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. Overall design: P. gingivalis cultured in the miedum; intracellular P. gingivlais; HUVEC cultured in the medium; HUVEC infected with P. gingivalis