Activation of the Cpx Envelope Stress Response Down-Regulates Expression of Several Locus of Enterocyte Effacement-Encoded Genes in Enteropathogenic Escherichia coli

Abstract

The Cpx two-component system regulates an extracytoplasmic stress response that functions to rid the envelope of misfolded and mislocalized proteins that may interfere with normal cellular processes. The Cpx pathway is also involved in pathogenesis. This study investigated the role of the Cpx response in enteropathogenic Escherichia coli (EPEC) type III secretion (T3S). It was determined that a functional Cpx pathway is not required for T3S but that pathway activation inhibits secretion by reducing the cellular pools of T3S substrates. The EPEC T3S system structural components, as well as a number of its substrates, are encoded on the locus of enterocyte effacement (LEE) pathogenicity island. Transcriptional fusions to the five major operons of the LEE were constructed and examined under Cpx pathway-activating conditions. Induction of the Cpx response caused a decrease in the transcription of several LEE operons, with the most pronounced effect on LEE4 and LEE5. Collectively, these two operons encode components of the T3S translocation apparatus, the bacterial adhesin intimin, and the translocated bacterial receptor Tir. These data show for the first time that activation of the Cpx envelope stress response in EPEC inhibits T3S of both translocators and effectors, likely through down regulation of LEE transcription. Coupled with recent findings, our results suggest that Cpx-mediated down regulation of virulence is a conserved theme in a number of bacterial pathogens.

Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

“…Given that reduced expression of bundlin does not account for the ability of EPEC cells to tolerate a double mutation of bfpB and bfpF, we hypothesized that the envelope stress response pathway was upregulated in UMD947 and protected the cell envelope from damage. BFP proteins encounter periplasmic Cpx pathway effectors such as DegP and DsbA once they cross the IM (48); indeed, some low level of Cpx activity is required for pilus biogenesis (49,82). To assess the activity of the cell envelope stress response system in wild-type and various mutant strains, we constructed a luciferase reporter for degP transcriptional activation, pNLP27-Cm, that is compatible with the mutants.…”

“…Luciferase reporter assays were performed as previously described (49,82). In four independent experiments, quadruplicate colonies were grown overnight and diluted 1:100 in DMEM with antibiotics and buffered with 0.1 M Tris (pH 7.5).…”

Dual temporal transcription activation mechanisms control cesT expression in enteropathogenic Escherichia coli

“…A LEE5-lux construct was tested in wild-type EPEC and Dler for light production, and as expected, its light-producing activity was severely reduced (Fig. 4b), as reported earlier MacRitchie et al, 2008). Therefore, under these experimental conditions, Ler and GrlA did not contribute to the observed activity of cesTp-lux, and GrlR did not repress cesTp-lux activity.…”

“…A promoterless lux plasmid construct (pJW15; MacRitchie et al, 2008) containing the luxCDABE cassette from Photorhabdus luminescens was used as a starting plasmid for all lux plasmid constructs in this study. This plasmid has been used before in EPEC for luciferase reporter studies (MacRitchie et al, 2008).…”

“…A promoterless lux plasmid construct (pJW15; MacRitchie et al, 2008) containing the luxCDABE cassette from Photorhabdus luminescens was used as a starting plasmid for all lux plasmid constructs in this study. This plasmid has been used before in EPEC for luciferase reporter studies (MacRitchie et al, 2008). A DNA fragment was PCR-amplified from EPEC genomic DNA with primers EB1 and EB2 (Table 2 has a complete list of primers used in this study) and then treated with BamHI and XhoI followed by cloning into BamHI-and Xho-treated pJW15 to generate E. Brouwers, I. Ma and N. A. Thomas cesTp1-lux.…”