Exportin 1 activity is critical for <i>O</i>. <i>tsutsugamushi</i> to promote p65 export from the nucleus, but not for the bacterium to inhibit NF-κB-dependent transcriptional activity.

<p>(A and B) <i>O</i>. <i>tsutsugamushi</i> mediates p65 nuclear export in an LMB-sensitive manner. HeLa cells were infected with <i>O</i>. <i>tsutsugamushi</i> at an MOI of 10 for 24 h or 72 h. Next, the cells were treated with LMB or vehicle control for 1 h after which they were fixed, screened with antibodies against <i>O</i>. <i>tsutsugamushi</i> TSA56 (<i>Ot</i>) and p65, and examined by confocal microscopy. (A) Representative fluorescence images of cells viewed for <i>Ot</i>, p65, and merged images plus DAPI for infected cells treated with LMB or vehicle control (Ctrl) are presented. (B) The mean percentage <u>+</u> SD of cells exhibiting p65 in the nucleus was determined for each condition. Triplicate samples of 100 cells each were counted per time point. n.s., not significant. Results are representative of three separate experiments. (C and D) <i>O</i>. <i>tsutsugamushi</i> inhibits NF-κB-dependent transcriptional activation in an LMB-insensitive manner. Stably transfected HeLa cells bearing four copies of the NF-κB response element upstream of the Luc gene were infected with <i>O</i>. <i>tsutsugamushi</i> for 24 h at an MOI of 10 after which they were incubated with LMB or vehicle control in the presence or absence of TNFα for 8 h. (C) Representative fluorescence images of LMB-treated infected cells viewed for TSA56 (<i>Ot</i>), p65, and merged images plus DAPI. (D) The cells were lysed followed by mixing with Luc substrate. Data are presented as the mean percentage <u>+</u> SD of Luc activity normalized to the Luc activity of uninfected vehicle control treated cells that were exposed to TNFα from triplicate samples. Statistically significant (***<i>P</i> < 0.001) values are indicated. n.s., not significant. Results are representative of three independent experiments.