EutR promotes recognition and adaptation to the intracellular environment.

<p>(<b>A-C</b>) Competition assays between Δ<i>eutR</i>::Cm^R (CJA007) and Δ<i>eutB</i> (CJA020) strains collected from (<b>A</b>) harvested spleens, (<b>B</b>) the peritoneal cavity, or (<b>C</b>) phagocytized cells at 6 h pi. Mice were intraperitoneally infected with 1:1 mixtures of the Δ<i>eutR</i> and Δ<i>eutB</i> strains. Each column represents a CI. Each column shows the geometric mean value ± 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

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

EutR enhances S. Typhimurium survival within macrophages.

<p>(<b>A</b>) Intramacrophage survival and replication of <i>S</i>. Typhimurium (AJK61) and the Δ<i>eutR</i> (CJA023) strains after 5 h phagocytosis in RAW macrophages (error bars represent the geometric mean value ± SE of 24 independent experiments). (<b>B</b>) Intramacrophage survival and replication of <i>S</i>. Typhimurium (CJA034), Δ<i>eutR</i> (CJA032), and Δ<i>eutR</i> complemented with <i>eutR</i> (<i>eutR</i>+) (CJA033) strains after 5 h phagocytosis in peritoneal exudate macrophages (PEMs) (error bars represent the geometric mean value ± SE of nine independent experiments). (<b>C</b>) Intramacrophage survival and replication of <i>S</i>. Typhimurium (AJK61), Δ<i>eutR</i> (CJA023) and Δ<i>eutB</i> (CJA028) strains after 5 h phagocytosis in PEMs (error bars represent the geometric mean value ± SE of six independent experiments). (<b>D</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 SPI-2 inducing medium without or with supplementation of 5 mM ethanolamine (EA). Each data point shows the average of three independent experiments. (<b>E</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 tissue culture medium without or with supplementation of 5 mM ethanolamine (EA). Each data point shows the average of three independent experiments. *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.005; ***, <i>P</i> ≤ 0.0005; <i>P</i> > 0.05 = ns.</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 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

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 regulates SPI-2 expression.

<p>(<b>A</b>), Schematic of SPI-2. (<b>B</b>) qRT-PCR analysis of SPI-2-encoded and associated (<i>sifA</i>) genes from RNA isolated from <i>S</i>. Typhimurium (AJK61) or the Δ<i>eutR</i> (CJA023) strains after 5 h phagocytosis in RAW macrophages. n = 3; error bars represent the geometric mean ± SD; <i>strB</i> was used as the endogenous control. (<b>C</b>) EMSAs of <i>ssrB</i> and <i>amp</i> (ampicillin) with EutR::MBP. (<b>D</b>) EMSAs of <i>ssrB</i> with MBP or EutR::MBP. Also, competition EMSAs with EutR::MBP. The assay was performed with increasing amounts of unlabeled <i>ssrB</i> promoter probe. A competition assay was also performed using the <i>kan</i> promoter as a negative control. The ratios represent hot:cold probe. (<b>E</b>) qPCR showing enrichment of <i>eutS</i>, <i>ssrB</i>, and <i>strB</i> from <i>in vivo</i> ChIP of EutR::MBP (n = 2). *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.005; ***, <i>P</i> ≤0.0005; <i>P</i> > 0.05 = ns.</p

The impact of ethanolamine on SPI-2 expression <i>in vitro</i>.

<p>(<b>A</b>) qRT-PCR of <i>ssrB</i> from RNA isolated from the <i>S</i>. Typhimurium (SL1344) grown in SPI-2 inducing medium with ethanolamine (EA) supplementation as indicated. Statistical significance is shown relative to cells grown without EA supplementation. (<b>B</b>) qRT-PCR of <i>ssrB</i> from RNA isolated from the <i>S</i>. Typhimurium (SL1344) grown in DMEM with EA supplementation as indicated. Statistical significance is shown relative to cells grown without EA supplementation. (<b>C</b>) qRT-PCR of <i>eutR</i> from RNA isolated from <i>S</i>. Typhimurium (AJK61) grown in DMEM with supplementation as indicated or after phagocytosis in RAW macrophages. Statistical significance relative to cells grown in DMEM is indicated. (<b>D</b>) qRT-PCR of <i>eutS</i> from RNA isolated from <i>S</i>. Typhimurium (AJK61) grown in DMEM with supplementation as indicated or after phagocytosis in RAW macrophages. Statistical significance relative to cells grown in DMEM is indicated. (<b>E</b>) qRT-PCR of <i>ssrB</i> from RNA isolated from the <i>S</i>. Typhimurium strain (AJK61) grown in DMEM with supplementation as indicated or after phagocytosis in RAW macrophages. Statistical significance relative to cells grown in DMEM is indicated. For all, n = 3; error bars represent the geometric mean ± SD; <i>strB</i> was used as the endogenous control. *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.005; ***, <i>P</i> ≤0.0005; <i>P</i> > 0.05 = ns.</p

Improving the Affinity of SL0101 for RSK Using Structure-Based Design

Enhanced activity of the Ser/Thr protein kinase, RSK, is associated with transformation and metastasis, which suggests that RSK is an attractive drug target. The natural product SL0101 (kaempferol 3-<i>O</i>-(3″,4″-di-<i>O</i>-acetyl-α-l-rhamnopyranoside)) has been shown to be an RSK selective inhibitor. However, the <i>K</i>_i for SL0101 is 1 μM with a half-life of less than 30 min <i>in vivo</i>. To identify analogues with improved efficacy we designed a set of analogues based on the crystallographic model of SL0101 in complex with the RSK2 N-terminal kinase domain. We identified an analogue with a 5″-<i>n</i>-propyl group on the rhamnose that has >40-fold improved affinity for RSK relative to SL0101 in an <i>in vitro</i> kinase assay. This analogue preferentially inhibited the proliferation of the human breast cancer line, MCF-7, versus the normal untransformed breast line, MCF-10A, which is consistent with results using SL0101. However, the efficacy of the 5″-<i>n</i>-propyl analogue to inhibit MCF-7 proliferation was only 2-fold better than for SL0101, which we hypothesize is due to limited membrane permeability. The improved affinity of the 5″-<i>n</i>-propyl analogue for RSK will aid in the design of future compounds for <i>in vivo</i> use