FIG 6.

Small-molecule inhibition of host cell SUMOylation decreases E. chaffeensis survival. (A) THP-1 cells were treated with DMSO (vehicle) or AA, a SUMO E1 ligase inhibitor, for 4 h and then infected with E. chaffeensis. Bacterial loads were determined by qPCR analysis of the dsb gene normalized to the GAPDH gene and compared to those in vehicle-treated infected cells (24 h, 48 h, or 72 h p.i.) (Student's t test; P < 0.05; n = 3). (B) Immunoblot analysis was performed with pan-SUMO antibody (or histone 3 antibody for a loading control) and THP-1 whole-cell lysate, with or without 25 μM AA, at 24-h time intervals up to 72 h posttreatment (n = 4). Pixel intensities (determined using ImageJ software) were normalized to the loading control (histone 3) and graphed as a percentage of the vehicle-treated level. Wilcoxon signed-rank statistical analysis was performed to determine differences from vehicle-treated cells (100%). *, P < 0.05; **, P < 0.005. (C) Bright-field images (magnification, ×40) of Diff-Quik-stained samples collected at 72 h p.i. demonstrate a decreased number of ehrlichial inclusions per cell following treatment with increasing concentrations of AA.