The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis

Abstract

Bacterial pathogens sense host cues to activate expression of virulence genes. Most of these signals are sensed through histidine kinases (HKs), which comprise the main sensory mechanism in bacteria. The host stress hormones epinephrine (Epi) and norepinephrine are sensed through the QseC HK, which initiates a complex signaling cascade to regulate virulence gene expression in enterohemorrhagic Escherichia coli (EHEC). Epi signaling through QseC activates expression of the genes encoding the QseEF 2-component system. QseE is an HK, and QseF is a response regulator. Here, we show that QseE is a second bacterial adrenergic receptor that gauges the stress signals Epi, sulfate, and phosphate. The qseEF genes are organized within an unusual operonic structure, in that a gene is encoded between qseE and qseF. This gene was renamed qseG, and it was shown to encode an outer membrane (OM) protein. EHEC uses a type III secretion system (TTSS) to translocate effector proteins to the epithelial cells that rearrange the host cytoskeleton to form pedestal-like structures that cup the bacterium. QseE, QseG, and QseF are necessary for pedestal formation. Although QseE and QseF are involved in the transcriptional control of genes necessary for pedestal formation, QseG is necessary for translocation of effectors into epithelial cells. QseG is an OM protein necessary for translocation of TTSS effectors that also works in conjunction with a 2-component signaling system that senses host stress signals.

Fig. 1.

Autoregulation of the qseEGFglnB operon. (A) The qseEGFglnB operon contains 2 σ⁷⁰ promoters upstream of qseE and glnB, and a predicted σ⁵⁴ upstream of qseG. (B) The qseE-lacZ fusions within WT and qseE, qseG, and qseF nonpolar mutants. (C) qRT-PCR analysis examining the expression of qseE, qseG, qseF, and glnB in WT and qseE, qseG, and qseF mutants. Error bars represent the standard deviation of 3 independent experiments. ∗, P < 0.05; ∗∗, P < 0.005.

Fig. 2.

Cellular localization of QseG. (A) Depiction of the localization of QseE, QseF, and QseG. (B) Membrane fractions showing that QseG localizes to the OM.

Fig. 3.

QseG is required for pedestal formation by EHEC. (A) FAS assays. Arrows indicate bacteria forming AE lesions. (Magnification: 100X.) (B) Western blots of whole-cell lysates (Top) and secreted proteins (Bottom) with anti-EspB, anti-EspA, and anti-Tir antisera. (C) Transcriptional regulation of espFu using an espFu-lacZ fusion. β-Galactosidase activity was measured in Miller units, and the triangle means the differences between qseG and WT were not statistically significant. (D) Detection of the EspA filament by using immunofluorescence with anti-EspA antibody and FITC-labeled secondary antibody. An EPEC espA mutant was used as a negative control, and the espA mutant expressing the EHEC EspA was used as a positive control. (Magnification: 100X.) (E) Translocation of Tir to host cells by using tir-cyaA fusions. WT levels of translocation were set at 100%. (F) Expression of the LEE genes by using qRT-PCR. Error bars in C, E, and F indicate the standard deviation of 3 independent experiments.

Fig. 4.

QseE autophosphorylation in response to various agonists. (A) Schematic of QseE's orientation in the liposome. (B) QseE phosphorylation in response to nitrogen, iron, Epi, AI-3, AI-2, and fumarate. (C) QseE's response to ammonium sulfate and glutamine. (D) QseE's response to phosphate and sulfate sources. (E) QseE's response to ammonium sources containing phosphate or sulfate counter ions. (F) Transcription of espFu-lacZ in WT and qseE in the absence and presence of phosphate and sulfate. ∗, P < 0.05; ∗∗, P < 0.005.