Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli

Shimizu, Takeshi; Ohta, Yuko; Tsutsuki, Hiroyasu; Noda, Masatoshi

doi:10.1016/j.gene.2011.01.006

Cited by 25 publications

(32 citation statements)

References 45 publications

(38 reference statements)

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“…The Cpx system of E. coli is activated upon binding to hydrophobic surfaces, and this activation requires the outer membrane lipoprotein NlpE of E. coli (14). Therefore, to determine whether NlpE is involved in the surface signal sensing of hydrophobic surfaces in EHEC, we constructed EHEC chromosome-plasmid hybrid bioluminescent (C-P) reporter strains (6). For this purpose, we used a C-P reporter system utilizing the Photorhabdus luminescens luxCDABE operon that had previously been constructed to monitor the expressions of Shiga toxin genes in EHEC in real time (6).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, to determine whether NlpE is involved in the surface signal sensing of hydrophobic surfaces in EHEC, we constructed EHEC chromosome-plasmid hybrid bioluminescent (C-P) reporter strains (6). For this purpose, we used a C-P reporter system utilizing the Photorhabdus luminescens luxCDABE operon that had previously been constructed to monitor the expressions of Shiga toxin genes in EHEC in real time (6). The luxCDABE genes of P. luminescens were cloned and divided into a luxCDAB cassette and a luxE gene.…”

Section: Resultsmentioning

confidence: 99%

“…The pathogenesis of EHEC infections is known to involve the production of Shiga toxins (Stxs) that are similar to the Shiga toxin produced by Shigella dysenteriae type I (5). Production of Stxs in EHEC is enhanced upon contact with intestinal epithelial cells (6). The released Stxs act on intestinal epithelial cells to redistribute nucleolin so that they can bind to the bacterial surface protein intimin and promote attachment (7).…”

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The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion

2016

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Although the adhesion of enterohemorrhagic Escherichia coli (EHEC) is central to the EHEC-host interaction during infection, it remains unclear how such adhesion regulates virulence factors. Adhesion to abiotic surfaces by E. coli has been reported to be an outer membrane lipoprotein NlpE-dependent activation cue of the Cpx pathway. Therefore, we investigated the role of NlpE in EHEC on the adhesion-mediated expression of virulence genes. NlpE in EHEC contributed to upregulation of the locus of enterocyte effacement (LEE) genes encoded type III secretion system and to downregulated expression of the flagellin gene by activation of the Cpx pathway during adherence to hydrophobic glass beads and undifferentiated Caco-2 cells. Moreover, LysR homologue A (LrhA) in EHEC was involved in regulating the expression of the LEE genes and flagellin gene in response to adhesion. Gel mobility shift analysis revealed that response regulator CpxR bound to the lrhA promoter region and thereby regulated expressions of the LEE genes and flagellin gene via the transcriptional regulator LrhA in EHEC. Therefore, these results suggest that the sensing of adhesion signals via NlpE is important for regulation of the expression of the type III secretion system and flagella in EHEC during infection. Enterohemorrhagic Escherichia coli (EHEC) consists of multiple serotypes, among which O157:H7 is the one most commonly linked to epidemic and sporadic disease in humans throughout Japan, North America, and parts of Europe. EHEC infections are a primary cause of hemorrhagic colitis and hemolytic-uremic syndrome (HUS), and central nervous system complications are an important predictive factor for HUS and mortality in children (1-3). Since EHEC colonizes primarily in the human large intestine, the adhesion of EHEC to the large intestine wall is an important initial step in colonization.EHEC O157:H7 possesses an array of toxins and adhesins (cell adhesion proteins) that play a role in intestinal colonization (4). The pathogenesis of EHEC infections is known to involve the production of Shiga toxins (Stxs) that are similar to the Shiga toxin produced by Shigella dysenteriae type I (5). Production of Stxs in EHEC is enhanced upon contact with intestinal epithelial cells (6). The released Stxs act on intestinal epithelial cells to redistribute nucleolin so that they can bind to the bacterial surface protein intimin and promote attachment (7). Moreover, proteins associated with the locus of the enterocyte effacement (LEE)-encoded type III secretion system, including EspA, intimin, and Map, are required for the intimate adherence and histopathological changes known as attaching and effacing (A/E) lesions (8, 9). The type III secreted proteins of EHEC are involved in the initial adherence to Caco-2 cells (10). Promoters of these genes are activated upon bacterial contact with eukaryotic cells (11,12). In addition, evidence has been presented to support a role for EHEC O157:H7 flagella in epithelial colonization, not merely via motility/chemotaxis but a...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion

2016

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“…A strain containing a single-copy chromosome-plasmid luxE reporter fusion was constructed using a protocol adapted from that of Shimizu et al (40). To make a grvAB-luxE fusion, the kan cassette and flanking FLP recombination target sites were amplified from pKD4 (34) using primers pKD4forward/SacI and pKD4reverse/BamHI.…”

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confidence: 99%

“…Kanamycin resistance cassettes were removed using FLP recombinase as described previously (34), and the grvAB-luxE construct was validated using a combination of restriction mapping and DNA sequencing (MWG Operon). The lux operon genes luxCDAB were constitutively expressed in trans in TW14359 grvAB-luxE from pluxCDAB3 (40,41).…”

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Global Regulator of Virulence A (GrvA) Coordinates Expression of Discrete Pathogenic Mechanisms in Enterohemorrhagic Escherichia coli through Interactions with GadW-GadE

et al. 2016

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Global regulator of virulence A (GrvA) is a ToxR-family transcriptional regulator that activates locus of enterocyte effacement (LEE)-dependent adherence in enterohemorrhagic Escherichia coli (EHEC). LEE activation by GrvA requires the Rcs phosphorelay response regulator RcsB and is sensitive to physiologically relevant concentrations of bicarbonate, a known stimulant of virulence systems in intestinal pathogens. This study determines the genomic scale of GrvA-dependent regulation and uncovers details of the molecular mechanism underlying GrvA-dependent regulation of pathogenic mechanisms in EHEC. In a grvA-null background of EHEC strain TW14359, RNA sequencing analysis revealed the altered expression of over 700 genes, including the downregulation of LEE-and non-LEE-encoded effectors and the upregulation of genes for glutamate-dependent acid resistance (GDAR). Upregulation of GDAR genes corresponded with a marked increase in acid resistance. GrvA-dependent regulation of GDAR and the LEE required gadE, the central activator of GDAR genes and a direct repressor of the LEE. Control of gadE by GrvA was further determined to occur through downregulation of the gadE activator GadW. This interaction of GrvA with GadW-GadE represses the acid resistance phenotype, while it concomitantly activates the LEE-dependent adherence and secretion of immune subversion effectors. The results of this study significantly broaden the scope of GrvA-dependent regulation and its role in EHEC pathogenesis. IMPORTANCEEnterohemorrhagic Escherichia coli (EHEC) is an intestinal human pathogen causing acute hemorrhagic colitis and life-threatening hemolytic-uremic syndrome. For successful transmission and gut colonization, EHEC relies on the glutamate-dependent acid resistance (GDAR) system and a type III secretion apparatus, encoded on the LEE pathogenicity island. This study investigates the mechanism whereby the DNA-binding regulator GrvA coordinates activation of the LEE with repression of GDAR. Investigating how these systems are regulated leads to an understanding of pathogenic behavior and novel strategies aimed at disease prevention and control.

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Nitric oxide‐enhanced Shiga toxin production was regulated by Fur and RecA in enterohemorrhagic Escherichia coli O157

et al. 2017

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Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Nitric oxide (NO), which acts as an antimicrobial defense molecule, was found to enhance the production of Stx1 and Stx2 in EHEC under anaerobic conditions. Although EHEC O157 has two types of anaerobic NO reductase genes, an intact norV and a deleted norV, in the deleted norV‐type EHEC, a high concentration of NO (12–29 μmol/L, maximum steady‐state concentration) is required for enhanced Stx1 production and a low concentration of NO (~12 μmol/L, maximum steady‐state concentration) is sufficient for enhanced Stx2 production under anaerobic conditions. These results suggested that different concentration thresholds of NO elicit a discrete set of Stx1 and Stx2 production pathways. Moreover, the enhancement of Shiga toxin production in the intact norV‐type EHEC required treatment with a higher concentration of NO than was required for enhancement of Shiga toxin production in the deleted norV‐type EHEC, suggesting that the specific NorV type plays an important role in the level of enhancement of Shiga toxin production in response to NO. Finally, Fur derepression and RecA activation in EHEC were shown to participate in the NO‐enhanced Stx1 and Stx2 production, respectively.

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Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli

Cited by 25 publications

References 45 publications

The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion

The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion

Global Regulator of Virulence A (GrvA) Coordinates Expression of Discrete Pathogenic Mechanisms in Enterohemorrhagic Escherichia coli through Interactions with GadW-GadE

Nitric oxide‐enhanced Shiga toxin production was regulated by Fur and RecA in enterohemorrhagic Escherichia coli O157

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