The LysR-type transcriptional regulator QseD alters type three secretion in enterohemorrhagic Escherichia coli and motility in K-12 Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 responds to the host-produced epinephrine and norepinephrine, and bacterially produced autoinducer 3 (AI-3), through two-component systems. Further integration of multiple regulatory signaling networks, involving regulators such as the LysR-type transcriptional regulator (LTTR) QseA, promotes effective regulation of virulence factors. These include the production of flagella, a phage-encoded Shiga toxin, and genes within the locus of enterocyte effacement (LEE) responsible for attaching and effacing (AE) lesion formation. Here, we describe a new member of this signaling cascade, an LTTR heretofore renamed QseD (quorum-sensing E. coli regulator D). QseD is present in all enterobacteria but exists almost exclusively in O157:H7 isolates as a helix-turn-helix (HTH) truncated isoform. This "short" isoform (sQseD) is still able to regulate gene expression through a different mechanism than the full-length K-12 E. coli "long" QseD isoform (lQseD). The EHEC Delta qseD mutant exhibits increased expression of all LEE operons and deregulation of AE lesion formation. The loss of qseD in EHEC does not affect motility, but the K-12 Delta qseD mutant is hypermotile. While the lQseD directly binds to the ler promoter, encoding the LEE master regulator, to repress LEE transcription, the sQseD isoform does not. LTTRs bind to DNA as tetramers, and these data suggest that sQseD regulates ler by forming heterotetramers with another LTTR. The LTTRs known to regulate LEE transcription, QseA and LrhA, do not interact with sQseD, suggesting that sQseD acts as a dominant-negative partner with a yet-unidentified LTTR.

FIG. 1.

In EHEC 86-24, the intact qseD operon encodes a truncated QseD protein. (A) Cartoon representation of the qseD operon (untranslated region shaded) and the surrounding genes. (B) Comparison of the qseD sequences of EHEC 86-24 and K-12 E. coli, where boxed regions include the ribosome binding site (RBS) and the translational start site, and boxed and shaded regions include the stop codon, the alternative RBS, and the translational start codon in EHEC 86-24. (C) RT-PCR using the P₁/P₂ primer set and either cDNA (1), gDNA (2), or RNA (3) harvested and/or synthesized from EHEC 86-24 as a PCR template. (D) Cartoon representation of the full-length QseD (lQseD) and truncated QseD (sQseD) protein products from K-12 E. coli and EHEC 86-24, respectively. In EHEC, loss of the translated helix-turn-helix (HTH) domain does not appear to affect translation of the cofactor recognition/oligomerization domain (Co-In./Oligo.). (E) qRT-PCR of qseD expression levels in DMEM at lag phase (OD₆₀₀ of 0.2), mid-log phase (OD₆₀₀ of 0.5), late log phase (OD₆₀₀ of 1.0), and stationary phase (OD₆₀₀ of 1.5) in K-12 E. coli and EHEC 86-24.

FIG. 2.

QseD regulates the qseD operon and surrounding genes in E. coli. qRT-PCR of the qseD operon (iadA, yjiG, and yjiH) and the surrounding genes (kptA, yjiC, and yjiD) in EHEC 86-24, the ΔqseD mutant, the ΔqseD mutant complemented in trans with qseD (86-24), and the ΔqseD mutant complemented in trans with qseD (K-12) grown in DMEM (OD₆₀₀ of 1.0).

FIG. 3.

QseD affects motility in K-12 E. coli but not in EHEC 86-24. (A) Heat maps generated from microarray analysis depicting the differential regulations of the flagellar regulon in K-12 ΔqseD versus WT K-12 E. coli. (B) Motility plates of WT K-12 E. coli and WT EHEC 86-24 and their corresponding ΔqseD mutants, ΔqseD mutants complemented in trans with qseD (K-12), and ΔqseD mutants complemented in trans with qseD (86-24). Graphs plotting the measurement of the diameters of motility halos of three separate experiments are shown. (C) qRT-PCR of flhD, motA, and fliC in WT K-12 E. coli, the ΔqseD mutant, the ΔqseD mutant complemented in trans with qseD (K-12), and the ΔqseD mutant complemented in trans with qseD (86-24) grown in LB (OD₆₀₀ of 1.0). (D) Western blots of FliC from WT K-12 E. coli and WT EHEC 86-24 and their corresponding ΔqseD mutants, ΔqseD mutants complemented in trans with qseD (K-12), and ΔqseD mutants complemented in trans with qseD (86-24).

FIG. 4.

QseD regulates the LEE pathogenicity island but not Stx in EHEC 86-24. (A) qRT-PCR of ler, escV, escC, and espA in WT EHEC 86-24, the ΔqseD mutant, the ΔqseD mutant complemented in trans with qseD (86-24), and the ΔqseD mutant complemented in trans with qseD (K-12) grown in DMEM (OD₆₀₀ of 1.0). (B) qRT-PCR of stx_2a in WT EHEC 86-24, the ΔqseD mutant, the ΔqseD mutant complemented in trans with qseD (86-24), and the ΔqseD mutant complemented in trans with qseD (K-12) grown in DMEM (OD₆₀₀ of 1.0). (C) FAS assays depicting formation of AE lesions on HeLa cell monolayers by WT EHEC, the EHEC ΔqseD mutant, and the EHEC ΔqseD mutant complemented in trans with qseD (86-24). The bottom panel shows the EHEC ΔqseD mutant at 3× zoom.

FIG. 5.

K-12 E. coli lQseD binds to the ler promoter. Shown are EMSAs of the ler promoter with purified QseA (A), K-12 E coli lQseD (B), EHEC sQseD (C), and an attempt to prevent QseA binding of ler by heterodimer formation with sQseD (D).

FIG. 6.

EHEC 86-24 sQseD and K-12 E. coli lQseD do not interact with QseA. Shown are representative yeast two-hybrid nonselective (His⁺) plates and selective (His⁻) plates, depicting the potential protein-protein interactions of the LysR-like proteins QseA (A), sQseD (B), and LrhA (C).

FIG. 7.

Model of the regulatory role of lQseD and sQseD in EHEC 86-24. In non-O157:H7 EHEC strains, the presence of full-length “long” QseD (lQseD) represses ler and LEE transcription. In the absence of lQseD, an as-yet-unidentified LysR protein (LysR-X) activates LEE transcription. However, in O157:H7 EHEC strains, the presence of truncated “short” QseD (sQseD) represses ler and LEE transcription, presumably through dominant-negative interactions with LysR-X, resulting in incomplete DNA remodeling and transcriptional activation.

FIG. 8.

Evolution and prevalence of the various isoforms of QseD in E. coli. Shown is a cartoon representation of the evolution of EPEC and EHEC from the prototypical nonpathogenic K-12 ancestor. Solid (gray) strains represent the presence of lQseD, and open (white) strains represent the presence of sQseD (designed using original data from reference 18).