Figure 2.

Growth of E. chaffeensis is reversibly inhibited by the class III PtdIns3K inhibitor 3-MA. (A) 3-MA does not inhibit internalization of E. chaffeensis. The percentage of intracellular bacteria vs. total cell-associated bacteria was determined in THP-1 cells incubated with 3-MA or RPMI 1640 medium control (RPMI) at 2 h p.i. by scoring 100 E. chaffeensis bacteria in each group after 2 rounds of immunofluorescence labeling. Data are presented as the mean ± standard deviation of triplicate samples (not significantly different by the Student t test, P > 0.05). (B and C) 3-MA added at 1 h or 1 d p.i. inhibits E. chaffeensis infection. 3-MA was added to THP-1 cells at a final concentration of 2 mM, and infection was assessed at 3 d p.i. by Diff-Quik staining to determine the percentage of infected cells (B) and the number of bacteria per cell (C). Data are presented as the mean ± standard deviation of triplicate assays. *, Significantly different by the Tukey HSD test (P < 0.05). (D) 3-MA reversibly inhibits E. chaffeensis replication in THP-1 cells. i to iii, E. chaffeensis in THP-1 cells without 3-MA treatment at 23, 29, and 81 h p.i., respectively. iv to vi, E. chaffeensis in THP-1 cells treated with 10 mM 3-MA at 23 h p.i. for 6 h (iv) and incubated an additional 52 h with (vi) or without (v) 3-MA. Arrows indicate E. chaffeensis as shown by Diff-Quik staining. (E) Cells in panel (D) (iv) were immunostained with anti-P28. White arrows indicate large vacuoles containing condensed bacteria. Merged/DIC, fluorescence image merged with differential interference contrast (DIC) image. Deconvolution microscopy. Scale bar: 10 μm.