<i>Orientia tsutsugamushi</i> uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation

<div><p><i>Orientia tsutsugamushi</i> causes scrub typhus, a potentially fatal infection that threatens over one billion people. Nuclear translocation of the transcription factor, NF-κB, is the central initiating cellular event in the antimicrobial response. Here, we report that NF-κB p65 nuclear accumulation and NF-κB-dependent transcription are inhibited in <i>O</i>. <i>tsutsugamushi</i> infected HeLa cells and/or primary macrophages, even in the presence of TNFα. The bacterium modulates p65 subcellular localization by neither degrading it nor inhibiting IκBα degradation. Rather, it exploits host exportin 1 to mediate p65 nuclear export, as this phenomenon is leptomycin B-sensitive. <i>O</i>. <i>tsutsugamushi</i> antagonizes NF-κB-activated transcription even when exportin 1 is inhibited and NF-κB consequently remains in the nucleus. Two ankyrin repeat-containing effectors (Anks), Ank1 and Ank6, each of which possess a C-terminal F-box and exhibit 58.5% amino acid identity, are linked to the pathogen’s ability to modulate NF-κB. When ectopically expressed, both translocate to the nucleus, abrogate NF-κB-activated transcription in an exportin 1-independent manner, and pronouncedly reduce TNFα-induced p65 nuclear levels by exportin 1-dependent means. Flag-tagged Ank 1 and Ank6 co-immunoprecipitate p65 and exportin 1. Both also bind importin β1, a host protein that is essential for the classical nuclear import pathway. Importazole, which blocks importin β1 activity, abrogates Ank1 and Ank6 nuclear translocation. The Ank1 and Ank6 regions that bind importin β1 also mediate their transport into the nucleus. Yet, these regions are distinct from those that bind p65/exportin 1. The Ank1 and Ank6 F-box and the region that lies between it and the ankyrin repeat domain are essential for blocking p65 nuclear accumulation. These data reveal a novel mechanism by which <i>O</i>. <i>tsutsugamushi</i> modulates the activity and nuclear transport of NF-κB p65 and identify the first microbial proteins that co-opt both importin β1 and exportin 1 to antagonize a critical arm of the antimicrobial response.</p></div

Ank1 and Ank6 inhibit NF-κB-dependent transcriptional activation in an exportin 1-independent manner.

<p>Stably transfected HeLa cells bearing four copies of the NF-κB response element upstream of the Luc gene were transiently transfected to express the indicated Flag-tagged proteins for 16 h. The cells were treated with LMB or vehicle control in the presence or absence of TNFα for 4 h followed by lysis and mixing with Luc substrate. Data are presented as the mean percentage <u>+</u> SD of Luc activity normalized to the Luc activity of control cells expressing Flag-BAP and exposed to TNFα from triplicate samples. Statistically significant (**<i>P</i> < 0.01; ***<i>P</i> < 0.001; ****<i>P</i><0.0001) values are indicated. n.s., not significant. Results are representative of three independent experiments.</p

Schematics of Ank1 and Ank6 mutant proteins.

<p>Schematics of full-length wild-type Ank1 and Ank6 are presented atop the left and right column, respectively, below each of which are schematics in which deleted (△) amino acids and corresponding domains are indicated. The amino termini consisting of residues 1 through 17 are represented by red boxes. Each annotated ankyrin repeat (AR) is denoted by a blue-filled arrow bordered by a solid line. The arrow filled with white dots over a blue background and bordered by a hatched line corresponds to the potential AR that is not annotated as an AR but exhibits 41.7% identity with Ank6 AR4. The yellow hexagon in Ank6 corresponds to a putative nuclear export sequence (NES) that is found in the C-terminal region of AR4. The corresponding amino acids of Ank1 (143–160) that align with the Ank6 putative NES but are not predicted to be an NES are denoted by a white hexagon. An orange box demarcates the intervening sequence region (ISR) corresponding to Ank1 residues 167 to 201 and Ank6 168 to 202. The C-terminal F-box of each Ank is indicated by a green box.</p

<i>O</i>. <i>tsutsugamushi</i> inhibits p65 nuclear accumulation.

<p>HeLa cells (A and B) or BMDMs (C and D) were infected with <i>O</i>. <i>tsutsugamushi</i> at an MOI of 1. At 2, 4, 8, 12, or 24 h, the cells were fixed and screened with antibodies against <i>O</i>. <i>tsutsugamushi</i> TSA56 (<i>Ot</i>) and p65 prior to examination by confocal microscopy. (A and C) Representative fluorescence images of cells viewed for <i>Ot</i>, p65, and merged images plus DAPI, which stains the nucleus, are presented. White arrows denote representative individual <i>O</i>. <i>tsutsugamushi</i> bacteria. (B and D) The mean percentage <u>+</u> SD of cells exhibiting p65 in the nucleus was determined at each time point. Triplicate samples of 100 cells each were counted per time point. Results are representative of three separate experiments.</p

Ank1 and Ank6 promote p65 removal from the nucleus in an exportin 1-dependent manner.

<p>HeLa cells were transfected to express Flag-tagged BAP, Ank1, Ank6, or IκBα SR. At 16 h, the cells were treated with LMB or vehicle control for 1 h. The media was replaced with media containing TNFα or vehicle for 30 min. The cells were then fixed, screened with antibodies specific for the Flag epitope and p65, and examined by confocal microscopy. Representative fluorescence images are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007023#ppat.1007023.s007" target="_blank">S7 Fig</a>. The mean percentage <u>+</u> SD of cells exhibiting p65 in the nucleus was determined. Quadruplicate samples of 100 cells each were counted per time point. Statistically significant (*<i>P</i> < 0.05; ***<i>P</i> < 0.001) values are indicated. n.s., not significant. Results are representative of three independent experiments.</p

<i>O</i>. <i>tsutsugamushi</i> inhibits NF-κB-dependent gene transcription.

<p>Stably transfected HeLa cells bearing four copies of the NF-κB response element upstream of the Firefly Luciferase (Luc) gene seeded in a 96-well plate were incubated with <i>O</i>. <i>tsutsugamushi</i> at an MOI of 10, 50, or 100 or mock control for 24 h. The cells were treated with TNFα or vehicle control for 8 h to promote NF-κB-dependent gene transcription followed by the addition of lysis buffer and Luc substrate. The plate was read in a luminometer for 10 s per well to quantify Luc activity. Data are presented as the mean percentage <u>+</u> SD of Luc activity normalized to the Luc activity of mock control cells exposed to TNFα from triplicate samples. Statistically significant (<i>***P</i> < 0.001; <i>****P</i> < 0.0001) values are indicated. Data are representative of three independent experiments.</p

<i>O</i>. <i>tsutsugamushi</i> inhibits TNFα-stimulated p65 nuclear accumulation.

<p>HeLa cells (A and B) or BMDMs (C and D) were infected with <i>O</i>. <i>tsutsugamushi</i> at an MOI of 50 or 10, respectively, or mock infected. At 24, 48, or 72 h, the cells were treated with TNFα or vehicle control (Ctrl) for 30 min after which they were fixed, screened with antibodies against <i>O</i>. <i>tsutsugamushi</i> TSA56 (<i>Ot</i>) and p65, and visualized by confocal microscopy. (A and C) Representative fluorescence images of cells viewed for <i>Ot</i>, p65, and merged images plus DAPI, which stains the nucleus, are presented. (B and D) The mean percentage <u>+</u> SD of cells exhibiting p65 in the nucleus were determined at each time point. Triplicate samples of 100 cells each were counted per time point. Statistically significant (*<i>P</i> < 0.05; **<i>P</i><0.01; ***<i>P <</i> 0.001; ****<i>P</i> < 0.0001) values are indicated. n.s., not significant. Data are the mean <u>+</u> SD of three independent experiments performed in triplicate.</p

p65 levels are unchanged in <i>O</i>. <i>tsutsugamushi</i> infected cells.

<p>RAW264.7 (A and B) or HeLa cells (C and D) were infected with <i>O</i>. <i>tsutsugamushi</i> at an MOI of 25 or 10, respectively, or mock infected. (A) Whole cell lysates were analyzed by Western blotting with antibodies to <i>O</i>. <i>tsutsugamushi</i> TSA56 and p65. The blots were stripped and reprobed with antibody against GAPDH or β-actin as a loading control. (B and D) Mean normalized ratios <u>+</u> SD of p65:GAPDH (B) and p65:β-actin (D) from three separate experiments were calculated using densitometry. Statistically significant (<i>*P</i> < 0.05) values are indicated. n.s., not significant. The arrowhead and asterisk in A and B denote the expected size for TSA56 and a non-specific host cell-derived band, respectively.</p

Ank1 and Ank6 each interact with p65.

<p>HeLa cells ectopically expressing Flag-tagged BAP, Ank1, or Ank6 and were treated with TNFα or vehicle for 30 min. Whole cell lysates were incubated with Flag antibody-conjugated agarose beads to immunoprecipitate (IP) Flag-tagged proteins and their interacting proteins. Resulting Western blots were probed with p65 antibody to determine if any Flag-tagged protein co-immunoprecipitated endogenous p65. As a control to ensure that the conditions allowed for p65 and IκBα to interact, a parallel experiment was performed in which lysates of TNFα or vehicle control stimulated Flag-p65 expressing HeLa cells were incubated with the Flag antibody coated beads followed by Western blot analysis of the resulting eluate with antibody against endogenous IκBα. Expression of each Flag-tagged protein of interest was confirmed by subjecting input lysate to Western blotting using Flag antibody. Data presented are representative of three independent experiments.</p

Identification of the Ank1 and Ank6 domains that are essential for optimal translocation into the nucleus.

<p>HeLa cells were transfected to express Flag-tagged versions of Ank1, Ank6, or the indicated deletion mutant thereof. At 16 h, the cells were fixed, screened with Flag tag antibody, stained with DAPI, and examined by confocal microscopy. (A and B) Representative fluorescence images of cells viewed for Flag-tagged Ank1 (A) or Ank6 proteins (B) with and without DAPI. (C) The mean percentage <u>+</u> SD of transfected cells exhibiting Flag signal accumulation around the periphery of nuclei was determined. Triplicate samples of 100 cells each were counted per condition. Data presented are indicative of three experiments with similar results. Statistically significant (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001) values are indicated. n.s., not significant. Indicators of statistical significance relative to values obtained for Flag-Ank1, Flag-Ank1△ISR, Flag-Ank6, and Flag-Ank6△ISR are colored red, black, blue, and black, respectively.</p