Next Generation Sequencing Facilitates Quantitative Analysis of P. gingivalis and HUVEC Transcriptomes

Purpose: This study provides the transcriptional landscape of intracellular P. gingivalis as well as that of endothelial cells infected by this bacteria based on dual RNA sequencing. Methods: HUVECs were seeded in 6-well plates and cultured to 80% confluence. After washing with phosphate-buffered saline (PBS), cell were infected with P. gingivalis at a multiplicity of infection (MOI) of 100 for 2 h at 37 °C in 5% CO2, unless otherwise stated. Non-adherent bacteria were removed by washing with PBS, and the cell-adherent bacteria were killed using gentamicin (300 μg/mL) and metronidazole (200 μg/mL) for 1 h at 37 °C in 5% CO2.Total RNA was isolated from the samples using TRIzol® reagent.cDNA libraries for Illumina® sequencing were generated using the KC-DigitalTM Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). The library products corresponding to 200–250 bps were then enriched, quantified, and sequenced using a Nova-seq 6000 sequencer (Illumina, San Diego, CA, USA). Results: Using an optimized data analysis workflow, intracellular P. gingivalis showed a total of 573 DEGs compared with the control bacteria, and of these DEGs, 304 and 269 were upregulated and downregulated, respectively. A total of 838 DEGs were detected in P. gingivalis-infected HUVECs compared with their uninfected counterparts, and of these, 297 and 541 genes were upregulated and downregulated, respectively. Conclusions: This study firstly provides the transcriptional landscape of intracellular P. gingivalis as well as that of endothelial cells infected by this bacteria based on dual RNA sequencing. P. gingivalis cultured in the miedum; intracellular P. gingivlais; HUVEC cultured in the medium; HUVEC infected with P. gingivalis