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