire GFP filter. Cultures with transformed R. buchneri were noted three to four weeks later and subcultured one to twothrmonths post electroporation. After four serial transfers transformed R. buchneri were maintained in ambient air using medium containing 0.8 µg/mL rifampin. Rickettsia peacockii was transformed using shuttle vector pRAM18dSGA encoding spectinomycin resistance (aadA) and the fluorescent proteins mCherry (DNA 2.0) [27] or GFPuv [21,24]. Cell free R. peacockii were prepared using approximately 0.2 mL of sterile rock polishing grit (60/90 grit silicon carbide; Lortone, Inc., Mukilteo, WA, USA) in a 2 mL microfuge tube to which the cell suspension was added. The grit-cell suspension was vortexed for 30 s and the lysate filtered through a 2 µm syringe filter. Cell free R. peacockii were washed and electroporated as given above for R. buchneri, excepting cultures were incubated at 34 ◦C. Spectinomycin (10 µg/mL) was added three days later. Cultures were monitored for presence of fluorescent rickettsiae as described above, and subcultured two months post electroporation. Transformants were maintained using spectinomycin-supplemented medium as described for wild-type rickettsiae. We maintained both transfomants of both species by mixing infected cells or cell free rickettsiae with uninfected host cells. Centrifugation (13,600 rcf for 2.5 min; or 170 rcf, 3 min; 1 mL of suspension on a “dry” cell layer) of cell free R. buchneri with target host cells greatly enhanced the infection rate. Vet. Sci. 2016, 3, 34 4 of 13

2.5. Transposon Mutagenesis (HIMAR1 A7) of R. peacockii We used the 8423 bp plasmid pCis mCherry-SS HIMAR1 A7 to transform R. peacockii [20,22]. This cis-construct included both transposase and transposon encoded on a single plasmid, in order to improve efficiency of transformation. It encodes the A7 hyperactive mutant of the HIMAR1 transposase controlled by the Anaplasma marginale transcriptional regulator promoter, tr, that is well-expressed in both tick and mammalian cells [28], and a transposon carrying the Am tr promoter driving expression of mCherry or GFPuv and spectinomycin resistance. This promoter works efficiently in our hands for expression of transgenes in the genera Anaplasma, Ehrlichia, and Rickettsia. The coding genes are positioned between left and right HIMAR transposon repeats that are recognized by the transposase to facilitate excision followed by random insertion into genomic target sites containing TA dinucleotides [29]. 1 µg of himar1 plasmid DNA was mixed with 50 µL of host cell free R. peacockii and transferred to 1 mm gap electroporation cuvettes, incubated on ice for 15 min, and electroporated at 2.4 kV, 25 mF, 400 ohms, and ~8 ms. Electroporated rickettsiae were transferred to a 2 mL microfuge tube containing ISE6 cells (2 × 106 cells in 1.5 mL), centrifuged at 10,000 rcf for 5 min and incubated at room temperature for 15 min. Finally, the R. peacockii-ISE6 pellet was resuspended in 5 mL of medium, transferred to a flask (25 cm2) and incubated at 34 ◦C. Spectinomycin (5–10 µg/mL) was added two to three days later. Cultures with transformed R. peacockii were noted eight weeks later and subcultured three months post electroporation as described for wild-type rickettsiae. Transformants were maintained under continuous selection using spectinomycin.

2.6. Cloning and Sequencing of Transposon Integration Sites Genomic insertion sites were determined by plasmid rescue cloning as previously described [22,30]. Briefly, R. peacockii mCherry himar transformant genomic DNA was digested with EcoRI and HindIII (NEB, Beverly, MA, USA), purified by phenol/chloroform extraction, and ligated into dephosphorylated, EcoRI or HindIII-cut pMOD vector. The plasmid was transformed into E. cloni Elite electrocompetent cells (Lucigen, Middleton, WI, USA), and clones containing the transposon were selected on 50 µg/mL spectinomycin/streptomycin YT agar plates. Plasmid DNA from red-fluorescent, spectinomycin/streptomycin-resistant clones was isolated with the High Pure Plasmid Isolation kit (Roche, Madison, WI, USA) as per manufacturer’s protocol, checked by restriction digest (EcoRI or HindIII) for presence of inserts, and sequenced with primers reading out from the transposon at the University of Minnesota Genomics Center Sequencing and Analysis Facility.

2.7. Microscopy Cultures were observed weekly by phase-contrast microscopy to assess culture confluency and the presence and relative abundance of rickettsiae. Cultures were periodically sampled for the presence of rickettsiae using cells deposited onto slides by means of a cytocentrifuge (Cytospin; Shandon, Pittsburgh, PA, USA), fixed in methanol and stained with Giemsa. Cell layers were additionally monitored for presence of fluorescent rickettsiae by epifluorescence microscopy using an inverted Nikon Diaphot fitted with a Sapphire GFP filter or a TRITC Filter (Rhodamine)/Dil/Cy3 (Chroma Technology, Bellows Falls, VT, USA). Suspended cells on microscope slides were examined using a Nikon Eclipse E400 fitted with FITC and TRITC filters.

2.8. Preparation of Genomic DNA for PFGE and Southern Blot Analysis Cell free rickettsiae were prepared as described above, embedded in agarose (1%, InCert low melting point) and lysed in situ with proteinase K and sodium lauryl sarcosine in 0.5 M EDTA [21]. Released DNA was separated by pulsed-field gel electrophoresis (PFGE) on a Chef Mapper XA System (Bio-Rad) with 0.5× TBE using the Auto setting in CHEF mode. Chef Mapper XA System parameters were: DNA size range of 10 kbp to 100 kbp, a gradient of 6 V/cm, an angle of 120◦, a linear ramping factor, a calibration factor of 1, an initial switch time of 0.47 s, and a final switch time of 8.53 s. The total run time was 20.18 h. Vet. Sci. 2016, 3, 34 5 of 13

For Southern blotting, pulsed-field gels were depurinated and DNA transferred onto a Zeta Probe GT genomic membrane (Bio-Rad) [21]. To detect R. buchneri plasmids, blots were hybridized at 55 ◦C overnight with digoxigenin-labeled parA probes specific for pREIS1, 2, 3, and 4. To detect the native plasmid pRPR in R. peacockii, blots were hybridized with digoxigenin-labeled pRM6, which encodes the conserved chaperonin Hsp2 on the Rickettsia monacensis plasmid pRM (Baldridge et al. 2010). To detect gfpuv encoded on the shuttle vector pRAM18dRGA (R. buchneri) or pRAM18dSG (R. peacockii) ◦ in transformants, blots were labeled with digoxigenin-labeled gfpuv, washed at 55 C, and reacted with anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (Anti-Digoxigenin-AP, Fab Vet. Sci. 2016, 3, 34 5 of 13 fragments; Roche) and detected with CDP-Star (Roche). Blots were exposed to Kodak X-OMAT AR film, or fluorescenceFor Southern from blotsblotting, was pulsed-field captured withgels were the Infinity depurinated 3 camera and DNA with transferred Infinity Analyze onto a SoftwareZeta versionProbe 5.0 GT (Lumenera genomic membrane Corporation, (Bio-Rad) Ottawa, [21]. ON, To detect USA). R. Membranes buchneri plasmids, to be re-usedblots were were hybridized stripped by rinsingat 55 briefly °C overnight with Milli-Q with digoxigenin-labeled water, washing twice parA at 37probes◦C with specific 0.2 for M sodiumpREIS1, hydroxide/0.1%2, 3, and 4. To detect SDS for 15 min,the and native rinsing plasmid for pRPR 5 min in at R. room peacockii temperature, blots were in hybridized 2× SSC. Beforewith digoxigenin-labeled re-hybridization withpRM6, a newwhich probe, absenceencodes of prior the signalconserved was chaperonin verified by Hsp2 adding on detectionthe Rickettsia reagent monacensis and re-exposureplasmid pRM of (Baldridge membranes et al. to film. 2010). To detect gfpuv encoded on the shuttle vector pRAM18dRGA (R. buchneri) or pRAM18dSG (R. 3. Resultspeacockii) in transformants, blots were labeled with digoxigenin-labeled gfpuv, washed at 55 °C, and reacted with anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (Anti-Digoxigenin- 3.1. PlasmidAP, Fab Transformation fragments; Roche) of R. and buchneri detected and with R. peacockii CDP-Star (Roche). Blots were exposed to Kodak X- OMAT AR film, or fluorescence from blots was captured with the Infinity 3 camera with Infinity RickettsiaAnalyze Software buchneri versionand R. 5.0 peacockii (Lumenerawere Corporatio successfullyn, Ottawa, transfected ON, USA). with theMembranes shuttle vectorto be re-used pRAM18d carryingwere genes stripped for rifampinby rinsing or briefly spectinomycin with Milli-Q resistance water, washing and expression twice at 37 of a°C fluorescent with 0.2 M proteinsodium [21]. Tohydroxide/0.1% transform R. SDS buchneri for 15 tomin, express and rinsing a fluorescent for 5 min protein,at room temperature we used a 10,302-bpin 2× SSC. shuttleBefore re- vector pRAM18dRGAhybridization [21 with] based a new in probe, part absence on an 18,497-bp of prior signal plasmid, was verified pRAM18, by adding present detection in C. R.reagent amblyommii and AaR/SCre-exposure [31]. Clone of membranes ISO7-B8 to was film. electroporated with pRAM18dRGA and seeded onto an IRE11 cell layer. Rifampin selection was maintained for six weeks, and discontinued when no fluorescent 3. Results rickettsiae were detected. One month later, we noted small clusters of cells infected with rickettsiae expressing3.1. Plasmid GFPuv Transformation (Figure1, Panelof R. buchneri A). Transformed and R. peacockiiR. buchneri replicated slowly within tick cells, and required two months to grow and spread within the cell layer before the first transfer to fresh Rickettsia buchneri and R. peacockii were successfully transfected with the shuttle vector cells was done. Transformed R. buchneri are presently in the 23rd subculture and can be transferred pRAM18d carrying genes for rifampin or spectinomycin resistance and expression of a fluorescent onceprotein a month [21]. by seeding infected cells (Figure1, Panels B, C and D) onto a fresh IRE11 cell layer at a 1:5 dilution,To indicatingtransform R. that buchneriR. buchneri to expresshas a doublingfluorescent time protein, of a we week used or a more. 10,302-bp shuttle vector InpRAM18dRGA contrast, Rickettsia [21] based peacocki in partwas on an readily 18,497-bp transformed plasmid, pRAM18, using apresent shuttle invector C. R. amblyommii carrying genes codingAaR/SC for spectinomycin [31]. Clone ISO7-B8 resistance was electroporated and the far-red with fluorescent pRAM18dRGA protein and mKATE seeded (pRAM18dSFA[MCS])onto an IRE11 cell (Figurelayer.2), orRifampin GFPuv selection (pRAM18dSGK[MCS]) was maintained for [ si21x]. weeks, Both and red discontinued and green when fluorescent no fluorescentR. peacockii transformantsrickettsiae replicatedwere detected. faster One than monthR. buchneri later, we. Coloniesnoted small of clusters transformants of cells infected were noted with inrickettsiae cell layers at threeexpressing weeks and GFPuv transfers (Figure were 1, made Panel oneA). Transformed month post electroporation.R. buchneri replicated Transformants slowly within were tick maintained cells, by subculturingand required transformed two months to cells grow onto and a spread fresh cellwithin layer the 1:50 cell (0.1layer mL before per the 5 mL first culture) transferevery to fresh two to cells was done. Transformed R. buchneri are presently in the 23rd subculture and can be transferred three weeks. We were unable to obtain R. peacockii transformants with the shuttle vector pRAM18dRGA once a month by seeding infected cells (Figure 1, Panels B, C and D) onto a fresh IRE11 cell layer at a and rifampin1:5 dilution, selection. indicating that R. buchneri has a doubling time of a week or more.

FigureFigure 1. Cont.Cont .

Vet. Sci. 2016, 3, 34 6 of 13

Vet. Sci. 2016, 3, 34 6 of 13 Vet. Sci. 2016, 3, 34 6 of 13

Figure 1. Images of Rickettsia buchneri ISO7 clone B8 transformed with shuttle vector pRAM18dRGA

to express GFPuv. Transformants were isolated and grown in Ixodes ricinus embryonic cell line IRE11. Panel (A) Island of IRE11 cells containing transformed R. buchneri expressing GFPuv. Image collected 2.5 months after electroporation and rifampicin selection. Infected cells visualized using a fluorescein isothiocyanate (FITC) filter; Panel (B) Phase contrast microscopic appearance of IRE11 cells heavily infected with transformed R. buchneri. Transformed R. buchneri were in the 23rd serial transfer when 6 imaged. Transformants were maintained in cell cultures at high density (1–5 × 10 cells/mL) and were diluted for this image. Infected cells detach; (C) Same field as shown in panel (B) but visualized using Figure 1. Images of Rickettsia buchneri ISO7 clone B8 transformed with shuttle vector pRAM18dRGA fluorescenceFigure 1. Images microscopy of Rickettsia withbuchneri FITC filter;ISO7 (D clone) Composite B8 transformed image made with shuttleby merging vector images pRAM18dRGA shown in to express GFPuv. Transformants were isolated and grown in Ixodes ricinus embryonic cell line IRE11. Panelsto express (B,C GFP). Alluv .images Transformants taken with were a Nikon isolated Diaphot and grown fluorescence in Ixodes microscope. ricinus embryonic Bar equals cell line 40 µm IRE11. in Panel (A) Island of IRE11 cells containing transformed R. buchneri expressing GFPuv. Image collected allPanel panels.2.5 (A months) Island after of IRE11electroporation cells containing and rifampicin transformed selection.R. Infected buchneri cellsexpressing visualized GFP usinguv .a Imagefluorescein collected 2.5 monthsisothiocyanate after electroporation (FITC) filter; Panel and ( rifampicinB) Phase contrast selection. microscopic Infected appearance cells visualized of IRE11 using cellsa heavily fluorescein Inisothiocyanate contrast,infected withRickettsia (FITC) transformed filter; peacocki PanelR. buchneri. was (B) Phase readilyTransformed contrast transformed R. microscopicbuchneri wereusingappearance in thea shuttle23rd serial of vector IRE11 transfer cellscarrying when heavily genes codinginfected imaged.for withspectinomycin Transformants transformed wereR. buchneri resistancemaintained. Transformed in cellan dcultures R.the buchneriat highfar-red densitywere in(1–5fluorescent the × 10 23rd6 cells/mL) serial proteintransfer and were when mKATE (pRAM18dSFA[MCS])imaged.diluted Transformants for this image. (Figure were Infected maintained 2), cells or detach;GFPuv in cell (C) cultures Same(pRAM18dSGK[MCS]) field at as high shown density in panel (1–5 (B×[21].) but106 visualizedcells/mL)Both red using and and were green fluorescentdilutedfluorescence R. for peacockii this image. microscopy transformants Infected with cells FITC detach; replicatedfilter; (D (C) )Composite Same faster field thanimage as shown R. made buchneri inby panel merging. Colonies (B) images but visualized ofshown transformants in using Panels (B,C). All images taken with a Nikon Diaphot fluorescence microscope. Bar equals 40 µm in were fluorescencenoted in cell microscopy layers at withthree FITC weeks filter; and (D )transfers Composite were image made made one by mergingmonth post images electroporation. shown in all panels. TransformantsPanels (B,C were). All imagesmaintained taken by with subculturing a Nikon Diaphot transformed fluorescence cells microscope. onto a fresh Bar cell equals layer 40 1:50µm (0.1 in mL all panels. per 5 mL culture)In contrast, every Rickettsia two to peacocki three weeks.was readily We were transformed unable tousing obtain a shuttle R. peacockii vector transformantscarrying genes with the shuttlecoding vector for pRAM18dRGAspectinomycin andresistance rifampin an selection.d the far-red fluorescent protein mKATE (pRAM18dSFA[MCS]) (Figure 2), or GFPuv (pRAM18dSGK[MCS]) [21]. Both red and green fluorescent R. peacockii transformants replicated faster than R. buchneri. Colonies of transformants were noted in cell layers at three weeks and transfers were made one month post electroporation. Transformants were maintained by subculturing transformed cells onto a fresh cell layer 1:50 (0.1 mL per 5 mL culture) every two to three weeks. We were unable to obtain R. peacockii transformants with the shuttle vector pRAM18dRGA and rifampin selection.

Figure 2. Image of Ixodes scapularis cells (ISE6) infected with Rickettsia peacockii expressing the far red Figure 2. Image of Ixodes scapularis cells (ISE6) infected with Rickettsia peacockii expressing the far red fluorescent protein mKATE. Panel (A) Phase contrast microscopic appearance of ISE6 cells infected fluorescent protein mKATE. Panel (A) Phase contrast microscopic appearance of ISE6 cells infected with transformed R. peacockii. Transformed R. peacockii were in the 23rd serial transfer when image with transformed R. peacockii. Transformed R. peacockii were in the 23rd serial transfer when image was was collected. Transformant is maintained in cell layers seeded at high cell density (1–5 × 106 cells collected. Transformant is maintained in cell layers seeded at high cell density (1–5 × 106 cells per mL); per mL); (B) Same field as shown in panel B but visualized using fluorescence microscopy with TRITC (B) SameFigure field 2. Image as shown of Ixodes in panelscapularis B but cells visualized (ISE6) infected using with fluorescence Rickettsia peacockii microscopy expressing with the TRITC far red filter; filter; (C) Composite image made by merging images shown in Panels (A,B). All images taken using (C) Compositefluorescent image protein made mKATE. by merging Panel (A) images Phase contrast shown inmicroscopic Panels (A appearance,B). All images of ISE6 taken cells using infected a Nikon aDiaphot Nikonwith Diaphot fluorescence transformed fluorescence R. microscope. peacockii. microscope. Transformed Bar equals Bar R. 40 equals peacockiiµm in 40 all were µm panels. in theall panels.23rd serial transfer when image was collected. Transformant is maintained in cell layers seeded at high cell density (1–5 × 106 cells 3.2. Characteristics of Transformed R. buchneri and R. peacockii in Tick Cell Culture 3.2. Characteristicsper mL); (B of) Same Transformed field as shown R. buchneri in panel B and but visualized R. peacockii using in fluorescence Tick Cell Culture microscopy with TRITC filter; (C) Composite image made by merging images shown in Panels (A,B). All images taken using Transformants of both species displayed growth characteristics similar to those shown by wild Transformantsa Nikon Diaphot of both fluorescence species microscope. displayed Bar growth equals 40 characteristics µm in all panels. similar to those shown by wild type strains. Foci of cells infected with rickettsiae were readily evident in infected cell layers. In type strains. Foci of cells infected with rickettsiae were readily evident in infected cell layers. In contrast, the formation3.2. Characteristics of plaques, of Transformed a feature ofR. pathogenicbuchneri and rickettsiae,R. peacockii in was Tick not Cell observed. Culture Transformed symbionts grew in theTransformants cytoplasm, of usually both species within displayed clusters growth that expanded characteristics to eventuallysimilar to those fill theshown cytoplasm by wild and causedtype loss strains. of host Foci cell of pseudopodia. cells infected with Both rickettsiae species adhered were readily poorly evident to host in cells infected and cellcell freelayers. rickettsiae In accumulated in the culture medium between transfers. Vet. Sci. 2016, 3, 34 7 of 13 contrast, the formation of plaques, a feature of pathogenic rickettsiae, was not observed. Transformed symbionts grew in the cytoplasm, usually within clusters that expanded to eventually fill the cytoplasmVet. Sci. 2016 ,and3, 34 caused loss of host cell pseudopodia. Both species adhered poorly to host cells7 and of 13 cell free rickettsiae accumulated in the culture medium between transfers.

3.3.3.3. Identification Identification of of Shuttle Shuttle Vector Vector pRAM18dRGA pRAM18dRGA and Native Plasmids pREIS1-3 in R. buchneri TransformantTransformant and Clone B8 WeWe used used Pulsed Pulsed field field gel gel electrophoresis electrophoresis (PFGE) (PFGE) and and Southern Southern blots blots to detect to detect the presence the presence of the nativeof the pREIS native plasmids pREIS plasmids (pREIS1, (pREIS1,2 and 3) in 2 andthe B8 3) clone in the of B8 R. clonebuchneri of R.and buchneri confirmand the confirmpresence theof shuttlepresence vector of shuttle plasmids vector in the plasmids transformants. in the transformants. Cell free R. buchneri Cellfree wereR. embedded buchneri were in agar embedded plugs and in lysedagar plugswith proteinase and lysed K, with sodium proteinase lauryl K,sarcosin sodiume, and lauryl EDTA sarcosine, to release and plasmid EDTA toand release chromosomal plasmid DNA.and chromosomal PFGE separated DNA. plasmids PFGE and separated shuttle plasmidsvector from and the shuttle larger R. vector buchneri from chromosome. the larger R. Ethidium buchneri bromidechromosome. stained Ethidium PFGE bromidegels (Figure stained 3, PFGEPanels gels A (Figureand E)3 ,showed Panels Athe and presence E) showed of theI. scapularis presence mitochondriaof I. scapularis (m),mitochondria R. buchneri (m),chromosomeR. buchneri (C),chromosome and multiple (C), plasmid and multiplebands in plasmidboth the B8 bands clone in (lane both B8)the and B8 clonethe GFP (lane expressing B8) and transformant the GFP expressing (lane T). The transformant 10 kbp shuttle (lane vector, T). The pRAM18dRGA, 10 kbp shuttle was vector, not apparentpRAM18dRGA, in the ethidium was not apparent bromide in stained the ethidium PFGE bromidegel. Southern stained blotting PFGE gel.using Southern digoxigenin-labeled blotting using probesdigoxigenin-labeled revealed the location probes revealed of the pREIS the location plasmids of theand pREIS the shuttle plasmids vector and within the shuttle the gels vector (Figure within 3, Panelsthe gels B, (Figure C, D, and3, Panels F). Probes B, C, D,containing and F). Probes the parA containing gene of thepREIS1parA (55gene kbp), of pREIS1 2 (67 kbp), (55 kbp), and 3 2 (50 (67 kbp) kbp), wereand 3used (50 kbp)to detect were the used native to detect plasmids. the native In both plasmids. clone B8 In bothand clonethe transformant B8 and the transformantthe three pREIS the plasmidsthree pREIS were plasmids present wereand clearly present visible and clearly in stained visible blots in (Figure stained 3, blots Panels (Figure B, C,3 ,and Panels F). The B, C, pREISand F4). (34The kbp) pREIS is not 4 (34 present kbp) is in not clone present B8 [3 in] and clone the B8 southern [3] and the blot southern using the blot pREIS using parA the pREISprobe parAconfirmedprobe theconfirmed absence the of absence pREIS4 of in pREIS4 both inthe both parent the parent and the and transformant the transformant (Figure (Figure 3, 3Panel, Panel G). G). Monomers Monomers (arrows)(arrows) of of each each plasmid plasmid and and their their conformational conformational isomers isomers were were present present (asterisks). (asterisks). To To check check for for the the presencepresence ofof thethe shuttle shuttle vector vector (pRAM18dRGA) (pRAM18dRGA) in thein the transformant, transformant, the blotthe wasblot strippedwas stripped and re-probed and re- probedwith a probewith containinga probe containing the GFPuv thegene. GFPuv The smallergene. The 10 kbp smaller pRAM18dRGA 10 kbp pRAM18dRGA and its conformational and its conformationalisomers was clearly isomers present was inclearly the transformant present in the (T) tran butsformant not in clone (T) but B8 (Figurenot in clone3, lane B8 D). (Figure In summary, 3, lane D).these In resultssummary, indicate these that results the transformantindicate that the retained transformant the original retained 3 pREIS the plasmidsoriginal 3 found pREIS in plasmids clone B8, foundpREIS1-3, in clone and acquiredB8, pREIS1-3, the intact and acquired shuttle vector the intact pRAM18dRGA. shuttle vector pRAM18dRGA.

FigureFigure 3. 3. IdentificationIdentification of of shuttle shuttle vector vector pRAM pRAM18dRGA18dRGA and and native native plasmids plasmids pREIS1-3 pREIS1-3 in in R.R. buchneri buchneri transformanttransformant and and clone clone B8 B8 by by pulsed pulsed field field gel gel electr electrophoresisophoresis (PFGE) (PFGE) and and Southern Southern blot blot (SB) (SB) analysis. analysis. NativeNative R. buchneri (B8)(B8) andand pRAM18dRGA pRAM18dRGA (T) (T) plasmids plasmids in in REIS. REIS A.. PFGEA. PFGE gel; gel; B & B C. & SB C. of SB gel of in gel panel in panelA probed A probed with digoxygenin with digoxygenin labeled labeledparA probes parA probes specific specific for pREIS1 for pREIS1 and pREIS3, and pREIS3, respectively; respectively; D. SB of D.panel SB Aof probed panel withA probed GFPuv; with E. PFGE GFPuv; gel; FE. &PFGE G. SB gel; of gel F in& panelG. SB A of probed gel in with panel digoxygenin A probed labeled with digoxygeninparA probes specificlabeled forparA pREIS2 probes and specific pREIS4, for respectively. pREIS2 and Asterisks pREIS4, mark respectively putative. linearAsterisks monomer mark of each plasmid and arrows indicate their conformational isomers. IRE11 mitochondrial DNA—m. Chromosomal DNA—C. Linear DNA marker positions are to the left of panel A. Vet. Sci. 2016, 3, 34 8 of 13 Vet. Sci. 2016, 3, 34 8 of 13 putative linear monomer of each plasmid and arrows indicate their conformational isomers. IRE11 mitochondrial DNA—m. Chromosomal DNA—C. Linear DNA marker positions are to the left of 3.4. Identificationpanel A. of pRPR and pRAM18dSGA in R. peacockii and Transformants PFGE3.4. Identification and Southern of pRPR blots and demonstrated pRAM18dSGA the in R. presence peacockii ofand the Transformants native pRPR plasmid [11] in R. peacockii and confirmedPFGE the and presence Southern of blots shuttle demonstrated vector pRAM18dSGA the presence of plasmid the native in thepRPR transformant. plasmid [11] Ethidiumin bromideR. stainedpeacockii and PFGE confirmed gels (Figurethe presence4, Panels of shuttle A vect andor C)pRAM18dSGA showed the plasmid presence in the oftransformant. the R. peacockii chromosomeEthidium (C) bromide and multiple stained smallerPFGE gels bands (Figure in 4, both Panels the A native and C)R. showed peacockii the(lane presence RP) of and the the GFP expressingR. peacockii transformant chromosome (lane (C) T). and The multiple 10 kbp smaller shuttle bands vector, in both pRAM18dSGA, the native R. peacockii was (lane not apparent RP) and in the the GFP expressing transformant (lane T). The 10 kbp shuttle vector, pRAM18dSGA, was not R. buchneri ethidiumapparent bromide in the stained ethidium PFGE bromide gel.As stained with PFGE gel. As, Southernwith R. buchneri blots, usingSouthern digoxigenin-labeled blots using probesdigoxigenin-labeled revealed the location probes of revealed the pRPR the location plasmid of andthe pRPR the shuttleplasmid vectorand the withinshuttle vector the gels within (Figure 4, panels Bthe and gels D). (Figure A pRPR-6 4, panels probe B and was D). A used pRPR-6 to detect probe thewas nativeused to plasmiddetect the pRPR. native plasmid In both pRPR. untransformed In and transformedboth untransformedR. peacockii andthe transformed pRPR plasmid R. peacockii was the present pRPR and plasmid clearly was visible present in and stained clearly blots visible (Figure 4, Panels Bin andstained D). blots Monomers (Figure 4, (arrows) Panels B ofand each D). plasmidMonomers and (arrows) the moreof each predominant plasmid and conformationalthe more predominant conformational isomers were present (asterisks) in both pRPR and pRAM18dSGA. isomers were present (asterisks) in both pRPR and pRAM18dSGA. Again, these results indicate that the Again, these results indicate that the transformant retained its original plasmid, pRPR, and acquired transformantthe intact retained shuttle itsvector original pRAM18dSGA. plasmid, pRPR, and acquired the intact shuttle vector pRAM18dSGA.

Figure 4.FigureIdentification 4. Identification of plasmidsof plasmids pRPR pRPR andand pRAM18d in inuntransformed untransformed R. peacockiiR. peacockii (RP) and(RP) and transformanttransformant (T) by (T) PFGE by PFGE and and Southern Southern blot blot (SB)(SB) analysis. Panels Panels A and A andC. PFGE C. PFGE gels showing gels showing prominentprominent chromosomal chromosomal band band and and several several plasmidplasmid ba bands;nds; Panels Panels B and B andD. Southern D. Southern Blots of Blots gels of gels shownshown in panels in panels A and A C;and Panel C; Panel B. B. Southern Southern BlotBlot probed with with digoxygenin digoxygenin labeled labeled pRPR-6 pRPR-6 (probe (probe specific RP_p06 gene in pRPR). Arrow points to linear monomer of native 26 kb plasmid of R. peacockii; specific RP_p06 gene in pRPR). Arrow points to linear monomer of native 26 kb plasmid of R. peacockii; Panel D. Southern blot probed with digoxygenin labeled GFPuv probe. Note probe binding in lane T Panel D.only Southern and absence blot of probed reactivity with in RP digoxygenin lane. Arrows labeledmark putative GFPuv linear probe. monomer Note of probe each plasmid binding and in lane T only andasterisks absence indicate of reactivity their conformational in RP lane. isomers; Arrows Pane markl B. Note putative pRPR-6 linear probe monomer reactivity in of both each RP plasmid and and asterisksT. indicateLinear DNA their marker conformational positions are isomers;to the left of Panel panel B. A. Note pRPR-6 probe reactivity in both RP and T. Linear DNA marker positions are to the left of panel A. 3.5. Transposon Mutagenesis (HIMAR1 A7) of R. peacockii

3.5. TransposonWe Mutagenesistransformed R. (HIMAR1 peacockii A7)to express of R. peacockii either mCherry or GFPuv using the plasmid pCis mCherry-SS HIMAR1 A7 or pCis uv-SS HIMAR1 A7 [20,22]. As decribed above, both included the WeA. transformed marginale promoterR. peacockii tr drivingto expression express eitherof the fluorescent mCherry proteins or GFP GFPuvuv using or mCherry the plasmid and pCis mCherry-SSspectinomycin HIMAR1 resistance A7 or flanked pCis uv-SS by the HIMAR1 left and right A7 Himar1 [20,22]. transposon As decribed repeats. above, We grew both the included the A. marginale promoter tr driving expression of the fluorescent proteins GFPuv or mCherry and spectinomycin resistance flanked by the left and right Himar1 transposon repeats. We grew the rickettsiae in ISE6 cells under selection with the clinically irrelevant antibiotic spectinomycin and successfully isolated R. peacockii mutants that express GFP or mCherry (Figure5). PFGE followed by Southern blotting using mCherry and spectinomycin digoxygenin labeled probes revealed that the transposon were inserted into the chromosome. Further Southern blot analysis using infrequent cutting endonucleases (BglII, EcoRI, HindIII, HpaI, EcoRV, EcoRI, and HindIII) demonstrated a single insertion into the R. peacockii chromosome (data not shown). The genomic insertion site was Vet. Sci. 2016, 3, 34 9 of 13

rickettsiae in ISE6 cells under selection with the clinically irrelevant antibiotic spectinomycin and successfully isolated R. peacockii mutants that express GFP or mCherry (Figure 5). PFGE followed by Southern blotting using mCherry and spectinomycin digoxygenin labeled probes revealed that the Vet. Sci. 2016, 3, 34 9 of 13 transposon were inserted into the chromosome. Further Southern blot analysis using infrequent cutting endonucleases (BglII, EcoRI, HindIII, HpaI, EcoRV, EcoRI, and HindIII) demonstrated a single insertion into the R. peacockii chromosome (data not shown). The genomic insertion site was determined by plasmid rescue cloning [30]. Transposon insertion sites in the R. peacockii (R. peacockii determined by plasmid rescue cloning [30]. Transposon insertion sites in the R. peacockii (R. peacockii str. Rusticstr. Rustic complete complete genome) genome) Himar1 Himar1 transformant transformant mapped mapped to CP001227.1 bp bp 544,605/544,606. 544,605/544,606. This This is an intergenicis an intergenic insertion insertion downstream downstream of a hypothetical of a hypothetical protein protein gene gene (a predicted (a predicted transcriptional transcriptional regulator, bp 544,040–544,534)regulator, bp 544,040–544,534) and upstream and of upstream an aspartate of an kinaseaspartate gene kinase (bp gene 544,818–546,023) (bp 544,818–546,023) [11]. [11].

FigureFigure 5. PFGE 5. PFGE and and Southern Southern blot blot analysis analysis ofof R.R. peacockii mutantmutant obtained obtained by transposon by transposon mutagenesis mutagenesis (HIMAR(HIMAR A7). A7). Panel Panel A. A. PFGE PFGE comparison of transformant of transformant (T) with (T) wild with type wild R. peacockii type R. (RP). peacockii Black arrows(RP). Black arrowspoint point to position to position of linear of monomers linear monomers of the native of 26 the kbp native pRPR 26and kbp black pRPR asterisk and indicates black position asterisk of indicatesits positionconformational of its conformational isomers. Lane isomers.L contains 5 Lane kb ladder. L contains HP lanes 5 contain kb ladder. the 8.4 HPkb Himar1 lanes plasmid contain (pCis the 8.4 kb Himar1mCherry-SSplasmid HIMAR1 (pCis mCherry-SS A7) encoding HIMAR1mCherry and A7) spectinomycin encoding mCherry resistance and(aadA spectinomycin) genes. White arrows resistance point to position of supercoiled of pCis mCherry-SS HIMAR1 A7 and asterisks to its multimers; Panel B. (aadA) genes. White arrows point to position of supercoiled of pCis mCherry-SS HIMAR1 A7 and Southern blot probed with digoxygenin labeled mCherry probe. Note probe binding in lane T to the region asterisksof the to chromosome, its multimers; to supercoiled Panel B. and Southern multimeric blot forms probed of pCis with mCherry-SS digoxygenin HIMAR1 labeled A7 plasmid mCherry bands probe. Notein probe the HP binding lane, and in laneabsence T toof thereactivity region with of the 26 chromosome, kb pRPR; Panel to C. supercoiled Southern blot and probed multimeric with the forms of pCisdigoxygenin mCherry-SS labeled HIMAR1 spectinomycin A7 plasmid resistance bands gene. in Th thee probe HP lane,bound and in lane absence T to the of reactivityregion of the with the 26 kbchromosome, pRPR; Panel labeled C. Southern the himar1 blot plasmid probed in the with HP lane, the but digoxygenin did not react labeledwith the 26 spectinomycin kb pRPR in lane resistanceRP. gene.This The is probe consistent bound with in results lane Tshown to the in regionPanel B. of the chromosome, labeled the himar1 plasmid in the HP lane, but did not react with the 26 kb pRPR in lane RP. This is consistent with results shown in Panel B. In contrast to the R. peacockii transformed using shuttle vectors, the expression of mCherry and GFPuv was significantly lower in transposon mutants. Thus, mutants were visualized by fluorescence Inmicroscopy contrast tousing the aR. Nikon peacockii Eclipsetransformed E400 upright using or an shuttle Olympus vectors, spinning the disk expression DSU/BX60 of confocal mCherry and GFPuvmicroscopewas significantly (Figure 6). lower in transposon mutants. Thus, mutants were visualized by fluorescence microscopy using a Nikon Eclipse E400 upright or an Olympus spinning disk DSU/BX60 confocal microscopeVet. Sci. 2016 (Figure, 3, 34 6). 10 of 13

Figure 6. Images of Ixodes cells infected with Rickettsia buchneri and Rickettsia peacockii expressing Figure 6. Images of Ixodes cells infected with Rickettsia buchneri and Rickettsia peacockii expressing GFPuv and mCherry. Panel (A) Fluorescence microscopic appearance of IRE11 cells infected with GFPuv and mCherry. Panel (A) Fluorescence microscopic appearance of IRE11 cells infected with GFPuv expressing R. buchneri. Transformed R. buchneri were in the XX serial transfer when image was GFP expressing R. buchneri. Transformed R. buchneri were in the XX serial transfer when image uvcollected. Transformant is maintained in cell layers seeded at high cell density (1–5 × 106 cells per mL). 6 was collected.Rickettsiae Transformantwere visualized is maintainedusing fluorescence in cell layersmicroscopy seeded with at highFITC cellfilter; density (B) Fluorescence (1–5 × 10 cells

per mL).microscopic Rickettsiae appearance were visualized of ISE6 cells using infected fluorescence with mCherry microscopy expressing with R. peacockii FITC filter;. Rickettsiae (B) Fluorescence were microscopicvisualized appearance using fluorescence of ISE6 microscopy cells infected with with TRITC mCherry filter. All expressing images takenR. using peacockii an upright. Rickettsiae Nikon were visualizedEclipse using E400Diaphot fluorescence fluorescence microscopy microscope. with TRITC Bar equals filter. 5 Allµm. images Both images taken were using collected an upright at the Nikon Eclipsesame E400Diaphot magnification. fluorescence microscope. Bar equals 5 µm. Both images were collected at the same magnification. 4. Discussion In this communication, we report the successful genetic transformation of two rickettsiae widely recognized as non-pathogenic mutualistic symbionts, R. buchneri and R. peacockii. The beneficial effects of these rickettsiae on their host ticks range from protection of the tick from super infection by pathogenic rickettsiae [1] to nutritional supplementation [2]. We have demonstrated that fluorescent protein and antibiotic resistance genes can be delivered to R. buchneri and R. peacockii on shuttle vectors developed from R. amblyommi, a species that carries multiple native plasmids. We have also demonstrated that rickettsial symbionts can also acquire and maintain these low-copy-number plasmids, each rather unique in their genetic makeup, apparently without the loss of their native plasmids [2,21,30,32]. In addition, we have shown that himar1, a broad range Mariner element, was useful for the insertion of fluorescent protein and antibiotic resistance genes into the genome of R. peacockii. Rickettsial symbionts of ticks require tick cells for replication and selection of transformants that are rare and slow growing. They benefited greatly from the use of tick host cells that themselves grow slowly and can be maintained at high density for extended periods. While R. peacockii has a wider host cell range in vitro [33], R. buchneri is more fastidious and we have found only two Ixodes cell lines, ISE6 and IRE11, from Ixodes ticks that are suitable for isolating and growing R. buchneri. We have identified factors that hindered our progress and limited the number of transformants that we were able to isolate. Most noticeable was the low yield of transformants from a single electroporation. The low yield exacerbated the length of time required for transformants to replicate to detectable levels. Several weeks were required for the detection of fluorescent bacterial colonies by microscopy and there were few colonies within the cell layer. This indicated that very few of the symbionts had survived electroporation and/or acquired the transforming plasmid. This was especially apparent when we electroporated R. buchneri, a rickettsia having a long doubling time, with a shuttle vector. The isolation of himar1 transformed R. peacockii was met with limited success. The finding that the himar1 R. peacockii transformant population contained a single insertion site indicated that the population was clonal and arose from a single survivor of the electroporation and selection conditions that were used. Targeting specific genes via site directed mutagenesis and achieving “saturated mutagenesis” depends on our ability to define and improve important transformation parameters. Improved transformation conditions are necessary before we can apply genome-wide mutational analysis of rickettsial symbionts. The challenge that remains is to develop

Vet. Sci. 2016, 3, 34 10 of 13

4. Discussion In this communication, we report the successful genetic transformation of two rickettsiae widely recognized as non-pathogenic mutualistic symbionts, R. buchneri and R. peacockii. The beneficial effects of these rickettsiae on their host ticks range from protection of the tick from super infection by pathogenic rickettsiae [1] to nutritional supplementation [2]. We have demonstrated that fluorescent protein and antibiotic resistance genes can be delivered to R. buchneri and R. peacockii on shuttle vectors developed from R. amblyommi, a species that carries multiple native plasmids. We have also demonstrated that rickettsial symbionts can also acquire and maintain these low-copy-number plasmids, each rather unique in their genetic makeup, apparently without the loss of their native plasmids [2,21,30,32]. In addition, we have shown that himar1, a broad range Mariner element, was useful for the insertion of fluorescent protein and antibiotic resistance genes into the genome of R. peacockii. Rickettsial symbionts of ticks require tick cells for replication and selection of transformants that are rare and slow growing. They benefited greatly from the use of tick host cells that themselves grow slowly and can be maintained at high density for extended periods. While R. peacockii has a wider host cell range in vitro [33], R. buchneri is more fastidious and we have found only two Ixodes cell lines, ISE6 and IRE11, from Ixodes ticks that are suitable for isolating and growing R. buchneri. We have identified factors that hindered our progress and limited the number of transformants that we were able to isolate. Most noticeable was the low yield of transformants from a single electroporation. The low yield exacerbated the length of time required for transformants to replicate to detectable levels. Several weeks were required for the detection of fluorescent bacterial colonies by microscopy and there were few colonies within the cell layer. This indicated that very few of the symbionts had survived electroporation and/or acquired the transforming plasmid. This was especially apparent when we electroporated R. buchneri, a rickettsia having a long doubling time, with a shuttle vector. The isolation of himar1 transformed R. peacockii was met with limited success. The finding that the himar1 R. peacockii transformant population contained a single insertion site indicated that the population was clonal and arose from a single survivor of the electroporation and selection conditions that were used. Targeting specific genes via site directed mutagenesis and achieving “saturated mutagenesis” depends on our ability to define and improve important transformation parameters. Improved transformation conditions are necessary before we can apply genome-wide mutational analysis of rickettsial symbionts. The challenge that remains is to develop a homologous recombination system, but progress being made with other rickettsiae [34–36] may assist us. Our results demonstrate that studies on the experimental parameters influencing the isolation of R. buchneri and R. peacockii mutants are needed, as the reasons for the low yield remain to be identified. Low penetration of plasmid DNA into the symbionts, massive killing due to electroporation conditions and excessive antibiotic selection are potential factors that need to be examined. Recently, Driskell et al. [37] used fluorescence activated cell sorting to harvest cells infected with R. prowazekii expressing fluorescent reporter proteins. To reduce time and variability, frozen stocks of electrocompetent rickettsiae were used. They detected transformants as early as one week after electroporation. Shuttle vector (transforming DNA) concentration was identified as being critical to detecting transformants as soon as a few days after transfecting cells with R. prowazekii. Also needed is research on the effects of selection pressure (concentration and timing of antibiotic selection) and electroporation conditions. The use of non-pathogenic rickettsial symbionts, as opposed to R. prowazekii, a BSL3 pathogen, will further facilitate progress in this area. These mutant endosymbionts expressing fluorescent proteins will be useful for future studies on rickettsia-host interactions in vivo and in vitro. Shuttle vectors provide a way to introduce large and diverse constructs into rickettsial symbionts, circumventing the size limitations imposed by the himar1 transposon [29]. Furthermore, the shuttle vector system will also be useful for complementation assays aimed at examining the function of the wide array of genes that are defective in these two species. They will also enable paratransgenic studies using transformed rickettsial symbionts expressing Vet. Sci. 2016, 3, 34 11 of 13 anti-pathogen factors. Recent studies show that book lice (Liposcelis bostrychophila) and the tick Ixodes pacificus can be cured of endosymbiotic rickettsiae using heat or antibiotics [13,38,39], potentially setting the stage for future research aiming to reintroduce R. buchneri and R. peacockii transformants back into aposymbiotic host ticks.

5. Conclusions We have made progress in the genetic manipulation of two rickettsia species being used as models for mutualistic symbionts, R. buchnerii and R. peacockii. Transformed endosymbiotic rickettsiae maintained multiple plasmids, including shuttle plasmids carrying fluorescent protein and antibiotic resistance transgenes. Future studies are needed to define the experimental parameters that influence the isolation of R. buchneri and R. peacockii mutants.

Acknowledgments: This research was supported by National Institutes of Health grants R01 AI49424 and R01 AI081690 to U. G. M. (http://www.grants.nih.gov/grants/oer.htm). The funders had no role in study design, data collection, and analysis; decision to publish; or preparation of the manuscript. We thank Gerald Baldridge and Yumi Kumagai and Rod Felsheim for assistance in the R. peacockii research. Author Contributions: Ulrike Munderloh and Timothy Kurtti conceived and planned these studies. Timothy Kurtti, Ulrike Munderloh, Nicole Burkhardt and Chan Heu performed the transformations and did the PFGE analysis of transformant plasmids. Analysis of data was done by Timothy Kurtti, Ulrike Munderloh, Nicole Burkhardt, and Chan Heu. Timothy Kurtti and Ulrike Munderloh wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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