The NAG Sensor NagC Regulates LEE Gene Expression and Contributes to Gut Colonization by Escherichia coli O157:H7

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are human pathogens responsible for bloody diarrhea and renal failures. EHEC employ a type 3 secretion system to attach directly to the human colonic epithelium. This structure is encoded by the locus of enterocyte effacement (LEE) whose expression is regulated in response to specific nutrients. In this study, we show that the mucin-derived sugars N-acetylglucosamine (NAG) and N-acetylneuraminic acid (NANA) inhibit EHEC adhesion to epithelial cells through down-regulation of LEE expression. The effect of NAG and NANA is dependent on NagC, a transcriptional repressor of the NAG catabolism in E. coli. We show that NagC is an activator of the LEE1 operon and a critical regulator for the colonization of mice intestine by EHEC. Finally, we demonstrate that NAG and NANA as well as the metabolic activity of Bacteroides thetaiotaomicron affect the in vivo fitness of EHEC in a NagC-dependent manner. This study highlights the role of NagC in coordinating metabolism and LEE expression in EHEC and in promoting EHEC colonization in vivo.

Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing Pathogens

“…Recent work has also suggested that the regulator NagC directly influences LEE1 transcription and sugar catabolism in EHEC in response to the mucin-derived sugars N-acetylglucosamine (NAG) and N-acetylneuraminic acid (NANA). NagC directly binds the LEE1 regulatory region at a position that overlaps the Ϫ10 element of the P1 promoter (87) (Fig. 1B).…”

“…Once in the gut, microbiota-derived quorum sensing molecules (56, 76) (positive regulators of ler) provide an initial biogeographic signal as to where in the host the bacterium is located. However, virulence gene expression is repressed overall while in the lumen by the sensing of the microbiota-liberated sugars fucose (69), NAG, and NANA (87). This repression of the LEE is relieved as the bacterium moves laterally along the intestine in response to microbiota-derived butyrate and/or sensing of the host hormones adrenaline and noradrenaline (61,76).…”

Escherichia coliO157:H7 responds to phosphate starvation by modifying LPS involved in biofilm formation

“…The EDL933 Δ pst and Δ phoB mutants were previously constructed (46). The Δ waaH single mutant and Δ pst Δ phoB or Δ pst Δ waaH double mutants were generated by allelic exchange of phoB or waaH genes using a suicide vector in EDL933 wild-type strain or in EDL933 Δ pst mutant as previously described (47) with some modifications. Using the primers listed in Table S1, a tetracycline resistance cassette from pACYC184 was amplified and flanked by PCR with the 500 bp sequence adjacent to the phoB or waaH .…”

Regulation of waaH by PhoB during P i Starvation Promotes Biofilm Formation by Escherichia coli O157:H7

“…The Δpst and ΔphoB mutants were previously constructed (44). The ΔwaaH single mutant and Δpst ΔphoB or Δpst ΔwaaH double mutant were generated by allelic exchange of the phoB or waaH gene using a suicide vector in the wild type or in the Δpst mutant as previously described (45), with some modifications. Using the primers listed in Table S1 in the supplemental material, a tetracycline resistance cassette from pACYC184 was amplified and flanked by PCR with the 500-bp sequence adjacent to phoB or waaH.…”

“…RNA was extracted using a RiboPure-Bacteria kit (Ambion) by following the manufacturer's recommendations. cDNAs, standard curves, and quantitative PCR were performed as previously described (45). Results are presented as the ratios between the cDNA copy number of the gene of interest and the cDNA copy number of the housekeeping gene tus.…”