Name: an electroporation method to transform rickettsia spp. with a fluorescent protein-expressing shuttle vector in tick cell lines
Uploaded: 2022-10-11T14:20:00
Description: Watch this Scientific Journal Video about An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines at JoVE.com

We thank Timothy J. Kurtti and Benjamin Cull for their insightful discussions and suggestions. This study was financially supported by a grant to U.G.M. from the NIH (2R01AI049424) and a grant to U.G.M. from the Minnesota Agricultural Experiment Station (MIN-17-078).

1. Propagation and purification of R. parkeri from tick cell culture
NOTE: All cell culture procedures are to be performed in a class II biosafety cabinet.
<ol>
	<li>Preparing R. parkeri-infected tick cells
	<ol>
		<li>Grow ISE6 cells in 25 cm2 cell culture flasks at 34 &#176;C in 5 mL of L15C300 medium17, supplemented with 5% fetal bovine serum (FBS), 5% tryptose phosphate broth (TPB), and 0.1% lipoprotein concentrate (LP...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+shuttle+vectors+for+transformation+of+diverse+Rickettsia+species.">Development of shuttle vectors for transformation of diverse Rickettsia species.</a> PLoS One. 6 (12), 29511 (2011).</li><li>Welch, M. D., Reed, S. C., Lamason, R. L., Serio, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+an+epitope-tagged+virulence+protein+in+Rickettsia+parkeri+using+transposon+insertion.">Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion.</a> PloS One. 7 (5), 37310 (2012).</li><li>Oliver, J. D., Burkhardt, N. Y., Felsheim, R. F., Kurtti, T. J., Munderloh, U. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+characteristics+are+altered+for+Rickettsia+bellii+transformed+to+overexpress+a+heterologous+rickA+gene.">Motility characteristics are altered for Rickettsia bellii transformed to overexpress a heterologous rickA gene.</a> Applied and Environmental Microbiology. 80 (3), 1170-1176 (2014).</li><li>Kurtti, T. J., Burkhardt, N. Y., Heu, C. C., Munderloh, U. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+expressing+Rickettsia+buchneri+and+Rickettsia+peacockii+for+tracking+symbiont-tick+cell+interactions.">Fluorescent protein expressing Rickettsia buchneri and Rickettsia peacockii for tracking symbiont-tick cell interactions.</a> Veterinary Sciences. 3 (4), 34 (2016).</li><li>Oliver, J. D., Ch&#225;vez, A. S., Felsheim, R. F., Kurtti, T. J., Munderloh, U. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Ixodes+scapularis+cell+line+with+a+predominantly+neuron-like+phenotype.">An Ixodes scapularis cell line with a predominantly neuron-like phenotype.</a> Experimental and Applied Acarology. 66 (3), 427-442 (2015).</li><li>Grabowski, J. M., Gulia-Nuss, M., Kuhn, R. J., Hill, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+reveals+proteins+for+metabolism+and+protein+processing+associated+with+Langat+virus+infection+in+Ixodes+scapularis+(black-legged+tick)+ISE6+cells.">RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells.</a> Parasites &amp; Vectors. 10 (1), 1-4 (2017).</li><li>Al-Rofaai, A., Bell-Sakyi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+cell+Lines+in+research+on+tick+control.">Tick cell Lines in research on tick control.</a> Frontiers in Physiology. 11, 152 (2020).</li><li>Wang, X. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrion-dependent+apoptosis+is+essential+for+Rickettsia+parkeri+infection+and+replication+in+vector+cells.">Mitochondrion-dependent apoptosis is essential for Rickettsia parkeri infection and replication in vector cells.</a> mSystems. 6 (2), 01209-01220 (2021).</li><li>Berney, M., Hammes, F., Bosshard, F., Weilenmann, H. U., Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+interpretation+of+bacterial+viability+by+using+the+LIVE/DEAD+BacLight+Kit+in+combination+with+flow+cytometry.">Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry.</a> Applied and Environmental Microbiology. 73 (10), 3283-3290 (2007).</li><li>Riley, S. P., Macaluso, K. R., Martinez, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotransformation+and+clonal+isolation+of+Rickettsia+species.">Electrotransformation and clonal isolation of Rickettsia species.</a> Current Protocols in Microbiology. 39, 1-20 (2015).</li><li>Burkhardt, N. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+rickettsial+host+range+for+shuttle+vectors+based+on+dnaA+and+parA+genes+from+the+pRM+plasmid+of+Rickettsia+monacensis.">Examination of rickettsial host range for shuttle vectors based on dnaA and parA genes from the pRM plasmid of Rickettsia monacensis.</a> Applied and Environmental Microbiology. 88 (7), 00210-00222 (2022).</li><li>Kurtti, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rickettsia+buchneri+sp.+nov.,+a+rickettsial+endosymbiont+of+the+blacklegged+tick+Ixodes+scapularis.">Rickettsia buchneri sp. nov., a rickettsial endosymbiont of the blacklegged tick Ixodes scapularis.</a> International Journal of Systematic and Evolutionary Microbiology. 65, 965 (2015).</li><li>Munderloh, U. G., Kurtti, T. 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Here, we demonstrate a method for introducing exogenous DNA encoded on the shuttle plasmid pRAM18dSFA into rickettsiae using electroporation. In this procedure, cell-free rickettsiae were purified from host cells, transformed with a rickettsial shuttle vector, and released onto tick cells for infection. Also described is a confocal immunofluorescence procedure to detect red fluorescence protein-expressing R. parkeri in tick cells. Similar methods are applicable to other Rickettsia species and with furth...

Rickettsioses are caused by a broad range of obligate intracellular bacteria that belong to the genus Rickettsia (family Rickettsiaceae, order Rickettsiales). The genus Rickettsia is classified into four major groups based on phylogenetic characteristics1,2: the spotted fever group (SFG), which contains those rickettsiae that cause the most severe and fatal tick-borne rickettsioses (e.g., Rickettsia rickettsii, the causative agent of Rocky Mountain Spotted Fever), the typhus group (TG, e.g., Rickettsia prowazekii, the agent of epidemic typhus), the transitional group (TRG, e.g., Rickettsia felis, the causative agent of flea-borne spotted fever), and the ancestral group (AG, e.g., Rickettsia bellii).
Among the oldest known vector-borne diseases, rickettsioses are mainly acquired following transmission of the pathogens through the bites of infected arthropod vectors, including ticks, fleas, lice, and mites3,4. Although the discovery of effective antibiotics improved treatment outcomes, emerging and re-emerging epidemic rickettsioses continue to challenge traditional prevention and control strategies. Thus, a comprehensive understanding of rickettsia/host/vector interactions would ultimately establish a strong foundation for developing new approaches to prevent and cure these ancient diseases.
In nature, horizontal gene transfer (HGT) in bacteria occurs through conjugation, transduction, and transformation5. In vitro bacterial transformation utilizes these HGT concepts, although the intracellular nature of rickettsiae presents some challenges. The restricted growth conditions and poorly understood conjugation and transduction systems in different species of rickettsiae have prevented the application of conjugation and transduction methods in rickettsiae6,7,8. Compared with other obligate intracellular bacterial genera (e.g., Chlamydia, Coxiella, Anaplasma, and Ehrlichia), the genus Rickettsia differs with regard to the growth and replication strategies within the cell cytoplasm, which imposes specific challenges to the genetic modification of rickettsiae due to their unique lifestyle features9.
The initial hurdle to overcome when attempting the genetic modification of rickettsiae is to achieve successful transformation. Thus, designing a feasible approach with high transformation efficiency would be extremely valuable for developing genetic tools for rickettsiae. Here, we focus on electroporation, a broadly recognized transformation method that has been used to introduce exogenous DNA successfully into several species of rickettsiae, including Rickettsia prowazekii, Rickettsia typhi, Rickettsia conorii, Rickettsia parkeri, Rickettsia montanensis, Rickettsia bellii, Rickettsia peacockii, and Rickettsia buchneri10,11,12,13,14,15,16.
This paper describes a procedure for the electroporation of the R. parkeri strain Tate&#39;s Hell (accession: GCA_000965145.1) with the shuttle vector pRAM18dSFA derived from the Rickettsia amblyommatis strain AaR/SC plasmid pRAM18 engineered to encode mKATE, a far-red fluorescent protein, and aadA, conferring spectinomycin and streptomycin resistance13,15,20. Transformed R. parkeri are viable and stably maintained under antibiotic selection in tick cell lines. In addition, we show that the localization of transformed R. parkeri in live tick cells via confocal microscopy can be used to assess the quality of transformation rates in vector cell lines.

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	<tbody>
		<tr>
			<td>0.1 cm gap gene pulser electroporation cuvette</td>
			<td>Bio-Rad</td>
			<td>1652083</td>
			<td></td>
		</tr>
		<tr>
			<td>2 &#956;m pore size filter&#160;</td>
			<td>GE Healthcare Life Sciences Whatman</td>
			<td>6783-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Luer-lock syringe&#160;</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>60-90 silicon carbide grit&#160;</td>
			<td>LORTONE, inc</td>
			<td>&#160;591-056</td>
			<td></td>
		</tr>
		<tr>
			<td>absolute methanol&#160;</td>
			<td>Fisher Scientific</td>
			<td>A457-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptose phosphate broth&#160;</td>
			<td>BD</td>
			<td>260300</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin centrifuge Cytospin4</td>
			<td>Thermo Fisher Scientific</td>
			<td>A78300003</td>
			<td>The rotor is detatchable so the whole rotor can be put into the hood to load infectious samples</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit (10)</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td>used to obtain endotoxin-free pRAM18dSFA plasmid</td>
		</tr>
		<tr>
			<td>extended fine tip transfer pipet&#160;</td>
			<td>Perfector Scientific</td>
			<td>TP03-5301</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum&#160;</td>
			<td>Gemini Bio</td>
			<td>900-108</td>
			<td>The FBS batch has to be tested to make sure ISE6 cells will grow well in it.</td>
		</tr>
		<tr>
			<td>Gene Pulser II electroporator with Pulse Controller PLUS</td>
			<td>Bio-Rad</td>
			<td>165-2105 &#38; 165-2110</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore-Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Fiji</td>
			<td>National Institute of Health</td>
			<td></td>
			<td>raw image editing</td>
		</tr>
		<tr>
			<td>KaryoMAX Giemsa stain&#160;</td>
			<td>Gibco</td>
			<td>&#160;2021-10-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium</td>
			<td>Gibco</td>
			<td>41300039</td>
			<td></td>
		</tr>
		<tr>
			<td>lipoprotein concentrate&#160;</td>
			<td>MP Biomedicals</td>
			<td>191476</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Diaphot</td>
			<td>Nikon</td>
			<td></td>
			<td>epifluorescence microscope</td>
		</tr>
		<tr>
			<td>NucBlue Live ReadyProbes Reagent&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Disc Scanning Unit (DSU) confocal microscope&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petroff-Hausser Counting Chamber</td>
			<td>Hausser Scientific</td>
			<td>&#160;Chamber 3900</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Millipore-Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Vortex Genie 2</td>
			<td>12-812</td>
			<td></td>
		</tr>
	</tbody>
</table>

an electroporation method to transform rickettsia spp. with a fluorescent protein-expressing shuttle vector in tick cell lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

The morphology of R. parkeri in ISE6 cells under a light microscope after Giemsa staining are shown in Figure 1. In Figure 2, transformed R. parkeri expressing red fluorescence protein in ISE6 cells are shown using confocal microscopy. There is a substantial increase in the infection rate of transformed R. parkeri (red) in ISE6 cells (blue, corresponds to the nuclei) from (A) day 7 to (B) day 10 of inc...

Watch this Scientific Journal Video about An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines at JoVE.com

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

Functional studies of Rickettsia genes are crucial for understanding their role in pathogenesis, so we need dependable methods of altering the genes in a controlled fashion. Our protocol uses electroporation to introduce exogenous DNA into obligate intracellular bacteria of the genus Rickettsia, thus allowing us to study the effects of the introduced DNA. Demonstrating the procedure along with me today will be Lisa Price, a researcher from Dr.Munderloh and Dr.Kurtti's laboratory.To begin, prepare a 90 to 100%Rickettsia parkeri-infected ISE6 cell culture as described in the text. Then, to determine the infection rate by Giemsa staining, dilute the cell suspension to a ratio of 1:5 with complete medium and deposit 100 microliters of the cell suspension onto a glass microscope slide by centrifugation at 113 G for five minutes. After letting the slide air-dry, fix it an absolute methanol for five minutes at room temperature.Then, incubate the slide and Giemsa stain for 30 minutes at 37 degrees Celsius. Next, rinse the slide in water and once dry, view it under a light microscope with oil objectives. Determine the percentage of infected cells by counting the infected and non-infected cells.To prepare the cell-free Rickettsia parkeri, pour about 0.2 milliliters of sterile 60 to 90 silicon carbide grit into two two-milliliter sterile microcentrifuge tubes and set the tubes aside. From a previously inoculated Rickettsia parkeri-infected flask containing five milliliters of medium, remove two milliliters of the medium, taking care not to disturb the cell layer. Then, resuspend the cells with the remaining three milliliters of the medium.Divide this suspension equally between the two prepared silicone carbide grit tubes and vortex the tube at high speed for 30 seconds. Then, place them on ice. Next, prepare a sterile five-milliliter luer lock syringe by extending the plunger and insert the plunger end into a polystyrene base used for holding 15-milliliter conical tubes.Using a barrier two-milliliter Pasteur pipette operated with a rubber bulb, carefully remove the supernatant of the vortex cells without aspirating any grit. Then, insert the pipette tip into the syringe hub opening and eject the contents into the syringe with gentle pressure. Next, filter the Rickettsia parkeri culture into sterile 1.5-milliliter tubes through a sterile two-micrometer pore size filter fitted on the syringe hub and centrifuge the tubes at 13, 600 G for five minutes at four degrees Celsius.After removing the supernatant, wash the cell pellet two times with 1.2 milliliters of 300-millimolar ice-cold sucrose solution. Centrifuge the sample and remove the supernatant between washes. At the end of centrifugation, resuspend the pooled cell pellet with 100 to 150 microliters of ice-cold sucrose solution for two to three transformation samples, respectively.Then, divide the sample into 50-microliter aliquots in pre-chilled, sterile, 1.5-microliter microcentrifuge tubes. For transformation at three micrograms of endotoxin-free pRAM18sSFA plasmid DNA to each tube containing 50 microliters of Rickettsia parkeri on ice and stir gently with a pipette tip. Transfer the mixture into a pre-chilled sterile electroporation cuvette with a 0.1-centimeter gap size.Gently tap the cuvette to distribute the mixture evenly and incubate on ice for 10 to 30 minutes. Meanwhile, remove the medium from a fully-confluent previously cultured ISE6 cell culture flask and resuspend the cell layer by gently rinsing the cells with 1.5 milliliters of fresh medium containing sodium bicarbonate and HEPES buffer. After generating a homogenous cell suspension, transfer the whole volume into a single sterile two-milliliter microcentrifuge tube.Next, electroporate the Rickettsia parkeri and plasmid DNA mixture using an electroporator. After electroporation, use a sterile extended fine-tip pipette to transfer a small amount of the ISE6 cell suspension into the cuvette and gently pipette up and down to recover the electroporated Rickettsia parkeri. Then, transfer the contents of the cuvette to the two-milliliter tube containing the remainder of the ISE6 cell suspension.Next, centrifuge the sample at room temperature, first at 700 G for two minutes to pull down the ISE6 cells, and then at 13, 600 G for one minute to pull down the Rickettsia. Incubate the tube at 34 degrees Celsius for 15 minutes to one hour. After incubation, transfer the resuspended contents of one transformation into one 25 square-centimeter cell culture flask containing 3.5 milliliters of fresh medium with sodium bicarbonate and HEPES buffer.Rock the flask to spread the mixture evenly and incubate at 34 degrees Celsius. After 16 to 24 hours, add 10 microliters each of spectinomycin and streptomycin into the incubated flask. Rock the flask to mix the antibiotics evenly into the medium before returning the flask to the incubator.Successful propagation of wild-type Rickettsia parkeri and ISE6 cells was evident by Giemsa staining. The nuclei of the ISE6 cells stained dark purple with Giemsa in the intracellular as well as extracellular Rickettsi are visible. Transformation of Rickettsia parkeri expressing the red fluorescence protein in ISE6 cells was confirmed by confocal microscopy after seven days of incubation.A substantial increase in the infection rate was observed after 10 days. Keep everything sterile during the whole procedure. Pay attention to detail and be patient, as some species of Rickettsia take a long time to transform, especially slow growing endosymbionts.This protocol has enabled the study of multiple aspects of Rickettsia biology and their interactions with their arthropod vectors and mammalian hosts. For example, fluorescent protein-expressing Rickettsi have been used to track motility and symbiont tick cell interactions.

Research

JoVE Journal

Biology

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

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