Cell culture. Mus musculus mammary gland cancer cells (4T1) were grown at 37oC 5% CO2, in Roswell Park Memorial Institute (RPMI) media supplemented with 10% v/v Foetal Bovine Serum, 100 U/mL penicillin and 100 µg/mL of streptomycin (ThermoFisher). The cells were harvested with 0.5 ml/10 cm2 trypsin by centrifugation at 180 x g, washed with Phosphate Buffer Saline (PBS), and counted with a NucleoCounter® NC-100™ (chemometect, Copenhagen) following manufacturer’s instructions. The cells were fixed in 40 ml of 4% w/v buffered formalin for 24 h at room temperature (RT).
Bacterial growth conditions. Escherichia. coli K12 MG1655 carrying a P16Lux plasmid [38] was grown aerobically at 37oC to an OD600 of 0.8 in Luria-Bertani (LB) medium supplemented with 300 µg/ml Erythromycin and harvested by centrifugation at 3000 x g, for 10 min at 4oC, suspended to a 2X concentration in 4% w/v buffered formalin for 24 h at RT.
Counting fixed bacterial cells. Bacterial cell suspensions were counted following the instructions of the bacterial counting kit (Invitrogen). In brief, after fixation, a 10% aliquot was taken from this suspension and serially diluted (100X) with filtered sterilised 0.15M NaCl solution to obtain a cell density of approximately 1 × 106 cells in 989 µl of NaCl. Bacterial cells were stained with 1 µl of SytoBC and 10 µl (1 × 106) of counting beads were added to the suspension. Cells were counted in an LSR II Flow Cytometer (BD Biosciences, NJ, USA). The acquisition trigger was set to side scatter and set to 800.
Membrane permeabilisation assay. After fixation, 8 × 107 4T1 cells were harvested at 180 x g for 10 min, washed once with 20 ml of Tris Buffer Saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.6), and suspended to a final density of 2.5 × 106 cells per ml in TBS. Similarly, 5 × 109 E. coli cells, were harvested at 300 x g for 10 min, washed once with 20 ml of TBS and suspended to a final density of 2.5 × 107 cell per ml in TBS. 500 µl of the cell suspensions were aliquoted into 1.5 ml tubes and treated with a permeabilisation agent. Permeabilisation agents tested were: Triton X-100 (0.1% v/v), Tween-20 0.2% v/v, Saponin (0.1% w/v), Digitonin (0.5 µg/ml). All were acquired form Sigma-Aldrich. Concentrations used were as described for several protocols [6].
The cells were permeabilised for 25 min, at 25oC, shaking at 500 rpm. Permeabilised cells were washed once with TBS (centrifugation speeds as above) and blocked on ice with TBS + 1% w/v Bovine Serum Albumin (BSA) for 30 min. Blocked cells were exposed to 0.75 µg of Cyanine-5 (Cy5) or Phycoerythrin (PE) labelled Streptavidin (SAv-Cy5, MW = 60 KDa or SAv-PE, MW = 360 KDa) (Biolegend, CA, USA) for 30 min at 25oC, shaking at 280 rpm. Cells were washed with 1 ml of 0.15 M NaCl solution and resuspended in 350 µl of the same solution for analysis. Bacterial cells were also labelled with 1 µl of SytoBC (Invitrogen) for 5 min and analysed by flow cytometry in a BD LSRII. 4T1 Cells were identified and gated based on their Forward/Side scatter and E. coli cells were detected using the 488-1 (Fluorescein isothiocyanate - FITC), 525/50 filter for SytoBC and gated using the side scatter. Cy5 positive cells were detected with the red 670/14 filter. PE positive cells were detected with the yellow/green 780/60 filter. For each experimental replicate, 3 × 10,000 events were recorded for 4T1 cells and 3 × 100,000 for bacteria.
DNAse screening. A screen for selecting the DNAse that had highest activity in depleting DNA in a reaction buffer containing Saponin. DNAses tested: Recombinant DNAse I [1-2U, 1 µl] (Sigma-Aldrich), Turbo DNAse [2U, 1 µl] (Thermo-Fisher), Molysis DNase [2 µl] (Molzym GmbH & Co, Bremen, Germany), RQ1 DNAse [20U, 20 µl] (Promega), Benzonase [75 U, 0.3 µl] (Sigma-Aldrich). 5 × 106 4T1 cells, FF for 48 h were treated with 0.2% w/v Saponin and the DNAse tested. Reactions were set in reaction buffers provided or suggested by supplier for 20 min at 37oC. The reaction was stopped by either: the addition of Ethylenediaminetetraacetic acid (EDTA) for Benzonase, the supplied reaction Stop Buffer, or by incubating at 75oC (DNAse I). After which, cells were subject to DNA purification with the QIAamp DNA Mini Kit (QIAGEN). DNA yield was measured with Qubit™ dsDNA HS Assay Kit (Invitrogen). All reactions were performed in triplicate. A no-DNAse control was included. This was incubated under the same conditions with buffer supplied for DNAse I, but without the nuclease.
Saponin Titration. Different w/v saponin concentration (0.1%, 0.25%, 0.5%, 1%) were tested in 1 × 106 E. coli cells that were fixed, washed, permeabilised, blocked and imaged as described for membrane permeabilisation assay.
DNA depletion assay. Cells were fixed, washed and permeabilised as described for the membrane permeabilisation assay. 2.5 × 105 4T1 or 2.5 × 106 E. coli cells were permeabilised, blocked with 500 µl of 1% BSA in TBS + MgCl2 (20 mM Tris-HCL, 20 mM NaCl, 2 mM MgCl2, pH 8) for 30 min on ice. Blocked cells were treated with 1.5 µl (≥ 375 units) of Benzonase nuclease (Sigma-Aldrich) for 30 min at 37oC, shaking at 360 rpm. Treatment was stopped by the addition of 100 mM EDTA. The cells were washed once with TBS and suspended in 0.15M NaCl, where they were stained with 10 µM CytoPhase Violet (Biolegend) for 1 h at RT, shaking at 200 rpm in the dark. Bacterial cells were labelled with 100 µM of BacLight red (Invitrogen) for 15 min at RT, shaking at 200 rpm and analysed by flow cytometry. 4T1 cells were identified and gated based on their Forward/Side scatter and E. coli cells were detected using the 561 laser (Yellow/Green) 660/20 filter for BacLight red and gated using the side scatter. CytoPhase + cells were detected with the 355 (UV) laser and 450/50 filter.
Confirmation of host depletion (HD) strategy. The efficacy of the combined treatment was verified by qPCR in DNA purified from a mixed cell suspension, consisting of 1 × 107 E. coli cells and 1 × 104 4T1 cells. Cells were incubated for 30 min at 37oC, shaking at 360 rpm in TBS or the optimised HD buffer (0.2% Saponin, in TBS + MgCl2 (20 mM Tris-HCL, 20 mM NaCl, 2 mM MgCl2), pH 8) with or without 500 U of Benzonase. The treated cells were then processed for DNA purification following instructions of the QIAamp DNA FFPE Tissue Kit (QIAGEN) and the purified DNA analysed by qPCR.
Quantitative PCR (qPCR). Reactions were prepared using LUNA Universal qPCR master mix (NEB, USA) and 0.25 µM of each primer (Table 1). The thermal profile included a 1 min at 95oC initial denaturation, followed by 40 cycles of denaturation at 95oC x 10 sec, annealing for 15 sec at the temperature specified by NEB’s annealing temperature (Ta) calculator for Hot Start Taq, followed by 20–40 sec of extension at 68 oC. For each assay, a 5-point standard curve was made from log10 dilutions of gene blocks corresponding to species-specific genetic regions (Table 1), using an initial concentration of 107 copies. Primers and gene-blocks were acquired from IDT (Coralville, USA). Efficiency between 95% − 105% and R-square values > 0.995 were deemed as acceptable. All samples were run in triplicate.
Table 1
Primers used for qPCR analysis
Strain/Cell line | Gene/ Accession No | Primer/Probe sequence | F/R | Product size (bp) |
E coli MG1655 [CP032667] | IS5-like element IS5 family transposase AYG17556.1 [CP032667: 230175–231191] | 5’GCC GAA CTG TCG CTT GAT GA | F | 217 |
5’ATT TGT CTC AGC CGA TGC CG | R |
4T1 cells [ATCC® CRL-539™] Mus musculus [10090] | BetaActin AC144818.4 [NC000071.6: 73696 − 73082] | 5’GAT TAC TGC TCT GGC TCC TAG | F | 147 |
5’GAC TCA TCG TAC TCC TGC TTG | R |
Statistical analyses. Flow cytometry data was exported and analysed in FlowJo (BD, UK) and raw data exported to R. All statistical testing and visualisation was performed in the R environment (v3.6.3). Tests of means performed using paired samples T-Test, and visualisation performed using GGplot2 package (v3.2.1) within the R environment.