Bacterial aggregation facilitates internalin-mediated invasion strategy of Listeria monocytogenes
Bacterial aggregation facilitates internalin-mediated invasion strategy of Listeria monocytogenes
Bacterial cells for 16 samples across 4 different conditions (media vs. genotype) were harvested by centrifugation at 13000 x g for 3 mins. This experiment has two growth conditions (fresh and spent media) and two genotypes (wildtype L. monocytogenes and a actA-dC mutant strain). L. monocytogenes actA-dC mutant strain was constructed using the temperature sensitive shuttle plasmid pAUL-A as previously described (Wang, King et al. 2015), by deletion of the extracellular C terminal domain (amino acids-393-585) of ActA (Travier et al, 2013). L. monocytogenes were grown at 37°C shaking 200 rpm in Tryptone Soya Broth (TSB). Spent media was made by 1 seeding HeLA cells into an imaging dish in DMEM supplemented with FCS and L-glutamine at a cell density of 2.0 x 105 cells/ml and incubated overnight at 37°C 5% CO2 (v/v). The next day the DMEM was aspirated off the cells, they were washed with PBS and the media was replaced with serum-free DMEM (no FCS). The cells were incubated overnight at 37°C 5% (v/v) CO2. The next day this spent media was removed from the host cells and kept at 37°C until ready for use. 1 ml of spent media was added to a 75 cm2 imaging dish and the bacterial inoculum was added to a final bacterial cell density of 1.0 x 107 CFU/ml. Fresh media was the same serum free media that hadn't been incubated overnight on mammalian cells. The dish was sealed with parafilm and incubated at 37°C 5% CO2 (v/v). RNA extraction was carried out using Invitrogen's Purelink RNA Extraction Kit and Lysing Matrix E tubes (MP Biomedicals). Prior to starting the extraction, fresh lysozyme solution (consisting of 10 mg/ml lysozyme, 10 mM Tris-HCl pH 8.0 and 0.1 mM EDTA,) and a solution of 10% SDS (w/v) were prepared in RNAse-free water and the kit lysis buffer was supplemented with 10 μl of 2-mercaptoethanol per 1 ml of lysis buffer. Bacterial cells were harvested by centrifugation at 13000 x g for 3 mins. The supernatant was discarded and 100 μl of lysozyme solution was added to the pellet for cell resuspension. Next, 0.5 μl of SDS solution (10% v/v) and 350 μl of lysis buffer was added and vortexed sequentially. The lysate, added to a Lysing Matrix E tube, was homogenised in a rotor stator homogeniser (FastPrep FP120, 6.5 speed) for 45 secs. The tube was centrifuged for 5 mins at 2600 x g followed by transfer of the supernatant to a microcentrifuge tube (RNAse-free) and addition of 250 μl of absolute ethanol (100% v/v) to the tube. The solution mixture was vortexed to remove precipitate. The sample was then transferred to a spin cartridge and centrifuged at 12000 x g for 15 secs allowing RNA to bind to the spin column. The subsequent flow-through was discarded and 700 μl of Wash Buffer 1 was added to the column. This was then centrifuged again for 15 secs at 12000 x g, followed by discarding both the flow through and the collection tube. The column was then placed in a new collection tube. Wash Buffer 2 (500 μl) was added to the column and centrifuged at 12000 x g for 15 secs, again discarding the subsequent flow-through. The wash step was repeated with Wash Buffer 2 and flow-through was discarded again. The column was centrifuged at 12000 x g to remove any residual wash buffer. The collection tube was discarded and the column was transferred to a RNAse-free recovery tube; 50 μl of RNAse-free water was carefully added to the column membrane. RNA was eluted in this 1.5 ml tube with 1 min incubation at room followed by centrifugation at 12000 x g for 2 mins. All further experiments involving RNA were performed on ice. The Nanodrop spectrophotometer (ND-1000) was used for RNA quantification using 1 μl of the sample. Additionally, the Super RNAse Inhibitor (Invitrogen) was added to the samples at 1U/μl to inhibit RNAse activity. Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus. In addition, Illumina ran Listeria monocytogenes fasta files through their in house in-silico analysis program which predicted we would need some additional probes to efficiently deplete the rRNA from this species. These were purchased from from Integrated DNA Technologies, Inc.(https://eu.idtdna.com/) and added during the probe hybridization step.