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Background and aimsThe circadian clock orchestrates ~24-hour oscillations of gastrointestinal (GI) epithelial structure and function that drive diurnal rhythms in the composition, localization, and metabolism of gut microbiota. Here, we use experimental and computational approaches in enteroids to reveal reciprocal effects of microbial metabolites on intestinal epithelial timekeeping by an epigenetic mechanism.MethodsWe cultured 3D PER2∷LUCIFERASE andBmal1-ELuciferasejejunal enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 andBmal1were measured in enteroids cultured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolic profiles to identify phase-shifting metabolites.ResultsFiltrates from 3 of 7 ASF species (ASF360Lactobacillus intestinalis, ASF361Ligilactobacillus murinus, ASF502Clostridiumspp.) induced minimal alterations in circadian rhythms, whereas 4 ASF species (ASF356Clostridiumspp., ASF492Eubacterium plexicaudatum, ASF500Pseudoflavonifactorspp., ASF519Parabacteroides goldsteinii) induced profound, concentration-dependent phase delays. Random forest classification identified short chain fatty acids (SCFA: butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of “shifters”, i.e., ASF taxa whose metabolites induce phase delay. Experiments with SCFAs confirmed machine learning predictions, with a median phase delay of 6.2 hours. Pharmacological or botanical HDAC inhibitors generated similar phase delays. Further, mithramycin A, an inhibitor of HDAC inhibition, abrogated SCFA-induced phase delays by 20% (PBmal1-luciferaseenteroids.ConclusionsGut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms, in part, by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Ethanolamine (EA) metabolism is a trait associated with enteric pathogens, including enterohemorrhagic Escherichia coli O157:H7 (EHEC). EHEC causes severe bloody diarrhea and hemolytic uremic syndrome. EHEC encodes the ethanolamine utilization ( eut ) operon that allows EHEC to metabolize EA and gain a competitive advantage when colonizing the gastrointestinal tract. The eut operon encodes the transcriptional regulator EutR. Genetic studies indicated that EutR expression is induced by EA and vitamin B 12 and that EutR promotes expression of the eut operon; however, biochemical evidence for these interactions has been lacking. We performed EA-binding assays and electrophoretic mobility shift assays (EMSAs) to elucidate a mechanism for EutR gene regulation. These studies confirmed EutR interaction with EA, as well as direct binding to the eutS promoter. EutR also contributes to expression of the locus of enterocyte effacement (LEE) in an EA-dependent manner. We performed EMSAs to examine EutR activation of the LEE. The results demonstrated that EutR directly binds the regulatory region of the ler promoter. These results present the first mechanistic description of EutR gene regulation and reveal a novel role for EutR in EHEC pathogenesis.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome throughout the world. A defining feature of EHEC pathogenesis is the formation of attaching and effacing (AE) lesions on colonic epithelial cells. Most of the genes that code for AE lesion formation, including a type three secretion system (T3SS) and effectors, are carried within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). In this study, we report that a putative regulator, which is encoded in the cryptic E. coli type three secretion system 2 (ETT2) locus and herein renamed EtrB, plays an important role in EHEC pathogenesis. The etrB gene is expressed as a monocistronic transcript, and EtrB autoregulates expression. We provide evidence that EtrB directly interacts with the ler regulatory region to activate LEE expression and promote AE lesion formation. Additionally, we mapped the EtrB regulatory circuit in EHEC to determine a global role for EtrB. EtrB is regulated by the transcription factor QseA, suggesting that these proteins comprise a regulatory circuit important for EHEC colonization of the gastrointestinal tract.

Growth of a microorganism in a host is essential for infection, and bacterial pathogens have evolved to utilize specific metabolites to enhance replication in vivo. Now, emerging data demonstrate that pathogens rely on microbiota-derived metabolites as a form of bacterial-bacterial communication to gain information about location within a host and modify virulence gene expression accordingly. Thus, metabolite-sensing is critical for pathogens to establish infection. Here, we highlight recent examples of how the foodborne pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) exploits microbiota-derived metabolites to recognize the host intestinal environment and control gene expression that results in controlled expression of virulence traits.