Assessment of Intracellular Growth of Coxiella Mutants within Eukaryotic Cells

1. Generation of a library of green fluorescence protein, GFP-tagged Coxiella transposon mutants

Manipulate Coxiella burnetii RSA439 NMII in a biosafety containment level 2 (BSL-2) in a microbial safety cabinet (MSC) in compliance with local rules. If compatible with the bacterial model used, repeat steps from 1.4.1 to 1.4.4 to increase the probability of obtaining clonal mutants. A typical mutant library is composed (at least) of a number of mutants that is equal to three times the number of coding sequences annotated in the genome of the organism used.

1. Preparation of electrocompetent Coxiella RSA439 NMII:
   1. Prepare 1x acidified citrate cysteine medium-2 (ACCM-2): 13.4 mM citric acid, 16.1 mM sodium citrate, 3.67 mM potassium phosphate, 1 mM magnesium chloride, 0.02 mM calcium chloride, 0.01 mM iron sulfate, 125.4 mM sodium chloride, 1.5 mM L-cysteine, 0.1 g/L Bacto Neopeptone, 2.5 g/L casamino acids, 1 g/L methyl beta cyclodextrin, 125 ml/L Roswell Park Memorial Institute (RPMI). Adjust pH to 4.75 and filter sterilize (do not autoclave).
      NOTE: Liquid ACCM-2 is stable at 4 °C for approximately 1 month.
   2. Inoculate 100 ml of ACCM-2 with 2 x 10⁶ genome equivalent (GE)/ml of Coxiella RSA439 NMII (from a bacterial stock previously generated and quantified as in step 1.5) from -80 °C stocks and distribute the bacterial suspension in 75 cm² cell culture flasks with vented caps (10 – 15 ml of bacterial suspension per flask). Grow for 7 days at 37 °C in a humidified atmosphere of 5% carbon dioxide (CO₂) and 2.5% oxygen (O₂).
   3. Pool the resulting bacterial suspension in 50 ml tubes and centrifuge at 3,900 x g for 1 hr at 4 °C.
   4. Discard the supernatant and resuspend the pellet in 30 ml of 10% glycerol. Centrifuge at 3,900 x g for 1 hr at 4 °C.
   5. Resuspend the pellet in an adequate volume of 10% glycerol (typically 2 ml) and aliquot 50 µl in 500 µl tubes. Keep resuspended bacteria on ice during the whole process.
      NOTE: At this stage, bacteria are electrocompetent and one aliquot is sufficient to perform one electroporation. The bacterial suspensions can be stored at -80 °C for 6 months or be directly used for electroporation of plasmid deoxyribonucleic acid (DNA).
2. Electroporation of competent Coxiella with transposon- and transposase-encoding plasmids:
   1. Pre-cool a 0.1 cm electroporation cuvette for 10 min on ice. Mix 50 µl of electrocompetent Coxiella with 10 µg transposon plasmid and 10 µg of transposase plasmid. Ensure that plasmid concentration is higher than 500 µg/ml to minimize dilution of the glycerol.
   2. Electroporate using the following setup: 18 kV, 500 Ω, 25 µF. Ensure that the resulting time constant is comprised between 9 and 13 msec.
   3. Immediately add 950 µl of RPMI, resuspend the electroporated bacteria and transfer to a screw cap tube and keep at room temperature.
   4. Take 200 µl of the electroporated bacteria and add 3 ml of ACCM-2 supplemented with 1% heat-inactivated Fetal Bovine Serum (FBS) into 6-well plates. Add 88 µl of dimethyl sulfoxide (DMSO) to the remaining volume of electroporated bacteria (to reach a final concentration of 10% DMSO) and store at -80 °C.
      NOTE: for the following protocol, the transposable element and the transposase are encoded by two different plasmids (pITR-CAT-GFP and pUC19-Himar1C9, respectively). Both plasmids lack a Coxiella-specific replication origin, making them suicide plasmids when electroporated in Coxiella. This ensures stable transposon insertions. The transposable element contains a Chloramphenicol resistance cassette under the regulation of the Coxiella promoter p1169 for selection and the gfp gene under the regulation of the Coxiella promoter p311 to tag the generated mutants with GFP.
3. Selection of the transposon mutants:
   1. Incubate the 6-well plates inoculated as described above (1.2.4) overnight at 37 °C in a humidified atmosphere of 5% CO₂ and 2.5% O₂. Add the appropriate antibiotics (375 µg/ml kanamycin or 3 µg/ml chloramphenicol). Incubate the bacterial culture for 3 additional days in the conditions described above.
4. Isolation of individual mutants:
   1. Preparation of solid ACCM-2 plates and plating of Coxiella transposon mutants
      NOTE: The following instructions are for 1 Petri dish, several dilutions of bacterial cultures have to be tested to assess the optimal inoculation volume for colony isolation.
      1. Heat 10.5 ml of 0.5% agarose in a microwave and let it cool in a 55 °C water bath. Heat 11.25 ml of 2x ACCM-2 (pH 4.75) at 37 °C.
      2. Prepare bottom agarose:
         1. Mix 10 ml of melted 0.5% agarose with 10 ml of 2x ACCM-2 and add the appropriate antibiotics (375 µg/ml kanamycin or 3 µg/ml chloramphenicol).
         2. Pour immediately into the Petri dish. Keep the Petri dish unlidded, let the medium cool for 30 min, and air-dry for 20 min.
      3. Prepare top agarose:
         1. Mix 1.25 ml of 2x ACCM-2 with 0.75 ml of water in a 5 ml polystyrene tube, add the appropriate antibiotics (375 µg/ml kanamycin or 3 µg/ml chloramphenicol) and incubate at 37 °C.
         2. Add the bacterial culture (typically 1 to 100 µl) and vortex for 5 sec.
         3. Add 0.5 ml of melted agarose, mix, and immediately pour on the bottom agarose.
         4. Allow to cool for 20 min, replace the lid on the Petri dish, and incubate at 4 °C for 20 min to facilitate agarose solidification.
         5. Air dry for 20 min unlidded in an MSC. Grow plates at 37 °C in a humidified atmosphere of 5% CO₂ and 2.5% O₂ for 6 to 7 days.
   2. Add DMSO to the remaining bacterial cultures in order to reach a final concentration of 10% DMSO and store at -80 °C.
   3. Assess the optimal dilution as follows: ensure that colonies are 0.5 to 1 mm in diameter and are properly isolated to avoid cross-contamination. Thaw remaining bacterial cultures from point 1.4.2 and plate at the appropriate dilution on ACCM-2 agar as described in 1.4.1.2 and 1.4.1.3. Incubate for 6 to 7 days as described in 1.4.1.3.5.
   4. Once colonies are detectable, collect them by cutting the end of a 1 ml tip, picking the plug containing isolated colonies, and dispersing the colony by pipetting in 1.5 ml of ACCM-2 containing the appropriate antibiotics (375 µg/ml kanamycin or 3 µg/ml chloramphenicol) in a 24-well plate. Amplify individual colonies for 6 days in the conditions described in 1.3.1. On day 3 of incubation, disperse the bacterial clumps by pipetting each culture.
   5. Store each mutant suspension in 2D barcoded screw cap tubes in 96-well plates in 10% DMSO at -80 °C.
5. Evaluation of bacterial concentration:    
   NOTE: The following protocol can be applied to obtain the growth curves of bacterial mutants replicating in an axenic medium (see 1.4.4).
   1. Standard curve preparation:
      1. Prepare a 2 µg/ml stock solution of double-stranded DNA (dsDNA) (typically a random plasmid of known size and concentration) in 1x Tris- ethylenediaminetetraacetic acid, EDTA (TE). Prepare 10-fold serial dilutions from the stock solution to obtain concentrations ranging from 2 µg/ml to 2 ng/ml. Dispense 50 µl of each concentration to single wells of a 96-well microplate with black walls and bottom (see Table of Materials).
      2. Dilute the dsDNA quantitation reagent 1:200 in 1x TE buffer and add 55 µl of the diluted reagent to each sample in the 96-well microplate. Mix well using a plate shaker and incubate for 2 to 5 min at room temperature, in the dark.
      3. Measure the samples' fluorescence using a fluorescence microplate reader and filters for standard fluorescein wavelengths (excitation ~480 nm, emission ~520 nm).
      4. Plot the plasmid concentration range against the fluorescence intensity readings.
   2. Bacterial suspension quantitation:
      1. Dispense 5 µl of 10% Triton X-100 per well in a 96-well microplate with black walls and bottom (see Table of Materials). Add 50 µl of the bacterial suspensions to each well and incubate for 10 min at room temperature, on a plate shaker.
      2. Dilute the dsDNA quantitation reagent 1:200 in 1x TE buffer and add 55 µl of the diluted reagent to each sample in the 96-well microplate. Mix well using a plate shaker and incubate for 2 to 5 min at room temperature, in the dark.
      3. Measure the samples' fluorescence using a fluorescence microplate reader and filters for standard fluorescein wavelengths (excitation ~480 nm, emission ~520 nm).
      4. To obtain the bacterial DNA concentration, plot the fluorescence readings in the chart obtained at point 1.5.1.4. Divide the DNA concentration by the mass of the Coxiella genome (2.2 fg) to obtain bacterial concentrations. Express results in Genome Equivalent/ml.
      5. Discard mutants exhibiting a significant growth defect in ACCM-2.

2. Single Primer Colony Polymerase Chain Reaction (PCR), Sequencing, and Annotation

NOTE: The following protocol is for the DNA amplification of 96 samples, a multichannel pipette is recommended for the following steps. Column purification of PCR products using magnetic beads and DNA sequencing with a transposon-specific primer (2.3) is subcontracted to an external company.

1. Ensure that the amplification primer is designed in order to hybridize between 100 and 200 base pairs upstream of the inverted tandem repeat (ITR), to obtain PCR products covering the transposon insertion site on the Coxiella genome. Prepare 3 ml of PCR mix (1x high fidelity buffer, 200 µM deoxynucleoside triphosphate (dNTPs), 1 µM amplification primer, 20 U/ml high fidelity DNA polymerase) and dispense 29 µl per well in a 96-well PCR plate set on ice. Transfer 1 µl of each mutant in the stationary phase in ACCM-2 to the PCR mix.
2. Run PCR with initial denaturation (98 °C, 1 min), 20 high stringency cycles (98 °C, 10 sec; 50 °C, 30 sec; 72 °C, 90 sec), 30 low stringency cycles (98 °C, 10 sec; 30 °C, 30 sec; 72 °C, 90 sec) and 30 high stringency cycles (98 °C, 10sec; 50 °C, 30 sec; 72 °C, 90 sec) followed by a final extension at 72 °C for 7 min.
3. Purify PCR products using magnetic beads and sequence DNA with a transposon-specific primer. Design the transposon-specific primer with a predicted melting temperature comprised between 50°C and 75°C, a GC content between 40% and 60 %, a length between 18 and 25 nucleotides, and an annealing site downstream of the amplification primer hybridization site and at least 100 base pairs upstream of the first base pair of the transposon ITR.
4. Using sequence analysis software, load the complete, annotated genome of Coxiella burnetii 493 NMI. Use the "align to reference" function to load and align (blastn) the sequencing results and determine the site of transposition. Discard mutants with non-matching and/or displaying double reads. To monitor the saturation of the mutant library, keep a record of the occurrence of multiple transposon insertions at the same site.

3. Eukaryotic Cells Challenge with Coxiella Mutants and Monitoring of Intracellular Growth

NOTE: A multichannel pipette is recommended for the following steps. Infections were performed in triplicates in sterile 96-well microplates with black walls and flat transparent bottoms. wt Coxiella burnetii expressing GFP was provided by Dr. Robert Heinzen.

1. Grow Vero cells in RPMI without phenol red supplemented with 10% fetal bovine serum (FBS) in the absence of antibiotics (Complete RPMI media).
2. The day before infection, wash Vero cells from a confluent or sub-confluent cell culture flask with 10 ml of phosphate-buffered saline (PBS).
3. Detach Vero cells by adding 1 ml of trypsin EDTA solution to the cell culture flask and incubate for 3 to 5 min at 37 °C in a humidified atmosphere of 5% CO₂.
4. Resuspend cells in 10 ml of Complete RPMI media. Count cells and prepare a cell suspension of 10⁵ cells per ml.
5. Dispense 100 µl of the cell suspension in each well of a black 96-well plate with a flat transparent bottom.
6. Centrifuge for 5 min at 400 x g at room temperature (RT) to facilitate cell adhesion at the bottom of the wells and incubate overnight at 37 °C in a humidified atmosphere of 5% CO₂.
7. Thaw the 96-well plates containing the Coxiella mutants at RT and dilute 150 µl of bacterial suspension in 300 µl of RPMI without phenol red and FBS in a deep well 96-well plate.
8. Remove media from the microplate containing Vero cells and dispense 100 µl/well of diluted Coxiella mutants (MOI of 100). Use well A1 as negative (non-infected cells) control and wells A2 and A3 as positive controls (cells infected with wt Coxiella expressing GFP at multiplicities of infections (MOI) of 100 and 200).
9. Centrifuge the plate for 10 min at 400 x g at RT using an aerosol-tight centrifuge plate holder.
10. Incubate at 37 °C in a humidified atmosphere of 5% CO₂ for 2 hr then replace bacteria-containing medium with 100 µl/well of fresh, complete RPMI medium.
11. Measure GFP fluorescence every day for 7 days using a fluorescence microplate reader and filters for standard fluorescein wavelengths (excitation ~480 nm, emission ~520 nm). To avoid interference due to condensation and signal dispersion in the culture medium, use bottom excitation and emission recording on the microplate reader.

4. Preparation of Samples for Automated Image Acquisition

NOTE: The procedure is for one 96-well plate, scale up volumes accordingly. Steps from 4.2 may take advantage of a plate washer.

1. On the 7^th day post-infection, remove the medium from the plate and replace it with 50 µl/well of fresh, complete medium containing a cell-permeable fluorescent dye at the appropriate dilution (usually 1:1,000, to be optimized according to the cell line used). Incubate cells for 30 – 60 min at 37 °C in a humidified atmosphere of 5% CO₂.
2. Replace the medium with 50 µl/well of 4% paraformaldehyde (PFA) in PBS, incubate for 30 min at room temperature (RT) then remove the PFA-containing buffer and wash 3 times with PBS.
3. Remove PBS and dispense 50 µl/well of blocking solution (0.5% bovine serum albumin, 50 mM ammonium chloride, NH₄Cl in PBS, pH 7.4) supplemented with 0.05% saponin. Incubate at RT for 30 min.
4. Replace the blocking solution with 40 µl/well of fresh blocking solution supplemented with saponin (as above) and with an anti-lysosome-associated membrane glycoprotein-1 (anti-LAMP1) antibody at a 1:500 dilution. Incubate plate for 30 min at RT.
5. Remove the blocking solution and wash the 96-well plate 5 times with 100 µl/well of PBS.
6. Dispense 40 µl/well of blocking solution supplemented with saponin (as above), the appropriate fluorescently labeled secondary antibody (at a dilution of 1:1,000) to reveal the anti-LAMP1 antibody applied at step 4.4, and with Hoechst 33258 at 5 µg/ml. Incubate plate for 30 min at RT.
7. Remove the blocking solution and wash the 96-well plate 5 times with 100 µl/well of PBS. Leave the volume of PBS corresponding to the last wash in the 96-well plate, as the fixed cells should not dry out.
8. Image the plate immediately or store the plate at 4 °C, protected from light, for subsequent analysis.

5. Image Acquisition

1. Acquire images in the GFP (488 nm, bacteria), Hoechst 33258 (350 nm, host cell nuclei), red (~555 nm, cell membrane marker), and far red (~615 nm, LAMP1) channels using an epifluorescence automated microscope equipped with a 20X objective. Acquire 21 independent fields per well in order to image a minimum of 5,000 cells per sample. Apply autofocusing using the host cell nuclei channel as a reference. When working with bacterial pathogens infecting a low percentage of host cells, users can adjust the number of independent fields imaged per well, in order to obtain a minimum of 500 infected cells to analyze.