Investigation of colicin activity against intramacrophagic LF82 was measured by the gentamicin protection assay (Falkow et al. 1987). RAW 264.7 macrophages (2 x 105 /well) were seeded on to 24-well plates (Corning) and were grown for 18 h to confluency. Prior to infection of cells, bacteria were washed and re-suspended in antibiotic-free RPMI-1640 media and added to cells at a multiplicity of infection (MOI) of 50. After 2 h infected macrophages were washed with PBS and exposed to RPMI media containing gentamicin (100 µg ml-1) to kill extracellular bacteria that had failed to invade macrophages. After 1 h, the media was removed and
macrophages were then treated with culture media containing antimicrobial treatments in the presence of gentamicin (20 µg ml-1) at 37°C, 5% CO
2. For LF82 growth curves, media was free from antimicrobial treatments and was replaced every 24 h. Antimicrobial treatments used were colicin E9, colicin E1 and the antibiotic ciprofloxacin (100 µg ml-1). After incubation with antibiotic for desired time point, RAW264.7 cells were washed three times with sterile PBS. If colicin was added, macrophages were also treated with trypsin (0.25%) in EDTA for 5 min at 37°C. This was to ensure all macrophage bound colicin protein was
inactivated. The macrophages were then scraped from the surface of the plate and lysed with 2% Triton X-100 for 5 min. Recovered intracellular bacteria were
quantified by plating serial dilutions on LB agar containing ampicillin (50 µg ml-1). The plates were incubated overnight at 37°C and CFU counts were performed. Bacterial survival in RAW 264.7 cells (% survival) were calculated as a % of viable internalised bacteria relative to the number recovered from untreated cells infected with LF82, considered as 100%.
2.9.2 Colicin killing of cell-associated E. coli
To assess efficacy of colicin against LF82 associated with T84 monolayers, adhesion assays were performed. T84 cells were grown in 24 well plates for 3-5
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days at 37°C in 5% CO2 until an epithelial monolayer was formed. Prior to
adhesion assays T84 cell monolayers were washed with sterile PBS. LF82 were grown to OD600 = 0.6 in LB broth and resuspended in DMEM culture and added to cells at a multiplicity of infection (MOI) of 10 for 3 h at 37°C, 5% CO2. To assess antimicrobial killing of adherent bacteria, colicin E9 and the antibiotic ciprofloxacin were diluted in DMEM culture media to concentrations of 100 µg ml-1 and added to separate wells at 15, 30, 45 and 60 min post infection. After 3 h, monolayers were washed three times with sterile PBS to remove non-adherent bacteria and treated with trypsin (0.25%) in EDTA for 10 min at 37°C. The cell suspension was
removed by aspiration and homogenised by passing through a 22-gauge needle. Cells were lysed by sonication and cell-associated LF82 were serially diluted and plated on LB agar plates containing ampicillin (50 µg ml-1). Plates were incubated overnight at 37°C and CFU counts were performed. All adhesion assays were repeated in triplicate.
To assess inhibition of LF82 adhesion by colicin producing commensal E. coli, co- culture adhesion assays were performed using LF82 and E. coli BZB2104
(pColE1-K53). For co-culture assays, bacteria were added in equal volumes at a MOI of 10 for 3 h and number of cell-associated LF82 were determined as stated before.
2.9.3 Photomicroscopical analysis of T84 cell morphology
For photomicroscopy T84 cell monolayers were grown on glass coverslips in 6 well plates (Corning) for 5-7 days. Cells were infected with LF82 (MOI of 10) and when required were treated with colicin E9 (100 µg ml-1) 15 min post infection. After 3 h monolayers were washed three times with sterile PBS and fixed with 10% methanol in PBS for 10 min. Cells were washed again in sterile PBS as above and then Giemsa stained (20% (v/v) Giemsa in dH2O) for 20 min at room temperature. Cover slips were washed and mounted on slides using clear nail varnish solution. Images were captured using Zeiss Axioskop attached to QImaging Micropublisher 3.3 RTB camera at 40 x magnification.2.9.4 Confocal microscopy
In order to visualise colicin and bacteria associated with cells, RAW 264.7 macrophages were seeded (2 x 105/well) on glass cover slips in 6 well plates
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(Corning) and grown to confluency for 18 h at 37°C. Culture media used for infection contained 50 µg ml-1 kanamycin to select for growth of the GFP-
expressing LF82 strain. Where required, macrophages were infected with LF82- GFP (MOI of 50) for 2 h and exposed to culture medium (RPMI-1640, FBS, L- glutamine and kanamycin) containing gentamicin (100 µg ml-1) for 1 h to ensure killing of extracellular bacteria. Cells were treated with culture media containing colicin E9-RFP (100 µg ml-1) and gentamicin (20 µg ml-1) for the required time. Infected cells were washed three times in sterile PBS and treated with 0.25% trypsin in EDTA for 5 min at 37°C to inactive extracellular colicin E9-RFP. RAW 264.7 macrophages were washed as above to remove macrophage bound protein and fixed with 2% paraformaldehyde (PFA) in PBS and 0.5 M sucrose for 10 min at room temperature. Fixed cells were permeabilised with 0.5% Triton X-100 for 3- 5 min at room temperature. Actin cytoskeleton was stained using Alexa-647 labelled phalloidin (Life Technologies) and nuclear DNA was stained with 4',6- diamidino-2-phenylindole (DAPI) (Life Technologies). Cover slips were air dried and mounted on slides with Dako Mounting Medium (Dako UK). Slides were examined using a Zeiss LSM410 Laserscan Microscope equipped with periphera argon-UV laser. Image processing was performed using the LSM Image Browser software (Zeiss).
2.9.5 Drug treatment of macrophages
The following drugs were purchased from Sigma Aldrich and used as described previously (Marina-García et al. 2009). Cytochalasin B, that blocks phagocytosis, was used at 10 µg ml-1; polyinosinic acid, that blocks scavenger receptor mediated endocytosis was used at 50 µg ml-1; dynasore that blocks clathrin mediated
endocytosis was used at 80 µM; dimethylamyloride (DMA) that blocks pinocytosis was used at 500 µM and mannan that blocks mannose receptors was used at 1 mg ml-1. RAW 264.7 cells were treated with specific drug for 30 mins and then treated with colicin E9-RFP for 4 h and visualised by confocal microscopy as stated.
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2.9.6 Enzyme linked immunosorbent assay (ELISA) for detection
of tumour necrosis factor-α
RAW 264.7 macrophages were infected and treated as described. At various time points, post-infection supernatants were collected and assayed for TNF-α by cytokine ELISA kits (BioLegend). Optical density was determined at wavelength 450nm and cytokine concentration was determined as by manufacturer’s
instructions.
2.9.7 Lactate dehydrogenase hydrogenase cytotoxicity assay
For RAW 264.7 macrophages cytotoxicity was determined by LDH release.Lactate dehydrogenase (LDH) activity as a measure of cytoxicity was measured in cell supernatants according the manufacturer’s protocol (LDH Cytotoxicity Assay Kit, Abcam). LDH release was measured colormetrically according to
manufacturer’s protocol. % Cytotoxicity = (Compound treated LDH activity – Spontaneous LDH activity) / (Maximum LDH activity – Spontaneous LDH activity) x 100.