Redox, amino acid, and fatty acid metabolism intersect with bacterial virulence in the gut

Abstract

The gut metabolic landscape is complex and is influenced by the microbiota, host physiology, and enteric pathogens. Pathogens have to exquisitely monitor the biogeography of the gastrointestinal tract to find a suitable niche for colonization. To dissect the important metabolic pathways that influence virulence of enterohemorrhagic Escherichia coli (EHEC), we conducted a high-throughput screen. We generated a dataset of regulatory pathways that control EHEC virulence expression under anaerobic conditions. This unraveled that the cysteine-responsive regulator, CutR, converges with the YhaO serine import pump and the fatty acid metabolism regulator FadR to optimally control virulence expression in EHEC. CutR activates expression of YhaO to increase activity of the YhaJ transcription factor that has been previously shown to directly activate the EHEC virulence genes. CutR enhances FadL, which is a pump for fatty acids that represses inhibition of virulence expression by FadR, unmasking a feedback mechanism responsive to metabolite fluctuations. Moreover, CutR and FadR also augment murine infection by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. This high-throughput approach proved to be a powerful tool to map the web of cellular circuits that allows an enteric pathogen to monitor the gut environment and adjust the levels of expression of its virulence repertoire toward successful infection of the host.

Keywords: EHEC; cutR; enterohemorrhagic E. coli; fadL.

Fig. 1.

Screen for LEE regulators. (A) Schematic of the LEE operon arrangement. The LEE1-encoded Ler activates expression of all LEE operons. (B) Schematic of the screen for LEE regulators. BW25113 K-12 deletion strains were transformed with the LEE-encoding cosmid, pJAY1512. Strains were grown in DMEM under anaerobic conditions in 96-well plate format and evaluated by ELISA for EspB production. (Inset) Validation of EspB-directed ELISA procedure by addition of recombinant EspB to culture supernatants of BW25113. (C) Class makeup of the BW25113 deletion strains included in the screen. (D) Makeup of gene families found in the screen with a twofold cutoff in change in EspB production. Statistical significance was calculated as ANOVA with Dunnett’s post hoc test. (E) K-12 knockouts identified in genes previously known to regulate the LEE. (F) EspB ELISA of EHEC (86-24) deletion strains used for validation of screen results. *P < 0.05, **P < 0.01; nd, results below limit of detection.

Fig. 2.

Representative screen hits in C. rodentium pathogenesis. All experiments were conducted using conventional mouse feed. Survival curves of C3H/HeJ infected with 10⁹ cfu of WT, (A) ΔcutR, or (C) ΔfadR DBS770 C. rodentium or with PBS control (10 animals per infection group and 8 animals for PBS). Statistical significance calculated by Gehan–Breslow–Wilcoxon test. Weight of animals infected with (B) ΔcutR, or (D) ΔfadR DBS770, WT or mock (PBS). (E) Western for EspB from in vitro culture supernatants from WT, ΔcutR DBS770 and ΔcutR complemented (cultures grown in DMEM). (F) Western for EspB from in vitro culture supernatants from WT, ΔfadR DBS770, and ΔfadR complemented (cultures grown in DMEM). (G) qRT-PCR of C. rodentium LEE mRNAs from in vitro anaerobically grown WT, ΔcutR DBS770, and ΔcutR complemented strains (cultures grown in DMEM). (H) qRT-PCR quantification of C. rodentium LEE mRNAs from in vitro anaerobically grown WT, ΔfadR DBS770, and ΔfadR complemented strains (cultures grown in DMEM). (I) qRT-PCR of C. rodentium LEE mRNAs in murine cecum tissue of animals infected with WT, ΔcutR, and complemented strains. (J) qRT-PCR of C. rodentium LEE mRNAs in murine colon tissue of animals infected with WT, ΔcutR, and complemented strains. (K) qRT-PCR of C. rodentium LEE mRNAs in murine cecum tissue of animals infected with WT, ΔfadR, and complemented strains. (L) qRT-PCR of C. rodentium LEE mRNAs in murine colon tissue of animals infected with WT, ΔfadR, and complemented strains. *P < 0.05, **P < 0.01, ***P < 0.001. P.I., postinfection.

Fig. 3.

CutR regulation of LEE expression in EHEC. (A) qRT-PCR of LEE genes from WT, ΔcutR, and complemented EHEC strains grown anaerobically in the presence of cysteine (cultures grown in DMEM). (B) Western for EspB secreted from WT, ΔcutR, and complemented EHEC strains grown anaerobically in the presence of cysteine (cultures grown in DMEM). (C) Western for EspB from whole cell lysates of WT, ΔcutR, and complemented EHEC strains (cultures grown in DMEM). (D) Schematic of the cutR locus from E. coli, Shigella dysenteriae, C. rodentium, and S. Typhimurium. (E–G) qRT-PCR of LEE transcripts for (E) ler, (F) eae, and (G) espA from WT or ΔcutR grown anaerobically in DMEM either with or without cysteine. (H) Schematic representing a putative mechanism of cutR-dependent LEE regulation. CutR positively regulates the expression of yhaO, a serine transporter that positively regulates LEE expression via the LysR-type transcription factor YhaJ. (I) qRT-PCR of yhaO mRNA from WT, ΔcutR, and complemented strains (cultures grown in DMEM). (J) qRT-PCR to assess LEE expression from ΔcutRΔyhaO double mutant (cultures grown in DMEM). (K) qRT-PCR for complementation of LEE transcriptional phenotype by N-terminally V5-tagged CutR during preparation of cells for ChIP (cultures grown in DMEM). (L) ChIP-qPCR results for empty vector control or N-terminally tagged CutR. Probes are designed to amplify the promoter regions of yhaO (positive control), ybaO (cutR promoter), rpoZ (negative control), fadL, or overlapping fragments of the ler (LEE1) promoter, numbered from the proximal transcriptional start site. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

FadR LEE regulator in EHEC. (A) Western for EspB from supernatants of WT, ΔfadR, and complemented strains (cultures grown in DMEM). (B) qRT-PCR of LEE genes from WT, ΔfadR, and complemented strains (cultures grown in DMEM). (C) Diagram of EHEC LEE1 promoter segments used for EMSA probes. Probes are designed to have ∼80 bp of overlap between segments, extending from 967 bp upstream of the proximal LEE1 transcription start site to 155 bp downstream. (D) EMSA of His-tagged FadR and kanamycin probe (negative control), fadL promoter probe (positive control), and segments of the EHEC LEE1 regulatory region numbered from the proximal transcriptional start site. (E) EMSA of a Citrobacter LEE1 promoter fragment extending from 437 bp upstream to 104 bp upstream of the transcriptional start site. (F) Schematic of model of fadR- and cutR-dependent LEE regulation. **P < 0.01, ^##P < 0.0001.