Ethanolamine Signaling Promotes Salmonella Niche Recognition and Adaptation during Infection

Abstract

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.

Fig 1. EutR in pathogen-microbiota-host interactions.

(A) Schematic of the eut operon. (B) In vitro growth curve of S. Typhimurium WT (SL1344), ΔeutR (CJA009), or ΔeutB (CJA020) strains in LB without or with supplementation of 5 mM ethanolamine (EA). Each data point shows the average of three independent experiments. (C) qRT-PCR of eutR in WT or the ΔeutB (CJA020) S. 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); strB was used as the endogenous control. (D-F) Competition assays between (D) ΔeutB::Cm^R (CJA018) and WT strains; (E) ΔeutR::Cm^R (CJA007) and WT strains; or (F) ΔeutR::Cm^R (CJA007) and ΔeutB (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)). *, P ≤0.05; **, P ≤ 0.005; ***, P ≤0.0005; P > 0.05 = ns.

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.

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.

Fig 4. EutR regulates SPI-2 expression.

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

Fig 5. EutR enhances S. Typhimurium survival within macrophages.

(A) Intramacrophage survival and replication of S. Typhimurium (AJK61) and the ΔeutR (CJA023) strains after 5 h phagocytosis in RAW macrophages (error bars represent the geometric mean value ± SE of 24 independent experiments). (B) Intramacrophage survival and replication of S. Typhimurium (CJA034), ΔeutR (CJA032), and ΔeutR complemented with eutR (eutR+) (CJA033) strains after 5 h phagocytosis in peritoneal exudate macrophages (PEMs) (error bars represent the geometric mean value ± SE of nine independent experiments). (C) Intramacrophage survival and replication of S. Typhimurium (AJK61), ΔeutR (CJA023) and ΔeutB (CJA028) strains after 5 h phagocytosis in PEMs (error bars represent the geometric mean value ± SE of six independent experiments). (D) In vitro growth curve of S. Typhimurium WT (SL1344), ΔeutR (CJA009), or ΔeutB (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. (E) In vitro growth curve of S. Typhimurium WT (SL1344), ΔeutR (CJA009), or ΔeutB (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. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; P > 0.05 = ns.

Fig 6. EutR promotes recognition and adaptation to the intracellular environment.

(A-C) Competition assays between ΔeutR::Cm^R (CJA007) and ΔeutB (CJA020) strains collected from (A) harvested spleens, (B) the peritoneal cavity, or (C) phagocytized cells at 6 h pi. Mice were intraperitoneally infected with 1:1 mixtures of the ΔeutR and ΔeutB strains. Each column represents a CI. Each column shows the geometric mean value ± SE for each group (n = 2 litters (6–8 animals)). *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; P > 0.05 = ns.

Fig 7. EutR-associated signaling in vivo.

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

Fig 8. EutR in S. Typhimurium niche adaptation.

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