NF-κB DNA binding activity is reduced in IKKα and in IKKβ deficient cells.

<p>DNA binding activity of NF-κB was measured by gel shift assay. Indicated MEF cells were treated with TNFα for indicated times. Nuclear proteins were subject to gel shift assay for DNA binding analysis.</p

The role of IKKα and IKKβ in p65 phosphorylation and IκBα degradation in response to TNFα.

<p>MEF cells that are deficient for IKKα, IKKβ, or both IKKα and IKKβ (DKO) were treated with TNFα for the indicated times. NF-κB activity, as measured by IκBα degradation and p65 phosphorylation, is diminished in IKKα and IKKβ deficient MEF cells. IKKα and IKKβ DKO cells show no detectable p65 phosphorylation. Tubulin levels are shown as a loading control.</p

IKKα and IKKβ knock-down in Hela cells leads to diminished IκB degradation and p65 phosphorylation.

<p>HeLa cells were grown in 6-well plates and transfected with the indicated siRNA for 3 days. Western blots were performed on total cell extracts after treatment with TNF for the indicated times. Tubulin levels are shown as a loading control.</p

IKKβ kinase mutant inhibits TNF and IKKα-induced NF-κB-dependent reporter gene activity.

<p>WT and IKKβ null MEFS were transfected with the indicated vector construct and with the NF-κB-dependent luciferase reporter. Luciferase luciferase activity was measured 48 hr after transfected. Where indicated, cells were treated with TNF for 4 hrs. Relative luciferase values were calculated using a renilla control expression vector for normalization. Relative luciferase values are normalized to vector control samples.</p

IKKα and IKKβ each contribute to TNF-induced NF-κB activity in HeLa cells.

<p>HeLa cells, seeded in 24-well plates were transiently transfected with indicated siRNA constructs for 48 hr. Then media was replaced and cells were further transfected with NF-κB responsive 3x-κB luciferase and a control Renilla luciferase contructs. TNF was added (as indicated) and 24 hr later cells were lysed and dual lucifearse assay was performed. Luciferase readings in untreated and control vector transfected cells were normalized as 1.</p

Effect of ethanolamine and EutR on SPI-1.

<p>(<b>A</b>) qRT-PCR of <i>sipC</i> from WT <i>S</i>. Typhimurium (SL1344) grown in LB or LB supplemented with 5 mM ethanolamine (EA). (<b>B</b>) qRT-PCR of <i>sipC</i> from WT <i>S</i>. Typhimurium (SL1344) grown in DMEM or DMEM supplemented with ethanolamine (EA) as indicated. For (<b>A</b>) and (<b>B</b>), n = 3; error bars represent the geometric mean ± SD. Statistical significance is shown relative to cells grown without EA supplementation; <i>strB</i> was used as the endogenous control. (<b>C</b>) Invasion of HeLa cells by WT (SL1344) and the Δ<i>eutR</i> (CJA009) strains. Mean ± SE of nine independent experiments. (<b>D</b>) Invasion of HeLa cells by WT (SL1344) and the Δ<i>eutR</i> (CJA009) strains. Mean ± SE of six independent experiments with supplementation of 5 mM EA. **, <i>P</i> ≤ 0.005; <i>P</i> > 0.05 = ns.</p

EutR-associated signaling <i>in vivo</i>.

<p>(<b>A</b>) qRT-PCR analysis of <i>ssrB</i> expression in WT <i>S</i>. Typhimurium (SL1344) or the Δ<i>eutR</i> strain (CJA009) harvested from infected spleens. (<b>B</b>) qRT-PCR analysis of <i>eutR</i> or <i>eutS</i> expression in WT <i>S</i>. Typhimurium (SL1344) harvested from infected spleens compared to <i>S</i>. Typhimurium (SL1344) grown in tissue culture medium (DMEM). For (<b>A</b>) and (<b>B</b>), n = 2–3; error bars represent the geometric mean ± SD; <i>strB</i> was used as the endogenous control. *, <i>P</i> ≤ 0.05. nd = not detected.</p

EutR in <i>S</i>. Typhimurium niche adaptation.

<p>(<b>A</b>) EutR senses ethanolamine to activate transcription. (<b>B</b>) In the intestine, EutR promotes expression of the <i>eut</i> operon that encodes ethanolamine metabolism, thereby enhancing <i>S</i>. Typhimurium growth. (<b>C</b>) EutR expression in macrophages activates expression of genes in SPI-2, which are required for intramacrophage survival and dissemination.</p

Ethanolamine Signaling Promotes <i>Salmonella</i> Niche Recognition and Adaptation during Infection

<div><p>Chemical and nutrient signaling are fundamental for all cellular processes, including interactions between the mammalian host and the microbiota, which have a significant impact on health and disease. Ethanolamine is an essential component of cell membranes and has profound signaling activity within mammalian cells by modulating inflammatory responses and intestinal physiology. Here, we describe a virulence-regulating pathway in which the foodborne pathogen <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. Typhimurium) exploits ethanolamine signaling to recognize and adapt to distinct niches within the host. The bacterial transcription factor EutR promotes ethanolamine metabolism in the intestine, which enables <i>S</i>. Typhimurium to establish infection. Subsequently, EutR directly activates expression of the <i>Salmonella</i> pathogenicity island 2 in the intramacrophage environment, and thus augments intramacrophage survival. Moreover, EutR is critical for robust dissemination during mammalian infection. Our findings reveal that <i>S</i>. Typhimurium co-opts ethanolamine as a signal to coordinate metabolism and then virulence. Because the ability to sense ethanolamine is a conserved trait among pathogenic and commensal bacteria, our work indicates that ethanolamine signaling may be a key step in the localized adaptation of bacteria within their mammalian hosts.</p></div

EutR in pathogen-microbiota-host interactions.

<p>(<b>A</b>) Schematic of the <i>eut</i> operon. (<b>B</b>) <i>In vitro</i> growth curve of S. Typhimurium WT (SL1344), Δ<i>eutR</i> (CJA009), or Δ<i>eutB</i> (CJA020) strains in LB without or with supplementation of 5 mM ethanolamine (EA). Each data point shows the average of three independent experiments. (<b>C</b>) qRT-PCR of <i>eutR</i> in WT or the Δ<i>eutB</i> (CJA020) <i>S</i>. Typhimurium strains grown in Dulbecco’s Modified Eagle Medium (DMEM) or DMEM supplemented with 5 mM EA. n = 3; error bars represent the geometric mean ± standard deviation (SD); <i>strB</i> was used as the endogenous control. (<b>D-F</b>) Competition assays between (<b>D</b>) Δ<i>eutB</i>::Cm^R (CJA018) and WT strains; (<b>E</b>) Δ<i>eutR</i>::Cm^R (CJA007) and WT strains; or (<b>F</b>) Δ<i>eutR</i>::Cm^R (CJA007) and Δ<i>eutB</i> (CJA020) strains. Mice were orogastrically inoculated with 1:1 mixtures of indicated strains. Colony forming units (cfu) were determined at indicated time points. Each bar represents a competition index (CI). Horizontal lines represent the geometric mean value ± standard error (SE) for each group (n = 2 litters (6–8 animals)). *, <i>P</i> ≤0.05; **, <i>P</i> ≤ 0.005; ***, <i>P</i> ≤0.0005; <i>P</i> > 0.05 = ns.</p