Mutations in hns reduce the adherence of Shiga toxin-producing E. coli 091:H21 strain B2F1 to human colonic epithelial cells and increase the production of hemolysin

Scott, Maria E.; Melton‐Celsa, Angela R.; O'Brien, Alison D.

doi:10.1016/s0882-4010(03)00002-0

Cited by 20 publications

(12 citation statements)

References 29 publications

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“…Scott et al (30) performed a transposon mutagenesis in a Shiga toxin-producing E. coli (STEC) 091:H21 strain B2F1, which does not carry the LEE genes, to identify genes involved in binding to human colonic epithelial T84 cells. They found that several of the mutants displayed reduced adherence to T84 cells, and some of those mutants mapped to the hns gene.…”

Section: Discussionmentioning

confidence: 99%

Contribution of the Ler- and H-NS-Regulated Long Polar Fimbriae of Escherichia coli O157:H7 during Binding to Tissue-Cultured Cells

et al. 2008

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The expression of the long polar fimbriae (LPF) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by a tightly regulated process, and, therefore, the role of these fimbriae during binding to epithelial cells has been difficult to establish. We recently found that histone-like nucleoid-structuring protein (H-NS) binds to the regulatory sequence of the E. coli O157:H7 lpf1 operon and "silences" its transcription, while Ler inhibits the action of the H-NS protein and allows lpf1 to be expressed. In the present study, we determined how the deregulated expression of LPF affects binding of EHEC O157:H7 to tissue-cultured cells, correlating the adherence phenotype with lpf1 expression. We tested the adherence properties of EHEC hns mutant and found that this strain adhered 2.8-fold better than the wild type. In contrast, the EHEC ler mutant adhered 2.1-fold less than the wild type. The EHEC hns ler mutant constitutively expressed the lpf genes, and, therefore, we observed that the double mutant adhered 5.6-fold times better than the wild type. Disruption of lpfA in the EHEC hns and hns ler mutants or the addition of anti-LpfA serum caused a reduction in adhesion, demonstrating that the increased adherence was due to the expression of LPF. Immunogold-labeling electron microscopy showed that LPF is present on the surface of EHEC lpfA ؉ strains. Furthermore, we showed that EHEC expressing LPF agglutinates red blood cells from different species and that the agglutination was blocked by the addition of anti-LpfA serum. Overall, our data confirmed that expression of LPF is a tightly regulated process and, for the first time, demonstrated that these fimbriae are associated with adherence and hemagglutination phenotypes in EHEC O157:H7.

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Section: Discussionmentioning

confidence: 99%

Contribution of the Ler- and H-NS-Regulated Long Polar Fimbriae of Escherichia coli O157:H7 during Binding to Tissue-Cultured Cells

et al. 2008

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“…The STEC strain B2F1 used in this study has two copies of stx 2d-activatable , which are referred to below as stx 2d1-activatable and stx 2d2-activatable for clarity. The plasmid-cured derivative of B2F1 no longer produces detectable hemolytic activity (40).…”

Section: Methodsmentioning

confidence: 99%

Comparative Pathogenicity of Escherichia coli O157 and Intimin-Negative Non-O157 Shiga Toxin-Producing E. coli Strains in Neonatal Pigs

et al. 2003

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We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H ؊ strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysinencoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.Serotype O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause the majority of sporadic and multiperson outbreaks of bloody diarrhea in the United States (15). However, non-O157 STEC strains were the source of three such outbreaks of bloody diarrhea (1) and an apparent cluster of three cases of hemolytic-uremic syndrome (HUS) (5, 26) and may be responsible for 20% (2) or even 50% (13) of all STEC infections. Up to 10% of patients with hemorrhagic colitis due to infection with an O157 strain develop HUS, a sequela that includes serious kidney damage. The risk of HUS for patients infected with non-O157 STEC is not as clear, but it could be as high as that for patients infected with O157 strains (29).STEC can be grouped by the array of potential virulence factors that they express. All STEC strains produce one or more Stxs. There are two main groups of Stxs (also called verotoxins), Stx1 and Stx2, which have the same enzymatic activity and general structure but are not cross-neutralized by heterologous antisera (34). The Stx2 group contains variants that include Stx2c (39), Stx2d-activatable (whose toxicity is increased by elastase from intestinal mucus [31]) (23, 43), Stx2d-nonactivatable (36), Stx2e (associated with edema disease in weaned pigs [48]), and Stx2f (isolated from feral pigeons and from a child with diarrhea [33,38]).A number of STEC strains have the same general constellation of pathogenic factors as the O157:H7 isolates. These strains not only produce Stxs but also have the capacity to cause attaching and effacing (A/E) lesions in the intestines of animals or in tissue culture models (8). A/E lesion formation is mediated by products of the locus of enterocyte effacement (LEE) found in enterohemorrhagic E. coli O157 strains and some non-O157 STEC strains, such as all O26:H11 strains and most O111:H ؊ strains (20). The LEE...

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“…The global transcriptional regulator H-NS is known to bind to curved and AT-rich DNA sequences upstream of several defined UPEC virulence genes (85), including genes encoding toxins (86)(87)(88)(89) and autotransporter proteins (8,10,90). To investigate the effect of H-NS on vat transcription, the levels of Vat expression were compared by Western blot analysis of supernatant fractions prepared from WT CFT073, CFT073 vat, CFT073 vatX, CFT073 hns, and a CFT073 vatX hns double mutant (Fig.…”

Section: Transcription Of the Vat Gene Is Directly Repressed By H-nsmentioning

confidence: 99%

Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli

et al. 2016

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The vacuolating autotransporter toxin (Vat) contributes to uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator; we termed this gene vatX. The vat-vatX genes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, the hns gene was mutated in CFT073 (to generate CFT073 hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073 hns compared to that in wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein of Enterobacteriaceae (SPATE) secreted by UPEC during infection. IMPORTANCEUropathogenic Escherichia coli (UPEC) is the major cause of hospital-and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation of vat was demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream gene vatX (encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected with vatpositive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor. U rinary tract infections (UTIs) are one of the most common human infections, affecting 40 to 50% of women and approximately 12% of men globally (1). UTIs are ascending infections and can involve infection of the bladder (cystitis), kidneys (pyelonephritis), or dissemination into the bloodstream (urosepsis). Uropathogenic Escherichia coli (UPEC) strains ...

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Mutations in hns reduce the adherence of Shiga toxin-producing E. coli 091:H21 strain B2F1 to human colonic epithelial cells and increase the production of hemolysin

Cited by 20 publications

References 29 publications

Contribution of the Ler- and H-NS-Regulated Long Polar Fimbriae of Escherichia coli O157:H7 during Binding to Tissue-Cultured Cells

Contribution of the Ler- and H-NS-Regulated Long Polar Fimbriae of Escherichia coli O157:H7 during Binding to Tissue-Cultured Cells

Comparative Pathogenicity of Escherichia coli O157 and Intimin-Negative Non-O157 Shiga Toxin-Producing E. coli Strains in Neonatal Pigs

Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli

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