CRISPR-cas cas7 influences the host-pathogen interaction of Porphyromonas gingivalis

We investigated gene expression using a dual transcriptomic approach to understand if P. gingivalis ?cas7 responds differently than WT to the intracellular environment of THP-1 cells and if these changes impact host response. Total RNA was extracted from THP-1 cells infected with P.gingivalis WT or ?cas7 for dual-transcriptomics gene expression analyses. Overall design: Human monocyte/macrophage cell line THP-1 (ATCC, Virginia, USA) were cultured at 37°C in a 5% CO2 incubator in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, l-glutamine (2 mM), penicillin/streptomycin (100 U/100 µg/ml), HEPES (10 mM), sodium pyruvate (1 mM), glucose (4.5 mg/ml), sodium bicarbonate (1.5 mg/ml), and 2-mercaptoethanol (0.05 mM). For P. gingivalis infection assays, THP-1 cells were adjusted to 5 × 105 cells/ml and were treated with 100 ng/ml phorbol 12-myristate-13-acetate (PMA; Sigma-Aldrich, Missouri, USA) to induce differentiation into a macrophage-like state. These cells were added to each well of 24-well cell culture plates and allowed to differentiate into a macrophage-like state for 48 h. At this point, the cell culture medium was replaced with an antibiotic-free culture medium. P. gingivalis WT and ?cas7 mutant harvested from overnight TSBHK broth culture were washed with RPMI 1640 media, adjusted to an OD600 of 1.0 (approximately 1 × 109 CFU/ml), and added to PMA-activated THP-1 cells at a multiplicity of infection (MOI) of 100, in complete antibiotic-free RPMI-1640 medium. After 2 h of co-culture, the wells were washed with PBS to remove non-attached bacteria and incubated with complete RPMI-1640 media supplemented with gentamicin (300 µg/ml)/metronidazole (200 µg/ml) and incubated for 2h or 6h. Total RNA was extracted from THP-1 cells co-cultured with either P. gingivalis WT, ?cas7 for 2h or 6h.