Ethanolamine Signaling Promotes Salmonella Niche Recognition and Adaptation during Infection

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 Salmonella enterica serovar Typhimurium (S. 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 S. Typhimurium to establish infection. Subsequently, EutR directly activates expression of the Salmonella 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 S. 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.


Introduction
Chemical and nutrient signaling mediate diverse biological processes, and underlie interactions among the mammalian host, the resident microbiota, and invading pathogens [1].Ethanolamine is abundant in cell membranes, as a component of phosphatidylethanolamine as well as in modified lipid molecules such as N-acylethanolamines [2].These ethanolamine-containing compounds play important roles in mammalian cell signaling and influence diverse physiological effects, including cytokinesis, immunomodulation, food intake and energy balance [2][3][4].Ethanolamine is abundant in the intestinal tract due to the turnover and exfoliation of enterocytes and bacterial cells [5,6], and intracellular pools of ethanolamine are maintained by low and high affinity uptake systems as well as through internal recycling of phosphatidylethanolamine [7][8][9][10].
Bacterial pathogens compete for nutrients with the resident microbiota and rely on environmental cues to control virulence gene expression.Salmonella enterica serovar Typhimurium (S.Typhimurium) is a facultative intracellular pathogen and a leading cause of acute gastroenteritis, which can progress to systemic infection in susceptible individuals [11].S. Typhimurium encodes two type three secretion systems (T3SSs) that are important for pathogenesis.S. Typhimurium uses the T3SS encoded within the Salmonella pathogenicity island (SPI)-1 to invade intestinal epithelial cells and penetrate to the lamina propria [12].There, S. Typhimurium is taken up by macrophages, where it survives and replicates.Intracellular survival is mediated by the T3SS and effectors encoded in SPI-2 [13][14][15].Ethanolamine can serve as a carbon and/or nitrogen source for bacteria in the intestine as well as within epithelial cells [16,17].The aim of this work was to determine whether S. Typhimurium relies on ethanolamine as a signal to coordinate gene expression and augment virulence in vivo.Here, we show that the intramacrophage environment promotes expression of the ethanolamine utilization transcription factor EutR, which directly activates SPI-2.Moreover, we demonstrate that EutR signaling during systemic infection is specific to the intracellular environment and is important for robust S. Typhimurium dissemination.Altogether, our findings suggest that ethanolamine, an intrinsic component of bacterial and mammalian cell membranes, functions as a signal to modulate metabolism and virulence and suggest a new layer of complexity in chemical signaling that underlies pathogenicity.

EutR contributes to dissemination in vivo
Genes encoding for ethanolamine metabolism are clustered in the eut operon [18] (Fig 1A).In the Enterobacteriaceae, expression of this operon is regulated by the eut-encoded transcription factor EutR. EutR is constitutively expressed at low levels from its own promoter and binds to the promoter region immediately upstream of eutS.In the presence of ethanolamine and vitamin B 12 , EutR activates transcription of this operon [19,20].In enterohemorrhagic Escherichia coli (EHEC), EutR senses ethanolamine to activate virulence gene expression in vitro, independently of ethanolamine metabolism [19,21,22].To determine whether EutR influences S. Typhimurium disease progression during infection, we generated an eutR deletion strain (ΔeutR) that cannot sense ethanolamine as well as an eutB deletion strain (ΔeutB) that lacks the large subunit of the ethanolamine ammonia lyase, and thus is unable to catabolize ethanolamine.The eutR and eutB mutations did not result in a general loss of fitness, as the ΔeutR and ΔeutB strains exhibited no measurable growth defects in vitro (Fig 1B).Importantly, the eutB mutation is nonpolar as this mutant can respond to ethanolamine (Fig 1C).Subsequently, we performed competitive infections in which streptomycin-treated mice were orally infected with an equal mixture of wild type (WT) and ΔeutB (ΔeutB::Cm R ) strains or the WT and ΔeutR (ΔeutR::Cm R ) strains.S. Typhimurium infection presents as intestinal outgrowth, invasion of epithelial cells, and subsequent uptake by macrophages and dissemination to secondary lymphoid tissue.Therefore, to monitor the course of S. Typhimurium infection, we analyzed the number of recovered bacteria from the intestinal contents, the colon, and the spleen.At 2 and 4 days post infection (dpi), the ΔeutR and ΔeutB strains were significantly outcompeted by the WT strain in intestinal contents (Fig 1D and 1E).These data underscore the importance of ethanolamine metabolism in S. Typhimurium colonization of the intestinal tract, and these findings are consistent with previous work by Thiennimitr et al., who showed that ethanolamine metabolism provides a growth advantage to S. Typhimurium during intestinal colonization [17].
Defects in ethanolamine metabolism have been reported to result in mild or no attenuation during S. Typhimurium systemic infection [23,24]; however the role of EutR, specifically, in contributing to dissemination has not been investigated.Therefore, to assess this, we harvested the colons and the spleens of infected mice at 4 dpi.The ΔeutR and ΔeutB strains were both recovered at significantly lower numbers than WT from the colon and spleen; however, the competition indices measured from the spleen from the ΔeutR/WT infections were significantly greater than between WT and the ΔeutB strain (P = 0.002).These findings led us to hypothesize that EutR plays a more extensive role in S. Typhimurium pathogenesis that is distinct from its function to promote ethanolamine metabolism.
To test this, we performed competition infections between the ΔeutB and ΔeutR (ΔeutR:: Cm R ) strains.At 2 days post infection (dpi), the ΔeutR and ΔeutB strains were recovered at similar numbers from intestinal contents (Fig 1F ), indicating that at this initial stage of colonization, EutR functions to drive ethanolamine metabolism.However, at 4 dpi, which is a time point consistent with the progression to systemic infection [25], the ΔeutR strain was significantly outcompeted by the ΔeutB strain (Fig 1F).Significantly, although equal numbers of the ΔeutR and ΔeutB strains were recovered from the colon, the ΔeutR strain was significantly outcompeted by the ΔeutB strain in the spleen (Fig 1F).These data suggest that EutR, independent of its role in ethanolamine metabolism, is important to S. Typhimurium dissemination during infection.

EutR does not influence invasion of epithelial cells
To further explore how ethanolamine signaling contributes to S. Typhimurium dissemination, we examined S. Typhimurium virulence gene expression in vitro.To examine ethanolaminemediated expression of SPI-1, we measured expression of sipC, a SPI-1 encoded translocase that plays a role in invasion of epithelial cells [26].For this, we grew S. Typhimurium in LB, which induces SPI-1 expression [27] as well as in DMEM used for cell culture assays.In both cases, expression of sipC was slightly decreased when ethanolamine was included in the culture medium (Fig 2A and 2B and S1 Fig).To determine whether the ethanolamine-dependent decrease in sipC expression impacted S. Typhimurium invasion of epithelial cells, we infected HeLa cells with either WT S. Typhimurium or the ΔeutR strain.WT S. Typhimurium and ΔeutR invaded HeLa cells at nearly equivalent levels in the presence or absence of ethanolamine (Fig 2C and 2D).Because we did not observe EutR-dependent effects on epithelial invasion under the specified conditions, the influence of ethanolamine at this stage in S. Typhimurium dissemination was not pursued further.

Ethanolamine influences SPI-2 expression
Next, we investigated whether ethanolamine impacted SPI-2 expression.SsrB is a SPI-2 encoded transcriptional regulator that is required for expression of all the SPI-2-encoded genes, as well as for expression of effectors and virulence genes encoded outside of SPI-2 [28][29][30][31].To test the influence of ethanolamine, we measured expression of ssrB in low magnesium, minimal medium, a condition that induces SPI-2 expression [32] (S2 Fig) without supplementation or with supplementation of 250 μM or 5 mM ethanolamine.These concentrations were used because 250 μM ethanolamine was the lowest concentration with which we could readily detect EutR expression (S3 Fig), whereas 5 mM is similar to ethanolamine concentrations in the gastrointestinal tract [33].When 250 μM ethanolamine was added to the SPI-2 inducing medium, expression of ssrB was significantly increased compared to medium without supplementation, but unchanged when 5 mM ethanolamine was added (Fig 3A).These data suggest that ethanolamine may enhance the response of S. Typhimurium in adapting to the intramacrophage environment.Expression of SPI-2 is tightly regulated and is induced specifically in the intracellular environment [34], or in conditions that mimic the intracellular environment.In accordance, ssrB expression was not induced in DMEM or LB when ethanolamine was supplemented to the medium (Fig Macrophages play a significant role in the pathogenesis of S. Typhimurium infection by providing protected sites for intracellular replication and a means of dissemination [35].Robust expression of EutR requires ethanolamine as well as the cofactor vitamin B 12 [20].S. Typhimurium synthesizes vitamin B 12 under anaerobic conditions [36]; however, S. Typhimurium must acquire ethanolamine from the environment [18].Therefore, we investigated whether the intracellular environment induces eutR expression.For this, we infected macrophages with S. Typhimurium in the absence of any exogenous ethanolamine or vitamin B 12 .intrinsic ability of S. Typhimurium to withstand and disrupt host defense mechanisms [37,38], and these data revealed that EutR influences this critical aspect of S. Typhimurium virulence. SsrB is a response regulator that comprises a two component system with the sensor kinase SsrA (also referred to as SpiR) [13,15].SsrA autophosphorylates in response to the acidic environment of the Salmonella-containing vacuole (SCV) within host cells [39,40], which initiates a signaling cascade that promotes SsrB activity as well as ssrAB expression [41].Importantly, the ssrB gene contains its own promoter [41].The genetic data indicated that EutR influenced expression of ssrB and downstream targets, but that EutR did not impact ssrA expression (Fig 4B  Consistent with these results, there was a significant enrichment of the ssrB promoter when EutR-DNA interactions were analyzed using in vivo chromatin immunoprecipitation followed by qPCR (Fig 4E).As a positive control, we also measured enrichment of the eutS promoter, an established binding target of EutR [19], and observed similar enrichment of both targets (Fig 4E ); strB DNA was used as a negative control.Control of ssrB expression is complex and also includes activation by additional TCS PhoP/PhoQ and EnvZ/OmpR, which respond to signals within the Salmonella containing vesicle (SCV) [41,42].Our findings suggest that EutR-dependent activation of ssrB enables S. Typhimurium to integrate intrinsic information regarding the host cell through ethanolamine signaling with SCV-specific signals to coordinate efficient spatiotemporal expression of SPI-2 and SPI-2 associated effectors.To verify that the defect in the ΔeutR strain was not the result of a defect in ethanolamine metabolism, we assessed survival of the ΔeutB strain within PEMs.The ΔeutB strain was recovered at similar numbers to WT and at significantly higher numbers than the ΔeutR strain (Fig 5C ).Importantly, the ΔeutR mutant grows similarly to WT and ΔeutB strains in SPI-2 inducing medium and tissue culture medium with or without the addition of ethanolamine (Fig 5D and  5E), confirming that the decrease in intracellular survival is not a result of a EutR-dependent growth defect.These findings indicate that ethanolamine-associated signaling, but not catabolism, impacts S. Typhimurium survival within macrophages.Moreover, these findings, in conjunction with lack of eut operon induction within macrophages (Fig 3D ), reveals that S. Typhimurium relies on EutR to direct gene expression in a manner that is particular to a specific niche.

EutR provides a gauge of the intracellular environment in vivo
Next, we confirmed that EutR mediates dissemination specifically through intracellular survival in vivo.To further discriminate between ethanolamine-associated signaling and ethanolamine metabolism, we infected mice with equal numbers of the ΔeutR::Cm R and ΔeutB strains by intraperitoneal injection.At 6 h pi, the ΔeutR strain was recovered at significantly lower numbers compared to the ΔeutB strain from the spleen (Fig 6A).Furthermore, we assessed bacterial burden in the peritoneal cavity.At this site, there were no significant differences between the ΔeutR and ΔeutB strains in the total bacteria recovered (Fig 6B ), the majority of which were extracellular (S10 Fig) .However, the ΔeutR strain was recovered at significantly lower numbers compared to the ΔeutB strain in the phagocytized population of S. Typhimurium within the peritoneal cavity (Fig 6C).These findings reveal that EutR augments S. Typhimurium fitness during systemic infection.Our findings differ from a previous study that used a genetic screen to identify genes important for systemic virulence [43].Discrepancies may reflect differences in study design such as the age and genetic background of mice, route of infection, and/or duration of infection.Importantly, using in vitro and in vivo approaches, our data establish a genetic and functional role for EutR in S. Typhimurium systemic disease, and altogether, these results indicate that EutR contributes to the ability of S. Typhimurium to gauge and adapt to the intracellular environment in vivo.

EutR signaling during systemic infection
The in vitro studies identified targets of EutR-dependent gene regulation.To test our findings within the complexities of the in vivo environment, we assessed EutR-dependent regulation of

Conclusions
These findings reveal a novel signaling pathway critical for S. Typhimurium to enhance disease progression during infection.We propose a model in which S. Typhimurium relies on ethanolamine signaling through EutR to gauge distinct environments in the host and then modulate expression of genes encoding metabolism and virulence (Fig 8).The resident microbiota do not readily metabolize ethanolamine [33].Thus, to establish infection, S. Typhimurium sidesteps nutritional competition by respiring ethanolamine in conjunction with tetrathionate, an electron acceptor generated specifically during intestinal inflammation [17,44].Fermentation of ethanolamine provides very little growth [17]; hence, outside of the intestine, and in the absence of bacterial competition, S. Typhimurium preferentially utilizes alternative nutrients [23].This enables EutR to direct expression of traits necessary for dissemination and systemic infection.Additional experiments are necessary to determine what factors influence the transition from driving metabolism to influencing virulence in the intestine.Genes encoding ethanolamine utilization are widespread in pathogenic bacteria as well as in members of the resident microbiota [45], and the extracellular pathogen EHEC responds to ethanolamine to regulate virulence gene expression [22].Therefore, ethanolamine signaling may be a conserved strategy used by diverse pathogens to coordinate metabolism and virulence in response to distinct host environments.Our findings highlight a sophisticated mechanism in which S. Typhimurium exploits an abundant and essential molecule within the host to gain specific information about the localized environment and modulate gene expression to overcome bacterial and host resistance mechanisms.

Strains and plasmids
All strains and plasmids used in this study are listed in S1 Table .Luria-Bertani (LB), Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen), or minimal medium (described below) were used as indicated.Ethanolamine (Sigma) and/or vitamin B 12 (Sigma) were supplemented to the media as indicated in the main text.Unless indicated otherwise, 150 nM vitamin B 12 was added whenever ethanolamine was added to the growth medium.Antibiotics were used in the following concentrations: ampicillin (100μg/ml), streptomycin (100μg/ml), chloramphenicol (20μg/ml), and kanamycin (50μg/ml).Recombinant DNA and molecular biology techniques were performed as described previously [46].

Construction of isogenic mutants
S. Typhimurium SL1344 [47] and its derivatives were used in all experiments.The invG mutant (strain AJK61) was a gift from James Casanova and was constructed as previously described [48].Nonpolar eutR and eutB deletion strains were generated in WT and ΔinvG backgrounds using λ-red mutagenesis [49].Briefly, PCR products (obtained with primers listed in S2 Table ) were amplified from plasmid pKD3 or pKD4 with flanking regions matching eutR or eutB and then transformed into S. Typhimurium expressing the Red genes from plasmid pKD46.The resistance cassette was resolved with flippase from temperature-sensitive plasmid pCP20, which was then cured through growth at 42°C.Unresolved strains were used in the murine competition assays as indicated in the text and figure legends.All deletions were confirmed by sequencing.The eutR mutant was complemented with pCJA002.Plasmid pCJA002 was constructed by amplifying S. Typhimurium genomic DNA using primers specific to the eutR gene, including 206 nucleotides upstream of the ATG start site (listed in S2 Table ).Amplified DNA was digested with HindIII and BamHI and inserted into pGEN-MCS [50] (Addgene MTA).As controls, WT and the ΔeutR strains were transformed with empty pGEN-MCS vectors for use in the complementation experiments.The EutR::Flag strain was generated as described for the deletion strains, using pSUB11 as described [51].

RNA extraction and quantitative reverse transcription polymerase reaction (qRT-PCR)
For the in vitro studies, RNA was extracted from S. Typhimurium cells grown in culture medium as described or from phagocytized S. Typhimurium.Cells were resuspended in Trizol (Life Technologies) and RNA was purified using the RiboPure kit (Ambion).For the in vivo studies, spleens were harvested at 2 dpi and homogenized in 1 mL Trizol per 100 mg tissue [52].RNA was isolated using standard molecular biological procedures.Primer validation and qRT-PCR was performed as described previously [53] using primers listed in S2 Table .Briefly, RNA was extracted from three biological replicates, and qRT-PCR was performed in a one-step reaction using an ABI 7500 sequence detection system (Applied Biosystems).Data were collected using the ABI software Detection 1.2 software (Applied Biosystems).All data were normalized to the endogenous control strB (main text) or to 16S rRNA (RNA was diluted 1:1000) as previously performed [54,55].Controls were used as indicated in figure legends and analyzed using the comparative critical threshold (C T ) method.The Student's unpaired t test was used to determine statistical significance.
For epithelial cell infection bacterial cultures were grown under invasion-inducing conditions [27].Briefly, overnight cultures were diluted back 1:100 and grown without agitation in LB until late logarithmic phase (OD 600 of approximately 1.0) at 37°C.Bacterial cells were washed and resuspended in 1x phosphate buffered saline before infection.HeLa cells were placed in DMEM or DMEM supplemented with 5 mM ethanolamine and 150 nM vitamin B 12 .HeLa cells were infected at a multiplicity of infection (MOI) of 100 for 1 h and either lysed directly or treated with 100 μg/ml gentamicin for 30 min to kill any extracellular bacteria.Percent invasion was calculated as the number of intracellular bacteria as a percent of the directly lysed sample and normalized such that wild type was equal to 100%.
For macrophage assays, we used an invG mutant (deficient in cell invasion) as the WT strain, and we generated corresponding ΔeutRΔinvG and ΔeutBΔinvG strains (described above).These strains were used because invasive S. Typhimurium rapidly kills macrophages [57].Additionally, expression of invasion-associated genes are down-regulated after entry into host cells; therefore, this strain more closely mimics S. Typhimurium as it is encountered by professional phagocytes after penetration of the epithelial barrier [58].
Gentamicin protection assays were performed according to published methods [14,57,59,60].S. Typhimurium was grown overnight in LB, washed and re-suspended in PBS before incubation with macrophages (without the addition of ethanolamine or vitamin B 12 ) at an MOI of 50.After 30 min of incubation, extracellular bacteria were killed with 100 μg/ml gentamicin treatment for 30 min, before replacement with media containing 10 μg/ml gentamicin for the remainder of the assay.Cells were lysed at indicated time points in 1% Triton-X and colony forming units (cfu) determined by serial dilutions and plating onto LB agar.After internalization, cells were treated with gentamicin and lysed to enumerate viable intracellular bacteria at time 0 h.Survival was calculated as previously described [14].Briefly, the viable cfu at the indicated time points were determined as the percentage of this intracellular time 0 h population and normalized such that wild type was equal to 100%.For all assays, the Student t test was used to determine statistical significance.

Mouse studies
All experiments were approved by the Institutional Animal Care and Use Committee at the University of Virginia School of Medicine.For the colitis infections, female C57BL/6 (10-12 week old) mice were given a single dose of 20 mg streptomycin 24 h prior to infection [61].Mice were infected with an equal mixture of 5 x 10 8 cfu of the indicated strains.Fresh fecal pellets were collected daily, and mice were euthanized at 4 dpi to assess bacterial burden in the colon and spleen.Tissue samples were weighed, homogenized in 1 ml PBS, and bacterial numbers were quantified by plating serial dilutions of homogenates on MacConkey agar supplemented with streptomycin or with chloramphenicol.The competitive index was calculated as the ratio of ΔeutR to wild type (WT) or ΔeutB strains or the ratio of the ΔeutB to WT recovered normalized to the ratio in the inoculum.Statistical significance was determined by one-sample t test with an expected value of 1. Comparisons between competitive indexes were performed using the Mann-Whitney U test.
For the systemic competition experiments, mice were infected intraperitoneally (i.p.) with 1x10 5 cfu of the ΔeutR and ΔeutB strains.Spleens were harvested at 6 h and bacterial burden was assessed as described above.Bacterial burden in the peritoneal cavity was assessed as described [62].Briefly, following euthanasia, 5mL of PBS was injected into the peritoneal cavity, aspirated, and immediately placed on ice.Samples were split into two aliquots, one receiving 100 μg/ml gentamicin treatment.After 30 minutes on ice, samples were washed, lysed and plated as described above.For RNA analyses, mice were infected by i.p. with 1x10 4 cfu of WT or the ΔeutR strain, and spleens were harvested at 2 dpi.

Electrophoretic mobility shift assays (EMSAs)
Plasmid pDC24 or the empty vector pMAL-c5X was used for the EMSA assays.This plasmid was constructed by amplifying the eutR gene from the S. Typhimurium strain SL1344 with indicated primers (S2 Table ).The resulting PCR product was cloned into the Nco1/Sbf1 cloning site of vector pMAL-c5X.EutR was purified under native conditions as described [19].Briefly, the MBP-tagged EutR protein was purified by growing the E. coli strain NEBexpress cells (NEB) containing pDC24 at 37°C in LB with glucose (0.2% final concentration) and ampicillin (100 μg/ml) to an OD 600 of 0.5, at which point IPTG was added to a final concentration of 0.3 mM and allowed to induce overnight at 18°C.Cells were harvested by centrifugation at 4000 x g for 20 min and then resuspended in 25 mL column buffer (20 mM Tris-HCl; 200 mM NaCl; 1 mM EDTA) and lysed by homogenization.The lysed cells were centrifuged, and the lysate was loaded onto a gravity column (Qiagen) with amylose resin.The column was washed with column buffer and then eluted with column buffer containing 10 mM maltose.Fractions containing purified proteins were confirmed by SDS-PAGE and Western analysis, and the protein concentration was determined using a NanoDrop Spectrophotometer.PCR-amplified DNA probes (listed in text and described in S2 Table ) were generated as previously described [19,63].DNA probes were end-labeled with [γ-32 P ]-ATP (Perkin-Elmer) using T4 polynucleotide kinase (NEB) following standard procedures [64].End-labeled fragments were run on a 6% polyacrylamide gel, excised, and purified using the Qiagen PCR purification kit.
EMSAs were performed by adding purified EutR-MBP or MBP to labeled DNA in binding buffer (500 μg ml -1 BSA (NEB), 50 ng poly-dIdC, 60 mM HEPES pH 7.5, 5 mM EDTA, 3 mM dithiothreitol (DTT), 300 mM KCl, and 25 mM MgCl 2 ).Ethanolamine (1 mM) and vitamin nM B 12 (150 nM) were added to the reactions.Reactions were incubated for 25 minutes at 25°C.Then, a 1% Ficoll solution was added to the reactions immediately before loading the samples on the gel.The reactions were electrophoresed for approximately 6 h at 150 V on a 6% polyacrylamide gel, dried, and imaged with a phosphorimager (Molecular Dynamics).

Fig 2 .
Fig 2. Effect of ethanolamine and EutR on SPI-1.(A) qRT-PCR of sipC from WT S. Typhimurium (SL1344) grown in LB or LB supplemented with 5 mM ethanolamine (EA).(B) qRT-PCR of sipC from WT S. Typhimurium (SL1344) grown in DMEM or DMEM supplemented with ethanolamine (EA) as indicated.For (A) and (B), n = 3; error bars represent the geometric mean ± SD.Statistical significance is shown relative to cells grown without EA supplementation; strB was used as the endogenous control.(C) Invasion of HeLa cells by WT (SL1344) and the ΔeutR (CJA009) strains.Mean ± SE of nine independent experiments.(D) Invasion of HeLa cells by WT (SL1344) and the ΔeutR (CJA009) strains.Mean ± SE of six independent experiments with supplementation of 5 mM EA. **, P 0.005; P > 0.05 = ns.doi:10.1371/journal.ppat.1005278.g002

Fig 3 .
Fig 3.The impact of ethanolamine on SPI-2 expression in vitro.(A) qRT-PCR of ssrB from RNA isolated from the S. 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) qRT-PCR of ssrB from RNA isolated from the S. Typhimurium (SL1344) grown in DMEM with EA supplementation as indicated.Statistical significance is shown relative to cells grown without EA supplementation.(C) qRT-PCR of eutR from RNA isolated from S. 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.(D) qRT-PCR of eutS from RNA isolated from S. 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.(E) qRT-PCR of ssrB from RNA isolated from the S. 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; strB was used as the endogenous control.*, P 0.05; **, P 0.005; ***, P 0.0005; P > 0.05 = ns.doi:10.1371/journal.ppat.1005278.g003
Next, we tested the consequences of EutR-dependent activation of SPI-2 on S. Typhimurium fitness during macrophage infection.Following infection of RAW or peritoneal exudate macrophages (PEMs), the ΔeutR strain was recovered at significantly lower numbers compared to the WT strain (Fig 5A-5C and S9 Fig).Additionally, complementation of the ΔeutR strain with eutR expressed from the native promoter (eutR+) restored intracellular survival to WT levels during primary macrophage infection (Fig 5B).