The Ethanolamine Permease EutH Promotes Vacuole Adaptation of Salmonella enterica and Listeria monocytogenes during Macrophage Infection

Abstract

Ethanolamine is a ubiquitous and essential molecule within a host. Significantly, bacterial pathogens exploit ethanolamine during infection to promote growth and regulate virulence. The ethanolamine permease EutH is dispensable for growth in vitro under standard conditions, whereas EutH is required for ethanolamine utilization at low pH. These findings suggested a model in which EutH facilitates diffusion of ethanolamine into the bacterial cell in acidic environments. To date, the ecological significance of this model has not been thoroughly investigated, and the importance of EutH to bacterial growth under physiologically relevant conditions is not known. During infection, immune cells internalize invading bacteria within an acidic, nutrient-depleted vacuole called the phagosome. Here, we investigated the hypothesis that EutH promotes bacterial survival following phagocytosis. Our findings indicate that EutH is important for survival and replication of the facultative intracellular pathogens Salmonella enterica serovar Typhimurium and Listeria monocytogenes during prolonged or transient exposure to the phagosome, respectively. Furthermore, in agreement with EutH being important in the acidic environment, neutralization of the vacuole abolished the requirement for EutH. Significantly, consistent with a role for EutH in promoting intramacrophage survival, EutH was not required during S Typhimurium local intestinal infection but specifically conferred an advantage upon dissemination to peripheral organs. These findings reveal a physiologically relevant and conserved role for EutH in spatiotemporal niche adaptation during infection.

Keywords: Listeria; Salmonella; ethanolamine; macrophage; pathogenesis; vacuole.

FIG 1

Schematic of the eut locus in (A) S. Typhimurium or (B) L. monocytogenes.

FIG 2

EutH promotes S. Typhimurium survival within macrophages. (A) In vitro growth curve of the WT S. Typhimurium (SL1344) and the ΔeutH (CJA052) strains grown in LB broth. Each data point shows the average from three biological replicates. Error bars represent the mean ± standard deviation (SD). (B) Intramacrophage survival and replication of WT (CJA034), ΔeutR (CJA032), or eutR complemented (CJA033) strains in peritoneal exudate macrophages after 7 h postphagocytosis. n = 9 replicates per strain. (C) Intramacrophage survival and replication of WT (AJK61), ΔeutR (CJA023), or ΔeutH (CJA043) S. Typhimurium strains in RAW macrophages after 5 h postphagocytosis. n = 18 replicates per strain. (D) Intramacrophage survival and replication of WT S. Typhimurium (AJK61), ΔeutH (CJA043), and ΔeutH ΔeutR (CJA168) strains after 5 h postphagocytosis in BMDMs. n = 15 replicates per strain. (E) Intramacrophage survival and replication of WT S. Typhimurium (CJA034), ΔeutH (CJA087), and eutH (CJA184) complemented strains after 5 h postphagocytosis in BMDMs. n = 18 replicates per strain. (F) Intramacrophage survival and replication of ΔssaV, ΔssaV ΔeutR (CJA064), and ΔssaV ΔeutH (CJA172) S. Typhimurium strains after 5 h postphagocytosis in BMDMs. ssaV encodes an essential component of the T3SS-2, and the ΔssaV strain is completely defective for T3SS-2-mediated secretion (48). n = 9 replicates per strain. (G) Initial phagocytosis of S. Typhimurium WT (AJK61), ΔeutH (CJA043), and ΔeutH ΔeutR (CJA168) strains at time zero (see Materials and Methods). n = 24 replicates per strain. (H) In vitro growth curve of the S. Typhimurium WT (SL1344) and ΔeutH (CJA052) strains grown in SPI-2 inducing medium. Each data point shows the average from three biological replicates. Error bars represent the mean ± SD. For panels B to G, the mean and standard error of the mean (SEM) are shown. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005. P values of >0.05 are not significant (ns).

FIG 3

Bacterial survival following EA supplementation. (A) Intramacrophage survival and replication of WT S. Typhimurium (AJK61) after 5 h postphagocytosis in RAW macrophages without or with EA supplementation. n = 9. (B) Intramacrophage survival and replication of ΔeutR S. Typhimurium (CJA023) after 5 h postphagocytosis in RAW macrophages without or with EA supplementation. n = 9. (C) Intramacrophage survival and replication of ΔeutH S. Typhimurium (CJA043) after 5 h postphagocytosis in RAW macrophages without or with EA supplementation. n = 9. (D) Intramacrophage survival and replication of the eutH complemented strain (eutH+) of S. Typhimurium (CJA184) after 5 h postphagocytosis in RAW macrophages without or with EA supplementation. n = 12. For all values, the mean and SEM are shown. *, P ≤ 0.05. P values of >0.05 are not significant (ns).

FIG 4

EutH enhances S. Typhimurium fitness in the acidified SCV. (A) Intramacrophage survival and replication of the WT (AJK61) or ΔeutH (CJA043) strain in BMDMs without or with concanamycin A treatment. n = 9 replicates per strain per condition. (B) Intramacrophage survival and replication of the WT (AJK61) or ΔeutH (CJA043) strain in BMDMs without or with NH₄Cl. n = 9 replicates per strain per condition. The mean and SEM are shown. *, P ≤ 0.05; **, P ≤ 0.005. P values of >0.05 are not significant (ns).

FIG 5

EutH contributes to survival of WT L. monocytogenes during macrophage infection. (A) In vitro growth curve of WT (10403S) and ΔeutH (VK01) L. monocytogenes strains grown in LB broth. Each data point shows the average from three biological replicates. Error bars represent the mean ± SD. (B) Initial phagocytosis of WT (10403S) and ΔeutH (VK01) L. monocytogenes strains at time zero (see Materials and Methods). (C) Intramacrophage survival and replication of WT (10403S) or ΔeutH (VK01) L. monocytogenes strains in BMDMs at the indicated times postphagocytosis. 2 hpi, n = 18 replicates per strain; 5 hpi, n = 12 replicates per strain; 8 hpi, n = 9 replicates per strain. For each time point, WT survival and replication were set to 100%. (D) Intramacrophage survival and replication of WT (10403S) or ΔeutH (VK01) L. monocytogenes strains in BMDMs without or with concanamycin A treatment at 2 h postphagocytosis. n = 9 replicates per strain per condition. (E) Intramacrophage CFU per milliliter of the WT (10403S), ΔeutH (VK01), Δhly (DP-2161), or Δhly ΔeutH (VK06) strains at time zero and 2, 5, and 8 h postphagocytosis. n = 12 to 18 replicates per strain per time point. (F) The percentage of change in intramacrophage survival and replication over time is calculated from data shown in panel E. Each percentage is indicative of the change between the two time points listed and was calculated as (later time point – earlier time point)/(earlier time point). For panels B to E, the mean and SEM are shown. *, P ≤ 0.05; **, P ≤ 0.005. P values of >0.05 are not significant (ns).

FIG 6

Contribution of EutH to S. Typhimurium fitness in vivo. (A) Competitive indices of the ΔeutH (CJA046) versus WT (SL1344) strain during colitis. n = 7 or 8 mice. (B) Competitive indices of the ΔeutH (CJA052) versus ΔeutR (CJA007) strain during colitis. n = 8 mice. (C) Competitive indices in the spleen of the ΔeutH (CJA046) versus WT (SL1344) strain, the ΔeutH complemented (CJA192) (eutH+, which contains pGEN::eutS-eutH) versus WT (CJA182, which contains pGEN empty vector) strain, the ΔeutH (CJA052) versus ΔeutR (CJA007) strain, the ΔeutH (CJA043) versus ΔeutB (CJA018) strain, and the ΔssaV ΔeutH (CJA172) versus ΔssaV strain at 6 hpi following intraperitoneal injection. n = 8 to 11 mice per competition. The median and interquartile range are shown *, P ≤ 0.05. P values of >0.05 are not significant (ns).

FIG 7

EutH plays a spatiotemporal role in S. Typhimurium niche adaptation. (A) EutH is dispensable for EA utilization in the intestine. (B) EutH contributes to EutR-dependent signaling and S. Typhimurium survival and replication during systemic infection.