Genetic and Mechanistic Analyses of the Periplasmic Domain of the Enterohemorrhagic Escherichia coli QseC Histidine Sensor Kinase

Abstract

The histidine sensor kinase (HK) QseC senses autoinducer 3 (AI-3) and the adrenergic hormones epinephrine and norepinephrine. Upon sensing these signals, QseC acts through three response regulators (RRs) to regulate the expression of virulence genes in enterohemorrhagic Escherichia coli (EHEC). The QseB, QseF, and KdpE RRs that are phosphorylated by QseC constitute a tripartite signaling cascade having different and overlapping targets, including flagella and motility, the type three secretion system encoded by the locus of enterocyte effacement (LEE), and Shiga toxin. We modeled the tertiary structure of QseC's periplasmic sensing domain and aligned the sequences from 12 different species to identify the most conserved amino acids. We selected eight amino acids conserved in all of these QseC homologues. The corresponding QseC site-directed mutants were expressed and still able to autophosphorylate; however, four mutants demonstrated an increased basal level of phosphorylation. These mutants have differential flagellar, motility, LEE, and Shiga toxin expression phenotypes. We selected four mutants for more in-depth analyses and found that they differed in their ability to phosphorylate QseB, KdpE, and QseF. This suggests that these mutations in the periplasmic sensing domain affected the region downstream of the QseC signaling cascade and therefore can influence which pathway QseC regulates. IMPORTANCE In the foodborne pathogen EHEC, QseC senses AI-3, epinephrine, and norepinephrine, increases its autophosphorylation, and then transfers its phosphate to three RRs: QseB, QseF, and KdpE. QseB controls expression of flagella and motility, KdpE controls expression of the LEE region, and QseF controls the expression of Shiga toxin. This tripartite signaling pathway must be tightly controlled, given that flagella and the type three secretion system (T3SS) are energetically expensive appendages and Shiga toxin expression leads to bacterial cell lysis. Our data suggest that mutations in the periplasmic sensing loop of QseC differentially affect the expression of the three arms of this signaling cascade. This suggests that these point mutations may change QseC's phosphotransfer preferences for its RRs.KEYWORDS enterohemorrhagic E. coli, EHEC, LEE, QseC, two-component system E nterohemorrhagic Escherichia coli (EHEC) O157:H7 causes outbreaks of bloody diarrhea and hemolytic-uremic syndrome (HUS) worldwide. EHEC colonizes the colon, where it forms attaching and effacing (A/E) lesions on enterocytes. The hallmark of these lesions is the effacement of the microvilli and the rearrangement of the cytoskeleton to form a pedestal-like structure that cups the bacterium (1-3). A/E lesion formation requires the expression of a type three secretion system (T3SS) encoded

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

“…In contrast, the cooperating TCSs QseCB and QseEF sense and respond to the microbiota-derived quorum sensing molecule AI-3 and the host hormones adrenaline and noradrenaline to activate the transcription of the LEE genes (68,76,77) and allow colonization (78). Following autophosphorylation, QseC activates three RRs, namely, QseB (QseC's cognate RR), QseF, and KdpE (68), via a conserved 8-amino-acid motif in its periplasmic sensing domain (79). QseF is also phosphorylated by its cognate membrane-bound histidine kinase QseE (77).…”

“…QseB indirectly represses LEE4 and LEE5 via the sRNA GlmY while activating flagellar and motility genes (24,68,80). QseF promotes the expression of the Shiga toxin (68,79) and the T3SS effector TccP/EspF U , which is necessary for AE lesion formation in EHEC (81). Importantly, QseB (directly) and QseF (indirectly) also repress the FusKR TCS, alleviating fucose-mediated repression and thus shifting the regulatory balance toward the expression of the entire LEE (69).…”

Involvement of Two-Component Signaling on Bacterial Motility and Biofilm Development

“…The mutants also differed in their abilities to phosphorylate QseB, KdpE, and QseF. It was concluded that the mutations influenced the phosphotransfer preference of QseC (53).…”

“…Site-directed mutagenesis of the QseC periplasmic sensing domain yielded four mutants that all exhibited increased levels of phosphorylation but differed in their motility, LEE, and Shiga toxin expression phenotypes (53). The mutants also differed in their abilities to phosphorylate QseB, KdpE, and QseF.…”

Bacterial Chat: Intestinal Metabolites and Signals in Host-Microbiota-Pathogen Interactions

“…The QseBC two-component system (TCS) is found in Proteobacteria, including animal and plant pathogens (57,102,103). The QseC histidine sensor kinase increases its autophosphorylation in the presence of AI-3-Epi/NE molecules and relays this information to its cognate RR QseB and two noncognate RRs, QseF and KdpE (Fig.…”