Fig. 3

Time course of ‘Ca. B. cookevillensis’ infection in PMA-differentiated THP-1 macrophage-like cells. THP-1 cells were infected with ‘Ca. B. cookevillensis’ (CC99) for 1 h at an MOI of 10 and incubated for the designated amount of time. Following incubation, immunofluorescent staining of bacteria was conducted with rabbit anti-serum to ‘Ca. B. cookevillensis’ and a TRITC-conjugated secondary antibody prior to DAPI staining. At 6 h p.i., puncta of individual or small clusters of bacteria are visible in the cytoplasm. By 12–24 h, the bacterial inclusions appear perinuclear or co-localized with the nucleus. At 36–48 h, the bacteria-containing vacuoles are filled with bacteria, and appear to be enclosed within the nuclear envelope. Imaging was performed with laser scanning confocal microscopy. Bars, 5 μm